development of some useful bioaffinity based …ir.amu.ac.in/3241/1/t 5791.pdf · of some useful...
TRANSCRIPT
DEVELOPMENT OF SOME USEFUL BIOAFFINITY BASED TECHNIQUES
. * « " » • *
T H E S I S SUBMITTED FOR THE AWARD OF THE DEGREE OF
Bottor of $i)tlo£(opi)p IN
i \ BIOCHEMISTRY
BY
H I N A J A M I L
DEPARTMENT OF BIOCHEMISTRY FACULTY OF LIFE SCIENCES
ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)
2003
^ i
•%••
?*•
Pedt
JIJ^IS^
AN
"j\.=»^;«<
ompnfor iBBSiS
83ll\X^'''4i^'-.-- -ff* T"^- r'*.
^ % ^ a . u, - - ^
• • • " I ff >•:'•.:
iv.'
•5 •• ..» 7 r : "•»•
0 1 J j : i 2GG5 * :, » « .• J" If, -
T5791
JIMAlf AHIH
(A:
Phones: Int: Office—291 Ext. (0571)2700741
Telex; 564 - 230 AMU IN
DEPARTMENT OF BIOCHEMISTRY FACULTY OF LIFE SCIENCES
ALIGARH MUSLIM UNIVERSITY ALIGARH-202 0 0 2 (INDIA)
Dated.
Certificate
I certify that the work presented in this thesis entitled ''Development
of some useful Bioaffinity Based Techniques^' has been carried
out by Ms. Hina Jamil under my supervision. It is original in nature
and has not been submitted for any other degree.
1. M. Saleemuddin Professor
J^lcknorvlcdgcntents
D am yrojoundly gratejul and exceedingly obliged to my supervisor ^rojessor
c7W. Saleemuddin, who provided excellent guidance and indefatigable interest
at each and every stage oj this thesis. Hie gave me vision and new directions
without which it would have been impossible jor me to complete this research
work. The thought stimulating discussions that 3 had with him perfected many
concepts and enabled me in articulating the novel aspects oj this work.
D would like to express my deep sense oj gratitude to ^rojessor ^Masood
iJ^hmad, Chairman oj the department oj biochemistry who provided me all
the jacilities in pursuance oj my research.
The valuable comments and suggestions provided by all the jaculty members
instilled enthusiasm and ensured smooth continuation oj my thesis.
D am highly indebted to ^r Owais and ^ r ^izwan oj the department oj
biotechnology jor their active help and guidance in the experiments.
D would also like to express my genuine thanks to my seniors r . J^tia, ^ r
Tlljat, ^r Hiemlata and ^r JAthar who extended their cooperation and
guidance. (J^ token oj appreciation to my lab mates ^ana, Sonali, Sonish,
D^aghma, JArshi, Suhail, J^mjad, Hiashij, J^rivarasu, J^sjar, Showket,
JAzhar, Sandeep, Hiahkashan, ^ouzia. Dram, Drjana, ^asha and Shamila
jor their nice company. D am especially gratejul to ^awan jor being a wonderjul
lab colleague.
cMy heartjelt thanks to my jriends Samina, Shahid, %aleem and Sheema
jor their ajjable company and amicable help. The co-operation and help J got
jrom my jriend Sadaj is jar beyond one would expect jrom a colleague and an
intellectual companion.
D would like to gratejuUy acknowledge the help oj the library stafj oj
biochemistry department jor providing me numerous reference materials.
!2ielp rendered by ^Mr. LMujtaba JAhmed jor his excellent photographic
techniques, cMr. Saleemuddin jor transforming my scribbles into elegant
jigures, and i_Mr. ^aheer JAhmad jor expert word processing oj the
manuscript deserve words oj appreciation.
finally D would like, to thank my parents jor their unstinting support,
encouragement and patience through the so many long hours spent in carrying
out my research work, and jor teaching me the value oj hard work. D can
never jorget the love and ajjectionate treatment D got jrom my grand father
^adam Shree ^roj. iSM. Shaji, my husband, and jrom the youngsters,
J^qeel, Suhail, ^JAdeeba, JAmir, ^ainab and Hiumaira who qave me
moments oj joy despite the arduous schedule.
Ulina ^amil
CONTENTS
List of Abbreviations (i)
List of Figures (ii)
List of Tables (v)
1.0 Introduction
1.1 Immunohistochemistry 1
L2 Immunochemical techniques 2
1.2.1 Enzyme amplification systems 5
1.2.2 Immunogold techniques 7
1.2.3 Immunofluorescence 8
1.2.4 Immunoblotting/dot immunobinding 11
1.2.4.1 Immobilizing matrices in solid phase 12
irmnunoassays
1.2.4.2 Applications ofimmunoblotting and dot 14
immunobinding
1.3 Human serum albumin 17
1.3.1 Functions of albumin 18
1.4 Immimoaffinity based immobilization 19
1.5 Immunoglobulins 21
1.5.1 Immunoglobulin G 22
1.6 Porcine pepsin 22
1.7 Procedures for producing F(ab)'2 24
1.8 Use oflMAC for purification of immunoglobulins. 29
1.9 Objective of the work 32
2.0 Materials 33
3.0 Methods
3.1 Immunological Procedures 35
3.1.1 Preparationof the antigen 35
3.1.2 Immunization of rabbits 35
3.1.3 Immunodifflission 3 5
3.1.4 Isolation and purification of immunoglobulin G 36
from the antiserum
3.1.5 Ion exchange chromatography 36
3.1.6 Preparationof F(ab) monomer 37
3.1.7 Formation of F(ab)', 37
3.1.8 Purification of specific IgG. 37
3.1.9 ELISA 38
3.1.10 Immunoaffinity layering in ELISA 3 9
3.1.11 Dot blot/western blot 39
3.2 Colorimetric Analysis
3.2.1 Determination of protein concentration 40
3.2.2 Carbohydrate estimation 41
3.2.3 Aminogroup estimation 41
3.2.4 Assay of Pepsin 41
3.3. Chemical modification of Pepsin. 42
3.4 Immobilized Metal ion Affinity Chromatography (MAC) 42
3.5 Slab gelelectrophoresis
3.5.1 Polyacrylamide gel electrophoresis (PAGE) 42
3.5.2 SDS polyacrylamide gel elecgtrophoresis 43
(SDS PAGE)
3.5.3 Staining procedures 44
3.6 Spectral Analysis
3.6.1 UV absorption spectroscopy 44
3.6.2 Fluorescence spectroscopy 45
3.6.3 Circular dichroism spectroscopy 45
3.7 Size exclusion high performance liquid 46
chromatography (HPLC)
4.0 Results
4.1 Immunogenicity of HSA and cross reactivity of anti- 47
HS A antibodies.
4.2 Immunoaffmity layering
4.2.1 Immunodot analysis ofHSA by immunoaffinity 47
layering
4.2.2 Effect of time of incubation 50
4.2.3 Immunodot analysis ofHSA with intact 53
anti-HSA-IgG and F(ab)'2 derived there of.
4.2.4 Immunoaffinity layering using glutaraldehyde 5 5
crosslinked anti- HSA-IgG
4.2.5 Immunodot analysis of HSA using affinity purified 55
anti-HSA specific antibody.
4.2.6 Immunodot analysis ofHSA of human serum 58
using the multiple incubation assay
4.2.7 Studies of albumin aggregation using multiple 61
incubation assay
4.2.8 Multiple incubation assay in ELISA 63
4.2.9 Multiple incubation assay in ELISA using F(ab)', 63
5.0 Modification of Pepsin
5.1 Preparation of Pepsin modified with histidine 66
5.2 Properties of modified pepsin 67
5.2.1 Kinetic behaviour
5.2.1.1 Effect of substrate concentration 67
5.2.1.2 Effect of temperature 67
5.2.1.3 Effect of pH 67
5.2.2 Electrophoretic behaviour 6 7
5.2.3 Autolysis ofpepsin and modified pepsin 71
5.2.4 Spectral studies
5.2.4.1 UV spectroscopy 71
5.2.4.2 Fluorescence spectroscopy 71
5.2.4.3 Circular dichroism of native and modified pepsin 74
5.2.5 Binding ofnative and modified pepsin on 74
Cu^^-IDA Sepharose
5.2.6 ElutionofPepsin and modified pepsin bound to 74
Cu^'-IDA with EDTA.
5.3 Purification and pepsinization of IgG for F(ab)'2 formation.
5.3.1 Purification ofIgG from anti-HSA antiserum. 79
5.3.2 Molecular weight of IgG 79
5.3.3 Cleavage ofIgG with modified pepsin and 82
purification of FCab)' by IMAC
5.3.3.1 pH dependent cleavage of IgG 82
5.3.3.2 Temperature dependent cleavage of IgG 85
5.3.3.3 Time dependent cleavage of IgG 85
5.4 Binding of F(ab) monomer to Cu^*-IDA Sepharose 85
5.5 Isolation of F(ab)'2 from unfractionated serum 85
5.6 Size exclusion high performance liquid 89
chromatography (HPLC)
5.7 IsolationofF(ab)'2dimer in a single tube 89
5.7.1 IsolationofF(ab)'2 in a single tube from 93
various mammalian species.
5.8 Antigenicity of F(ab)'2 isolated from IgG 93
6.0 Discussion 97
7.0 Summary 113
8.0 Bibliography 116
LIST OF ABBREVIATIONS
IgG
F(ab)',
F(ab)
Fc
EDA
IMAC
S
SDA
c„ c. v„ V.
BSA
HSA
ELISA
BLEIA
PCFIA
NC
CNBr
PAGE
HRP
A?
EDTA
TCA
ADRE
Immunoglobulin G
Fragment antigen binding (divalent)
Fragment antigen binding (monovalent)
Fragment crystallizable
Imino diacetic acid
Immobilized metal afiinity chromatography
Suedbergunit
Sodium dodecyl sulphate
Constant heavy chain domain
Constant light chain domain
Variable heavy chain domain
Variable light chain domain
Bovine serum albumin
Human serum albumin
Enzyme linked immunosorbent assay
Bio luminescent enzyme immoassay
Particle concentration fluorescence immunoassay
Nitro-cellulose
Cyanogen bromide
Polyacrylamide gel electrophoresis
Horse raddish peroxidase
Alkaline phosphatase
Ethylene diamine tetraacetic acid
Trichloroacetic acid
Analyte dependent reporter enzyme
(i)
LIST OF FIGURES
Figure 1.1 Schematic representation ofimmunologic labeling methods. 4
Figure 1.2 A flow chart for the HRP mediated deposition of AP and HRP 6
in a mouse IgG assay
Figure 1.3 Diagrammatic representation ofvarious fluorescence immunoassays 9
Figure 1.4 Three dimensional structure of immunoglobulin molecule 23
Figure 1.5 Schematic representation of cleavage sites of IgG by papain 25
and pepsin
Figure 1.6 A diagrammatic representation of the principle of IMAC. 30
Figure 4.1 Titre determination ofthe raised antisera by ELIS A 48
Figure 4.2 (A) Ouchterlony double diffusion of HSA against anti-HSA 49
antiserum and IgG fractions isolated thereof (B) Ouchterlony
double dififiasion of anti-HS A-IgG against purified HS A,
human serum and BS A
Figure 4.3 Immunodot analysis of HS A using the antiHS A antibody and 51
HRP conjugate using multiple incubation assay.
Figure 4.4 Effect of incubation time on immunodot assay. 52
Figure 4.5 Immunodot analysis of HSA with intact anti-HSA-IgG and 54
F(ab)'2 thereof
Figure 4.6 Gel electrophoresis of glutaraldehyde treated anti-HS A IgG 5 6
Figure 4.7 Immunodot analysis of HSA by immunoaffinity layering using native 5 7
anti-HSA-IgG (A) and gluteraldehyde linked anti-HSA-IgG (B).
Figure 4.8 Immunodot analysis of HSA using affinity purified anti- 59
HSA-IgG on nitrocellulose strips by multiple incubation assay.
Figure 4.9 Immunodot analysis of HSA of human serum by immunoaffinity 60
layering.
Figure 4.10 Studies of albumin aggregation using multiple incubation assay 62
(ii)
Figure 4.11 Multiple incubation assay on ELISA plate 64
Figure 4.12 Multiple incubation assay on ELISA plate using FCab)' 65
Figure 5.1 Line Weaver Burk Plot for haemoglobin digestion by pepsin 68
and modified pepsin preparations.
Figure 5.2 Temperatute activity profiles of native and modified pepsin 69
Figure 5.3 pH activity profiles ofnative and modified pepsin preparations. 69
Figure 5- 4 (A) Gel electrophoresis ofnative and modified pepsin. 70
(B) Molecular weight determination of modified pepsin by weber
and Osbom procedure.
Figure 5.5 Autolysis ofnative and modified pepsin 72
Figure 5.6 Ultravoilet spectra ofnative and modified pepsin 73
Figure 5.7 Flourescence spectra ofnative and modified pepsin 73
Figure 5.8 Far UV-CD spectra ofnative and modified pepsin 75
Figure 5.9 Near UV-CD spectra ofnative and modified pepsin 76
Figure 5.10 Binding ofnative and modified pepsin to Cu^^-IDASepharose 77
Figure 5.11 Elution profile ofnative and modified pepsin for Cu^*-IDA Sepharose 78
Figure 5.12 Purification of IgG 80
Figure 5.13 (A) SDS-PAGEofIgG during various stages of purification 81
(B) Molecular weight determination by Weber and Osbom procedure
Figure 5.14 PepsinizationofIgG 83
Figure 5.15 pH dependent cleavage of IgG 84
Figure 5.16 Temperature dependent cleavage of IgG 86
Figure 5.17 Time dependent cleavage of IgG 87
Figure 5.18 Binding of F(ab) monomer to Cu^"-IDA 88
Figure 5.19 SDS-PAGEofserum digested with modified pepsin and binding 90
to IDA-Sepharose loaded with various metal ions
Figure 5.20 High performance gel filtration chromatography of IgG and 91
its pepsin digests
(iii)
Figure 5.21 High performance gel filtration chi-omatography of seruni and 92
its pepsin digests
Figure 5.22 Production of F(ab)'2 formation in a single tube 94
Figure 5.23 Preparation ofFCaby jfromlgG derived fi:om various mammals 95
Figure 5.24 Ouchterlony double diffusion of anti-HSA-IgG and anti-HSA- 96
IgG-F(ab)'2 against HSA.
Figure 6.1 Diagrammatic representation of the multiple incubation 99
procedure.
Figure 6.2 Diagrammatic representation of the procedure of 106
coupling histidine to lysine residue of pepsin with the
help of glutaraldehyde
Figure 6.3 Diagrammatic representation of the preparation of FCab)' 112
from IgG and its separation from uncleaved IgG, pepsin
and Fc fragment
(iv)
LIST OF T A B L E S
Table LI Mechanism of Protein binding to Cu(II), Ni(II) and Zn(II)-IDA: 31
Relative contributionis to apparent metal affinity from aminoacid
side chains.
Table 6.1 Summary of the observed improvements in the sensitivity of HS A 102
detection/quantitation by the multiple incubation assay
(V)
hi>
Introduction
1.0 Introduction
Analytical methods that employ antibodies or antigens as primary reagents,
designated as immunoassays, are now indispensable in a large number of clinical,
pharmaceutical and basic scientific investigations. The development of immunoassays
during the past decades has revolutionized the determination of drugs, hormones and
pathogens in clmical, agricultural and pharmaceutical samples as well as contaminants
in the environment. Of the wide range of immunoassays available, enzyme based
immunoassays are well suited for testing large number of samples, detection of very
small amounts of pathogens and for rapid on site assays that require little expertise.
Advantages offered by enzyme immunoassays include a high degree of sensitivity resulting
from the inherent magnification of the enzyme substrate reaction v^thout the need for
radioactivity. Immunoassays are often preferred mainly because of their remarkable
specificity, simplicity, rapidity and relatively low cost.
1.1 Immunohistochemistry
The identification of tissue antigens using specific antigen-antibody interactions
and subsequent microscopic visualization is known as immunohistochemistry or when
dealing wdth intact cells, as immunocytochemistry. For the first time this technique was
introduced as a fluorescence based method by Coons et al (1941) and Coons and
Kaplan (1950). In 1967 immunoenzyme histochemistry was founded, introducing
antibodies conjugated with the enzyme horseradish peroxidase (HRP). This enzyme
was capable of generating an insoluble coloured reaction product suitable for light
microscopic visualization (Nakane and Pierce, 1967; Nakane, 1968). HRP and alkaline
phosphatase from calf intestine or Escherichia coli are the most frequently used
enzymatic markers in immunohistochemistry (Avrameas, 1969a; Hanker e^o/., 1977;
Mason et al., 1978; Li et al., 1984). Glucose oxidase from Aspergillus niger
(Avrameas, 1969b; Clark era/., \9S2) and Escherichia coli P-galactosidase (Bondi
et al., 1982; Portsman et al., 1985) are also employed but only rarely.
Until 1980 the applicability of immunochemistry was still hampered by the
availability of limited number of traditionally raised polyclonal antisera which while
useful in majority of studies, suffered from the problem of heterogeneity and lack of
absolute specificity. The introduction of hybridoma technology for the production of
mouse or rat monoclonal antibodies (Kohler and Milstein, 1975) against a wide range
of human cellular epitopes should be acknowledged as the first major breakthrough in
the areaof immunohistochemistry. Since the early 1980s the number of applications of
immvmohistochemistry has increased drastically, and the technique has established itself
as an important medico-biological investigatory tool.
1.2 Immunochemical techniques
During the last two decades, strategies that improve the sensitivity/effectiveness
of the histochemical staining techniques have attracted considerable attention. Detection,
localization and quantitation of antigens/antibodies by immunoenzjmiatic techniques
necessitates the use of enzyme-protein conjugates and for the preparation of such
conjugates, a number of procedures using various crosslinking agents are available.
Useful procedure for the covalent coupling of the marker enzyme to antibody with
glutaraldehyde has been described (Avrameas, 1969b; Nakane, 1968). An alternative
method of covalent coupling of the enzyme to antibodies was given by Temyck et al
(1976) using p-benzoquinone, but the harsh reaction conditions during conjugation of
enzyme to antibodies and the large size of the antibody-enzyme complex after
glutaraldehyde conjugation were the major concerns. Sternberger et al (1970)
circumvented the problem of conjugation of antibodies with enzyme by introduction of
the non covalent immunological procedure of peroxidase anti-peroxidase (PAP)
complex. This involved a bridging antibody ie (sheep anti-mouse immunoglobulin) which
binds simultaneously to primary mouse monoclonal antibody and peroxidase anti-
peroxidase complex formed by two different mouse monoclonal antibodies (Fig 1.1
PAP and APAAP complex).
In an attempt to improve the sensitivity of tlie assays, the extremely high binding
constants of biotin (a water soluble vitamin) for the egg yolk protein avidin was exploited.
The dissociation constant (Kd) of the avidin-biotin complex is exceptionally high (10'^
M) as compared to a Kd of 10"* - lO''" M for antigen antibody complex and a Kd of
10" -10"' M for those of lectins with carbohydrates (Capra and Edmundsum, 1977).
The high aflfmity of avidin-biotin has been used for bacteriophage inactivation (Beeker,
1972), for gene and cellular mapping (Heggeness et al., 1977), for selective adsorption
of cells (Jasiewicz et al, 1976) and for immobilization of macromolecules (Manning et
al., 1977). Guedson et al (1979) described two procedures for employing avidin-
biotin interaction for linking enzymes to proteins. In the first procedure biotin labelled
antibody (or antigen), biotin labelled enzyme and native unlabelled avidin were used
(Fig 1.1 Avidin-biotin complex). The procedure was also referred as bridged avidin-
biotin (BRAB) technique. The second procedure involved the use of biotin labelled
antibody or (antigen) and enzyme labelled avidin (Fig 1.1 biotin/enzyme avidin), and
was given the name labelled avidin-biotin (LAB) technique. The effects of introducing
biotin into antibody showed that even after extensive substitution of amino groups in
antibody molecules by biotin, the antigen-binding capacity was not significantly modified
(Green, 1963; Avrameas, 1969a).
Another promising technique for improving the sensitivity/efficiency of
immunoenzymatic histochemical detection system is the dextran polymer technology of
DAKO A/S researchers (Bisgaard etal, 1993., Bisgaard and Pluzek, 1996). Both
enzymatic tracers and antibodies are coupled to a large dextran polymer, ensuring a
high enzyme tracer/antigen ratio. This technology is applied for primary antibodies
resulting in a one step procedure (DAKO EPOSTM), as well as for secondary antibodies
resulting in a two step staining procedure (DAKO EnVisionTM). These detection
systems provide good staining intensity, circumvent possible problems with endogenous
biotin (Sabattini et al., 1998; Vyberg and Nielson, 1998) and reduce the three-step
_t *l* Direct Indirect Enzyme-anti-enzyme
(PAP or APAAP)
s^ M Biotin/enzyme - avidin Avidin - biotin complex
A A . Y A « Primary Unlabelled Enzyme Anti-enzyme Enzyme Antibody secondaiy antibody linked
antibody secondary antibody
Avidin Biotin
Figure 1.1 Schematic representation of immunologic labeling methods.
streptavidin-biotin method to a two-step (EnVisionTM) or even a one-step (EPOSTM)
user friendly procedure.
1.2.1 Enzyme amplification systems
The recently introduced enzyme amplification methods show great promise in
enhancing the enzyme immunoassay detection limits (Bates, 1987). One type of enzyme
amplification method, the enzyme cascade (Blake et ai, 1984; Hall et ai, 1984; Harris,
1984) involves the use of one enzyme to activate a second enzyme, that inturn can
activate a third and so on, and the final enzyme in the cascade is used to generate a
signal. Therefore a single Analyte Dependent Reporter Enzyme (ADRE) can result in
multiple signal generating enzyme. Another type of enzyme amplification utilizes the
ADRE to generate a product which modulates and is cycled by enzymes of a signal
generating system. Every molecule of the product formed by ADRE is cycled by the
signal generating enzyme to produce many molecules of the colored product (Johannsson
etal., 1985, 1986; Self, 1985,1986).
Another novel signal amplification system termed as Catalyzed Reporter
Deposition (CARD) or biotin-tyramide amplification system was introduced by Bobrow
etal (1989) for immunoenzymatic assays. The method involves ADRE to catalyze the
deposition of additional reporter enzyme on to the solid phase of the assay. Fig 1.2
demonstrate the method utilizing an HRP ADRE to catalyze the deposition of additional
HRP and alkaline phosphatase with colorimetric detection. Signal amplification with
HRP was observed with the addition of the biotinylated phenolic compound followed
by streptavidin-HRP. Untill now the rather laborious biotin-tyramide amplification has
not been considered as a routine applied detection system in immunoenzyme
histochemistry. The above technique appears to be particularly usefial in improving the
poor staining intensities observed with some primary antibodies, and multi target non
radioactive in situ hybridization using a sequential double immunofluorescence method
(Kersten er a/., 1995; Raap etal., 1995).
Figure 1.2 A flow chart for the HRP mediated deposition of AP and HRP in a
mouse IgG assay
Coat solid phase with anti-mouse IgG
Incubate with mouse IgG
React captured mouse IgG with anti-mouse-IgG-HRP
React with BT
OPD
(HRP)
Streptavidin-AP
pNPP
(HRP-AMP AP)
Streptavidin-HRP
OPD
(HRP-AMP HRP)
1.2.2 Immunogold techniques
This technique uses colloidal gold labelled antibodies initially developed for
immunoeiectron microscopy (Faulk and Taylor, 1971). Colloidal gold labelled antibodies
may be used both directly and indirectly for the location of antigens with light,
transmission or scanning electron microscopy. The preparation of gold probes is a
simple two step procedure involving first the generation of a colloidal gold solution
containing particles of a narrow range of sizes from 5 to 40 nm. Then the primary or
species specific antibody, or protein A, is adsorbed on to the surface of the gold
particles, giving rise to immunogold or protein A gold probe respectively, which are
stable for long durations. The basic technique involves incubating cryostat or fixed
sections of specimen first with primary antibody and then with immunogold probe. The
labelling is highly specific and lends itself to quantitate merely by permitting the counting
of the number of probes present in a sample. Pereira et al (1994) and Li etal{\ 995)
used antibodies labelled with colloidal gold to identify and localize epitopes on the
virus particles captured on antibody coated electron microscopic grids. Protein A gold
preparations have also been used to localize Cryptosporidium antigens in the tissue
sections (Lumb er a/., 1989; McDonald era/., 1995).
As the small gold particles are not visible with light microscopy, Holegate et al
(1983) adopted the silver enhancement from Danscher (1981) for the first immuno
gold/silver staining techniques on tissue specimens. The technique involves the
localization of gold probes by a silver precipitation reaction making the particles visible
as brown/black deposits with light microscopy. It can be used for the enhancement of
ultra-small gold particles both in light and electron microscopy. A Special feature of
the silver precipitate end product is the possibility of dark field epi-polarization
microscopy (De Waele etal, 1988). The dark field option is believed to be one of the
most sensitive/efficient immunohistochemical detection methods available. Furthermore,
it formed the basis of a double staining method in combination with a fluorescent alkaline
phosphatase detection technique (Vander Loos and Becker, 1994).
1.2.3 Immunofluorescence
Fluorescence Immunoassay (FIA) combines the specificity of antibodies with
the sensitivity of fluorimetric assays, usually by using antibodies coupled to a fluorescent
chromophore. The location of specific antigens in a tissue or cell preparation may be
studied by staining with specific antibody conjugated to a fluorescent chromophore
and illuminating with ultra violet light. Fluorecein emits green light and Rhodamine emits
orange light, where as the weak natural fluorescence of some biological material occurs
in the blue region of the spectrum. Seto et al (2001) developed a high sensitivity
Bioluminescent Enzyme Immunoassay (BLEIA) for prostate specific antigen using
biotinylated firefly luciferase labelled antibody. Immunoflourescence may be performed
in a number of different ways, illustrated in Fig 1.3. Although clear and sharp localization
of antigens with single and double immunofluorescence techniques is beyond dispute
(Pryzwansky, 1982; Brandtzaege/a/., 1983, 1997; Brandtzaeg, 1998), but these
techniques still suffer from well known drawbacks: quenching of fluorescence signal at
excitation, fading of fluorescence signal upon exposure to room temperature or
occurrence of auto fluorescence caused by formaldehyde fixation. (Mesa-tejada, 1977;
Taylor, 1978,1980; Valnes and Brandtzaeg, 1982;NaiemeM/., 1982; Mason era/.,
1983). Despite many technical improvements in this regard, the stability of a fluorescence
signal has not come even close to that of permanent enzymatic reaction product.
Therefore, light microscopic immunohistochemical techniques using enzymatic
chromogens are perferred.
In medical biology, a new generation of improved fluorescent labels (fluorescein
isothiocyanate, FITC) and dyes have been introduced mainly evolving with fluoresent
activated cell sorter (FACS) techniques (Raap et al, 1995). Cryptosporidium oocytes
in water samples were identified using this technique. The technique involved the
separation of FITC - antibody conjugated cells from other components of suspension
Figure 1.3 Diagrammatic representation of various fluorescence immunoassays.
L Direct immunofluorescence or single layer technique
Indirect immunofluorescence or double layer technique
Simple sandwhich technique for detection of specific antibodies
Multiple sandwhich technique
Antigen \ \ A
Fluorescent labelled secondary antibody
Fluorescent labelled primary antibody
Unlabelled primary antibody
in FACS. The suspension is passed through a laser beam and cells are separated on
the basis offluorescence intensity (Vesey era/., 1990,1997).
Particle Concentration Fluorescence Immunoassay (PCFIA) is a relatively new
and rapid immunoassay technique with high sensitivity. PCFIA relies on the principle
that the number of free particles coated with antigen decrease during agglutination and
that the angle at which a beam of light is scattered is a function of particle size. Jolly et
a/ (1984) exploited this technique for the detection of murine monoclonal antibodies to
human IgG in hybridoma culture supematants. For the antibody detection, antigen
bound to submicron polystyrene particles is bound to its specific antibody, which
intum is reacted with flourescein labelled affinity purified goat anti mouse IgG. After
completion of the reaction and solid phase separated the total particle bound flourescein
is quantitated by front surface fluorimetry. Jolly ef a/ (1984) reported that the sensitivity
of the technique for antibody detection is comparable to that of ELISA.
Fluorometric determinations have come to be regarded as a highly sensitive
detection systems superior both to radio isotopic and enzyme labelled immunoassays
in recent years. However, conventional fluorometric assays are often limited by high
back ground and non specific fluorescence emitted by non target compounds in complex
mixtures. A successful altemative has recently been realized in time resolved fluorometry
(Soukka et al., 2001), where the desired signal is distinguished from the interfering
background by temporal resolution. Time resolution (measuring of the fluorescence
intensity after a certain delay time has elapsed from the excitation pulse) technology is
a strategy to avoid interference caused by short lived natural fluorescence of biological
material. Lanthanide chelates europium (III), (Eu^*), samarium (III), (Sm^*), terbium
(III), (Tb " ) and dysprosium (III), (Dy " ) are very effective labels in time resolved
fluorometry that exhibit long emission duration and a reasonably high quantum yield.
Butcher et al (2003) developed a competitive solid phase assay for the low
molecular weight metal binding protein, metallothionein. The assay was based on the
10
Dissociation Enhanced Lanthanide Flouro-Immunoassay (DELFIA) detection of anti
metallothionein antibody bound to solid phase metallothionein. The assay involves
binding and exchange of different lanthanide chelates followed by fluorescent detection.
1.2.4 Immunoblotting/dot immunobinding
Much of the early quantitative and qualitative measurement of immunochemical
reactions such as the percipitin reaction (Kabat and Mayer, 1961), radial
immunodifiusion (Mancini et al., 1964), farr assay (Wold et al., 1968), light Scattering
procedures (Gitlin and Edelhoch, 1951) were observed in solution. With the advent of
ELISA (Engvall and Perlmann, 1971) increasing number of assays are solid phase
based (Kemeny and chantler, 1988).
In recent years solid phase immnoassay techniques have found wide application
in basic research and clinical studies. The high sensitivity which can be obtained with
labelled ligands, and the ease of separation of bound from unbound reagents make this
type of inununoassay particularly attractive. More recently the development of methods
for transferring proteins from the gel phase of electrophoresis to a solid phase for
immuno reaction permitted the optimal combination of high resolution gel electrophoresis
with the sensitivity and sunplicity of solid phase immunoassays. The first practical method
for transfer of protein from gel electropherograms to a suitable solid phase was reported
by Renart etal{\979), who introduced the transfer by diffusion to activated cellulose
by covalent binding. Towbin et al (1979), described the transfer of proteins in an
electric field and non covalent binding to nitrocellulose from polyacrylamide gels. This
feature can also be used in immunoassay where the resolving power of electrophoresis
is not needed. Thus antigens can be applied in the format of dots or spots (Glenney et
al, 1980; Hawkes et al., 1982; Herbrink et al, 1982; Huet et al, 1982; Yen and
Webster, 1982). This technique of dot immunobinding permits the use of any desired
geometry, and has the advantages of permitting multiple simultaneous assays, high
sensitivity and simple operation (Hawkes, 1982; Gordon etal., 1983).
1.2.4.1 Immobilizing matrices in solid phase immunoassays
Among the numerous immobilizing matrices available. Nitro-cellulose (NC) is
the most widely used. It is produced by allowing thin films of nitric acid esterified
cellulose, solublized in organic solvent mixture (ether alcohol or acetic acid acetone),
to gel by the evaporation of the solvents. Meticulous regulation of the conditions
(temperature, time) of the drying processes determine the porosity of the membrane
(Gershoni and Palade, 1983). NC filters were originally described as surface filters for
micro filtration. They were intended for sieving by particle size thus retaining bacteria
on or near the surface of the filter matrix. However in the current use of surface filters
for blotting, they serve as depth filters in which the micromolecules concerned are
adsorbed throughout the filter matrix predominantly by chemical interactions rather
than actual mechanical sieving (Presswood, 1981). Hydrophobic effects probably play
a role in the interaction and indeed subsequent elution of the protein from the filter is
facilitated by the non ionic detergents Triton X-100 (Schneider, 1980). Some proteins
specially those of low molecular weights bind with low affinity to NC and may be lost
during transfer or subsequent processing (Kakita et al., 1982). To prevent such losses,
the transferred polypeptides can be crosslinked to the filter, thereby covalently stabilizing
the protein pattern (Gershoni et al., 1982). As suggested by Burnette (1981) less
porous matrix (0.2 instead of 0.45 um) may also prevent such losses. The presence of
cellulose acetate in nitrocellulose membrane filters seems to reduce their capacities to
bind protein (Towbin et al., 1979). Schaltman and Pongs (1980) reported the use of
cellulose acetate filters for protein blotting.
Other matrices used include Diazobenzyloxymethyl (DBM) modified cellulose
paper (Alwine et al., 1979). Upon transfer to DBM paper, negatively charged proteins
probably interact first electrostatically with the positively charged diazonium groups of
the paper. This interaction is followed by a slow second step in which essentially
irreversible covalent linkage via azo derivates is accomplished (Alwen et al., 1977).
Hence stable transferred protein patterns are obtained (Renart, 1979). However there
is a slight loss in resolution from gel to blot in this material due to its intrinsic coaiseness
as compared to membrane filters (Bumette, 1981). Glycine commonly used in transfer
buffers may interfere with DBM protein blotting (Kakita et al., 1982). Diazo paper,
diazophenylthioether (DPT) paper has been found to be as efficient but more stable
than DBM paper (Clarke/Of/., 1979; Reiser era/., 1981). Cellulose acetate activated
with CNBr has also been used and offers much the same advantages as the DBM
paper.
Two other materials commercially available are Gene screen and Zeta bind (ZB).
Zeta bind is a nylon matrix (a polyhexamethylene adipamine), referred to as nylon-66,
modified by the introduction of numerous tertiary amino groups during its manufacture.
Thus it offers not only the mechanical strength but also the potential of very significant
electrostatic interactions between the filters and the polyanions. As a result ZB has
higher capacity for proteins than NC (Gershoni and Palade, 1982). A drawback of ZB
is the possibility of high non specific protein binding during subsequent overlay analysis.
One common characteristic of nylon membranes are their high affinity for common
anionic dyes as coomassie brilliant blue or amido black (Bittner et al., 1980). This
characteristic interferes with subsequent staining of the filters, to the extent that no
convenient technique for the staining of proteins to these matrices has yet been
developed. But proteins on NC can be stained readily with amido black 1 OB (Towbin
et al., 1979), coomassie brilliant blue (Bumette er a/., 1981), aniline blue black (Bowen
et al., 1980), ponceau S (Muilerman, 1982), fast green (Reinhart, 1982), and toludine
blue (Towbin e/a/., 1982).
Several proteins bind tightly to NC and are not readily released. This makes it
one of the most useful solid matrix for wide range of applications (Harlow and Lane,
1988). It is commonly used in quantitative and qualitative dot assays both for antibodies
and antigens. It is also the most commonly used solid phase matrix for immunoblotting.
13
Protein antigen immobilized to NC often make extremely good immunogens (Steinitz
et al., 1991). The complex acts as an effective adjuvant by slowly releasing particulate
antigen, thus continuously stimulating the immune system.
1.2.4.2 Applications of immunoblotting and dot immunobinding
Protein blotting has proved to be a highly versatile technique and finding increasing
applications. Dot immunobinding is attractive for a variety of routine diagnostic
applications because the procedures permits the performance of multiple of assays on
on a single strip and detects antigens/antibodies with high sensitivity. Hawkes et al
(1982) demonstrated the feasibility of serological screening with a variety of antigens
from commercially available kits.
DNA-protein and RNA-protein interactions have been analyzed by protein blots
by Bowen et al (1980). Histone H2 associations with H3 and H4 have also been
demonstrated (Bowen et al, 1980). Fernandez - Pol (1982) identified the receptor of
the epidermal growth factor (EGF) by overlaying a transferred membrane pattern with
the hormone.
Muilerman et al (1982) demonstrated the detection of an inactive enzyme on a
protein blot. Phosphodiesterase I was run on a SDS-polyacrylamide gel and the protein
was blotted on to nitrocellulose filter, which was subsequently reacted with anti-
phosphodiesterase I. The filter was then incubated with the crude prepapartion containing
active enzyme which bound (via unoccupied sites) to the antibody already bound to
the inactivated subunit of the enzyme immobilized on the filter. The filter was then
tested for enzyme activity to detect the immunocomplexes. Olmsted (1981) used blots
to purify monospecific antibodies. Polypeptides resolved on SDS-polyacrylamide gels,
were blotted on to Diazophenylthioether (DPT) paper and the filters were overlaid
with serum containing polyclonal antibodies. The single band containing antigen-antibody
complexes were excised from the filters and monospecific antibody was eluted from
the excised strips by incubating them at low pH (Adair et al, 1982; Bell et al, 1982).
14
Hayman et al (1982) used protein blots to demonstrate specific whole cell-
polypeptide interactions. Human plasma was run on SDS-polyacrylamide gels and
transferred to nitrocellulose. Once quenched, the filters were incubated with normal
rat kidney cells which specifically bound to immobilized polypeptides presumably
involved in cell attachment. The cells were stained with amido black lOB and were
found to locate themselves at two discrete bands.
Symington ef a/ (1981) and Erickson et al (1982) have found that transfer of
'••C or S labelled polypeptides from gel to filters increases the efficiency of polypeptide
detection by autoradiography. Distance between the emitting radioisotope and the
surface of the photographic emulsion is a critical parameter, determining the efficiency
of autoradiography for low energy P-particles. Thus by transferring radioactively labelled
proteins from relatively thick (~ 1 mm) wet gels to a thin (100-200 |im) dry membrane
filter, greater sensitivity in autoradiography is achieved.
Assays were further developed for total IgE and two allergens from dust mite
and bee venom and showed that the system is specific for IgE and sufficiently sensitive
for their determination in serum (Derrer et al., 1984). The assay was reproducible
with coefficient of variation between 3 and 6 % and correlated well with commercial
assays for total IgE and the allergen-specific IgE. Walsh et al (1984) developed a
similar system for qualitative use with several allergens deposited on nitrocellulose discs.
Autoimmunity profile for connective tissue diseases measures anti-DNA
antibodies, rheumatoid factor and antibodies against sub cellular fractions of HeLa
cells (Gordan and Rosenthal, 1984b). The assay correlated well with commercial test
for rheumatoid and anti-nuclear factor and with clinical diagnosis.
Pappas et al (1983) applied dot immunobinding to visceral leishmanias. They
performed assays with serial dilutions of sera with single dots in micro-titre plate wells
and showed that, in spite of cross- reactivity with African Trypanosomiaisis, Chagas
15
disease and Lupus Erythromatosis, the tests were diagnostically useful and lend
themselves speciallj' well to field screening. They also found that far less antigen was
required per test than in a comparable enzyme irmnunoassay.
Dot immunobinding and inuniunoblotting were also used for detecting nanogram
quantities of small peptides (angiotensin I, angiotensin II and met-enkephalin) on divinyl
sulfone and gluteraldehyde activated nitrocellulose. Peptides were attached to the
membrane by reaction of the amino group with the free carbonyl forming peptide bonds,
analyzed with specific rabbit antibody against the peptide and visualized by HRP
conjugated anti-rabbit antibody (Lauritzen etal., 1990).
Kristoffersen et al (1994) used dot immimobinding assay to demonstrate and
characterize the functional activity of soluble Fc gamma receptors (FcR). Samples
containing soluble FcR were immobilized on a nitrocellulose membrane. Immune
complexes of HRP and rabbit IgG antibodies to HRP were allowed to react with
nitrocellulose bound FcR and the immune complexes were visualized by HRP developer.
For detecting mite antigens in house dust sample, Mistrello et al (1998) developed
a dot immunobinding assay, which uses a nitrocellulose dipstick spotted with specific
anti-mite antibodies that act as a capture matrix. The same antibodies act as a detecting
reagent when conjugated with colloidal dye particles and the test response is visible as
a colored spot. Solid phase immunoassays are sensitive, specific £ind low cost diagnostic
tests for detection of specific antigens in tears, urine and dermal fluid (Ngu et al,
1998).
Mangold etal{\ 999) detected the elevated levels of circulating antigen 85 by
dot immunobinding assay in wild animals with tuberculosis. The assay also provides an
important adjunct to intradermal skin testing for antemortem diagnosis of tuberculosis
in non domestic species.
For the rapid diagnosis of Tuberculosis Meningitis (TBM), Sumi et al (2000)
developed a dot immunobinding assay to measure circulating anti mycobacterial antibody
16
in the cerebrospinal fluid (CSF). Specific CSF-IgG to Mycobacterium tuberculosis
from TBM patient was isolated and coupled with activated CNBr Sepharose 4B. Using
an immunosorbent affinity column, a 14 KDa antigen present in the culture filtrates of
Mycobacterium tuberculosis was isolated and used in the dot immunobinding assay
to quantitate specific antimycobacterial antibodies in CSF specimens. Siddiqui et al
(2002) recently studied the selective binding of the melanoma cell TF- apoprotein to
haemoglobin using dot immunobinding assay and western blotting.
1.3 Human serum albumin
Serum albumin belongs to a multigene family of proteins that includes
a-fetoprotein (AFP) and human group- specific component (GC) or vitamin D binding
protein. It is relatively large multi-domain protein which, as the major soluble protein
constituents of the circulatory system, has many physiological fimctions. Serum albumin,
also known as plasma albumin was recognized as a principal component of blood by
Ancell(1839).
Originally, the protein was referred to as "albumen" derived from the latin word
albus meaning "white", after the white colour of flocculant precipitate produced by
various proteins. Serum albumin is located in nearly every tissue and body secretions.
About 40% of the total albumin is found in the circulatory plasma (Peters, 1970) where
as of the remaining 60%, about half resides in viscera and half in muscle and skin
(Rabilloud et al., 1988). Albumin also occurs in milk (Phillippy and Mac Carthy, 1979),
amniotic fluid (Bala et al., 1987), semen (Blumsohn et al., 1991) and mammary cyst
(Balbine^a/., 1991).
Albumin concentration in plasma in an average person is about 35 - 50 g/1 which
declines slightly with age (Cooper and Gardner, 1989) but is lower in newborns
(Cartlidge and Ruter, 1986) and as low as 20g/l in premature infants (Reading et al.,
1990). Albumin is produced by the liver at the rate of 0.7mg/g liver per hour (Peters,
17
1985). About 4 - 5% of albumin is daily replaced by hepatic synthesis (Olufemi et ai,
1990). Albumin concentration in plasma is maintained through transcriptional control
of the albumin gene by the anabolic hormones-insulin and somatotrophin (Hudson et
al, 1987). Albumin synthesis is also highly dependent upon the supply of dietary amino
acids (Kaysen et al., 1989).
1.3.1 Functions of albumin
Albumin contributes to about 80% of colloid osmotic blood pressure
(Lundsgaard, 1986) and 100% of the protein effect in the acid base balance of plasma
(Figge etal., 1991). It acts as a carrier for long chain fatty acids (Brodersen et al.,
1991; Cistola and Small, 1991), their acyl coenzyme A esters (Richard et al., 1990)
and mono acyl phospholipids (Robenson et al., 1989) and affects the activity of lipase,
esterases (Posner et al., 1987) and carnitine acyl transferase (Richards et al., 1991).
Albumin binds polyunsaturated fatty acids (Anel et al, 1989) and influences the stability
(Haeggstrom et al., 1983), biosynthesis (Heinsohn et al., 1987) and transformations
of prostaglandins (Dieter et ah, 1990). It binds weekly to cholesterol (Deliconstantinos
et al., 1986), bile acids (Malavolti et al., 1989), corticoid hormones (Watanabe et
al, 1991; Mendel etal., 1990) sex hormones (Pardridge, 1988) and is involved in
transport of thyroid hormones (Mendel et al., 1990). Albumin also helps in the transport
of pyridoxal phosphate (Fondae^a/., 1991), cysteinand glutathione (Joshi etal, 1987)
by forming a covalent bond with these ligands.
Albumin is responsible for the transport and store housing of many therapeutic
drugs in the blood stream (Lindup, 1987). Albumin is an important constituent of tissue
culture media (Barnes and Sato, 1980) and serves as a medium to support the growth
of bacteria, fungi and yeast (Callister et al., 1990; Morrill et al., 1990).
Albumin because of its low cost and availability in pure form has been used as a
standard for assays of total protein by the method of Lowry etal {\9S\), Biuret (Gomall
et al, 1949 and coomassie blue (Bradford, 1976) procedures for many decades. It is
also used in the standardization of other types of analyses and as reference for gel
electrophoresis, diffusion techniques, sedimentation in ultracentrifugation and in bilirubin
determination (Szabolcs and Francia, 1989). Fructosamine - albumin serves to
standardize protein bound fructose assays (Podzoiski and Wells, 1989).
1.4 Immunoaffinity based immobilization
Inmiimoaffmity chromatography is a powerful technique for the isolation of the
individual protein from complex mixtures. The procedure involves the attachment of
the antibodies to solid supports like Agarose, Sepharose or synthetic organic and
inorganic porous bead matrices (Grormian and Wilchek, 1987).
Immobilization involves the reaction of the surface nucleophilic group (mostly
primary amines) of the antibody with polysaccharide support activated by CNBr
(Coding, 1983) epoxy activation using epichlorohydrin (Pepper, 1992) or bisoxiranes
(Sunderberg and Porath, 1974). Most of these immobilization procedures result in
random immobilization of the antibody molecule on the support. Immobilization of the
antibody on an immobilized protein A column however, leads to the favourably oriented
attachment of the antibody (Gersten and Marchalonis, 1978; Solomon et al, 1986).
Oriented immobilization of antibodies on support via their oligossacharide chains which
are mainly located in the Fc region of the molecdule has been reported by Quash et al
(1978) and Fleminger et a/ (1990).
Immobilization of enzymes using anti-enzyme antibodies has long been studied
and reviewed (Shami et al., 1989) and is a versatile technique as specific antibodies
can be raised against any enzyme in suitable animals (Ehle and Horn, 1990). A variety
of approaches have been adopted for the immobilization of enzymes with the help of
antibodies. The simplest being the formation of enzyme- antibody complex for which
neither pure antibody nor pure enzyme is a prerequisite. Bumette and Schmidt (1921)
19
demonstrated for the first time that insoluble immuno-complex of catalase and anti
catalase antibodies retained complete enzyme activity and have marked stability against
various denaturing factors (Shami etai, 1991; Jafri and Saleemuddin, 1993).
A considerable number of enzymes have been immobilized on various immuno-
adsorbents prepared using polyclonal or monoclonal antibodies. It was observed that
binding of enzymes on immuno-adsorbents prepared using polyclonal antibodies is
relatively strong due to the binding of antigen to more than one kind of antibody (Hansen
and Beavo, 1982; Ernst-Carbrera and Wilchek, 1988). Monoclonal antibodies also
appears to have shorter half lives and are more labile compared to polyclonal antibodies
(Coding, 1985; de-Alwis and Wilson, 1989). Schramm and Pack (1992) studied antigen
antibody complex formation using monoclonal antibody for its possible use in optimising
enzyme immunoassays.
Procedures that result in the binding of large quantities of enzymes on supports
are advantageous in nearly all applications requiring immobilized enzymes and such
strategies are particularly helpful in the construction of enzyme based biosensors that
necessitate the fixation of large amount of enzymes on a relatively small area for maximum
sensitivity (Alvarez-Kaza and Bilitewski, 1993; Vanderberge/a/., 1994).
Farooqi etal(l999) described a procedure for the high yield immobilization of
enzymes with the help of specific anti-enzyme antibodies. They demonstrated that
immobilization of glucose oxidase and horseradish peroxidase was achieved by binding
the enzymes to a Sepharose matrix coupled with IgG isolated from anti-glucose oxidase
and anti-horse radish peroxidase sera, erspectively. The procedure further followed
alternate incubations with the IgG and the enzyme to assemble layers of enzyme and
antibody on the support. The immunoaffinity layered preparation obtained after six
binding cycles contained 25 times more immobilized enzyme over that bound initially.
20
1.5 Immunoglobulins
Immunoglobulins (Ig) are soluble glycoproteins that circulate freely in blood and
exhibit properties that contribute specially to immunity and protection against foreign
material. Antibodies which are present in gamma globulin fraction of serum are slowest
migrating proteins in an electrophoretic field (Porter, 1959; Kabat et al, 1961)).
Immunoglobulins possess two distinct type of polypeptide chains linked by both
covalent and non covalent bonds (Milstein and Pink, 1970). The smaller light chain
(MW 25,000) is characteristic of this whole group of proteins and is of two types -
kappa (k) and lambda (X|). The larger heavy chain (MW 50,000 - 77,000) is specific
for each class. Based on antigenic as well as structural properties of the heavy chain,
the immimoglobulins are classified as IgG, IgA, IgM, IgD, IgE, the heavy chains of
each class designated by greek letters y, 0(|, M, 5 and e respectively.
Aminoacid sequencing of immunoglobulin heavy and light chains showed that
both chains contain several homologous units of about 110 amino acid residues containing
interchain disulphide bonds approximately 60 residues apart (Kabat and Wu, 1970).
The first or the N - terminal domain on both light and heavy chain is highly variable in
terms of amino acid sequence and is designated as V^ or V^ accordingly. The structural
genes for Ig variable region exist in the genome as a multigene system (Hood and
Talmage, 1970). The subsequent domains on both chains are much more conserved in
amino acid sequence and are designated C^ or C^l, C^2, and C^3. Each domain has
evolved to fulfil a specific function. The major function of V^ and V^ domain is to
provide the antigen binding site. A region between Cj l and C^2 domains containing
the interchain disulphide bonds is known as the hinge region (Stanworth and Turner,
1986). This region which is a segment of the heavy chain is unique for each class and
subclass of immunoglobulins. Flexibility in this area permits the antigen binding sites to
operate independently.
21
1.5.1 Immunoglobulin G
IgG is the predominant immunoglobulin in blood, lymph, cerebrospinal fluid and
peritoneal fluid. The four chain structure of IgG was proposed by Porter (1959) and
its complete sequence elucidated by Edelman, (1969). They are multimeric proteins
with a molecular weight of 150KDa corresponding to a sedimentation coefficient of
7S. IgG is made up of two heavy gamma (y) chains and two light chains, either kappa
(k) or lambda (k) linked together by disulphide bonds (Fig 1.4). Based on minor
differences in the aminoacid sequence of heavy chains there are four IgG subclass in
humans namely IgGl, IgG2, IgG3 and IgG4. Mice also synthesize four subclasses of
IgG designated as IgGl, IgG2a, IgG2b and IgG3 (Edelman et al., 1970).
The constant region of the gamma chain is constructed from three homologous
units designated C^ l, C^, and C S and contains a stretch of aminoacid residues
between C jl and C^, in the hinge region which shows no homology with any of the
homologous units. This region is rich in proline residues and also contains the cysteine
residues that link the two heavy chains together (Edelman et al, 1970). This region
acts as a susceptible site for proteolytic cleavage, as the amino acid residues in this
region are exposed to the surrounding medium.
1.6 Porcine pepsin
Aspartic proteinases (EC 3.4.23.X) are a group of proteolytic enzyme which
operate at acidic pH and possesses remarkable homology in their tertiary structures
(Pitts et al., 1992). X-ray crystallographic data reveal that this group of enzymes share
a bilobal symmetry, with the two structural domains separated by a hinged substrate
binding cleft. Two active site aspartic residues occupy the cleft to which admittance is
restricted by a hinged flexible flap region (Suzuki et al, 1989). Although high in structural
homology, these enzyme differ markedly in their catalytic functions, indicating that their
activity is the result of subtle differences in protein structure.
22
Figure 1.4 Three dimensional structure of immunoglobulin molecule (a) and pictorial
representation of IgG (b).
23
(a)
Antigcn-hindii^ site
VL domain
VH Uoniain
Antigen-binding site
VL domain
Carbohydnttc chain
\ • '
. ^ HeavA chaias
<:n3
(b)
Antigen-binding site
VH domain
/
Hinge region C:DRS
Antigen-binding site
Carbohydrate
Pepsin is the principal proteolytic enzyme of vertebrate gastric juice and the first
enzyme to be studied and the second to the crystallized (Northrop, 1930). Consequently
pepsin is the best understood of this family of proteases and has been used as a model
to study related enzymes (Lin et al, 1983; Abad Zapatero et al., 1990).
1.7 Procedures for producing F(ab)'j
F(ab)'J are the bivalent antibody fragments obtained by cleaving the antibody
below the hinge region. The use of F(ab)'j fragments may in some immunological tests
systems is advantageous over the use of intact IgG mainly because non-specific binding
through the Fc part can be avoided. The standard procedure of F(ab)'^ isolation involves
IgG purification from whole serum by triple salt precipitation using sodium sulphate
(Kekwick, 1940), followed by chromatography on DEAE cellulose (Levy and Sober,
1960). Purified IgG digested with pepsin (Nisonoff e al., 1960) showed the formation
of a single large fragment F(ab)'j along with a heterogeneous assortment of small
peptides (Fig 1.5). F(ab)'2 was isolated by gel filtration through Sephadex G -150
(Jaquet and Cebra, 1965).
Madson and Rhodkey (1976) devised an alternative method to isolate functionally
active antibody F(ab)'j fragments from small amounts of serum. The fragments were
produced by digesting ammonium sulphate saturated serum with pepsin, and F(ab)'j
were then isolated through gel filtration chromatography. Total serum IgG F(ab)'j
recovered was 90% using the technique, as compared with the usual 20 - 25 % recovery
using standard isolation procedures. Poulsen and Hjort (1980) devised another method
for the preparation of F(ab)'j by pepsin digestion of whole serum proteins. They also
showed that most of the serum proteins could be degraded to small peptides which
were removed by dialysis.
Various methods for improved pepsinolysis of different immunoglobulins and
immunoglobulin G subclasses were also described. Lamoyi and Nisonoff (1983)
24
Figure 1.5 Schematic representation of cleavage of IgG by papain and pepsin
25
u
I (0
0)
u
i-1 0)
(0
[X4 Fl4
£
investigated the effect of peptic digestion on mouse monoclonal immunoglobulin of
each subclass of IgG. FCab)' fragments were obtained in good yield from IgGl,
IgG2, and IgG3. The relative rates of digestion were IgG3>IgG2>IgGl.
For human IgG there have been a number of investigations of proteolysis by
papain, pepsin and trypsin. The fragments produced thereby including Fc, F(ab) and
F(ab)'2 have been well characterized (Bennich and Johnsson, 1971). Haba and Nisonoff
(1990), studied the conditions for the preparation of F(ab) and F(ab)'2 fragments of
mouse IgE by proteolytic digestion. Pepsin and trypsin, each produced F(ab)'j
fragments with molecular weight of 130 KDa which yielded Fab fragment on further
digestion. The best yield of F(ab) were obtained with papain. Results of digestion
were in order: Pepsin > Trypsin > papain.
The most important subclass of mouse immunoglobulin IgGl is quite resistant to
pepsin cleavage (Gorini et al., 1969; Lamoyi and Nisonoff, 1983; Mariani et al.,
1991; Andrew and Titus, 1997). Wilson et al (2002) devised an improved method for
pepsinolysis of mouse IgGl molecules to F(ab)'j fi-agments. They reported that IgG
molecules can be rendered pepsin sensitive by treatment with peptide N-glycosidase
F (PNFase), an enzyme that removes N linked oligosaccharides from proteins.
F(ab)'J or F(ab) are widely used for the treatment of snake envenoming (Karlson-
Stiber, 1994; Jones et al., 1999; Theakston et al., 2000). Such products should
comprise only highly pure immunoglobulin fragments since Fc or other contaminating
protein firagments or their aggregates may lead to side effects (Hristova, 1968b; Bleeker
etal., 1987; 1988; 2000). Jones and Landon (2002) gave a novel process for enhanced
pepsin digestion and F(ab)'^ fragment production in high yield from serum. They showed
that trypsin was effective at digesting purified IgG but unsuitable for the direct digestion
of serum while Pepsin was highly effective at digesting all unwanted serum proteins to
low molecular weight fragments leaving the 100KDaF(ab)'2 intact. Low molecular
weight fragments were dialyzed out through a 30 KDa cut piperazine membrane, while
26
pepsin and high molecular weights peptides were removed using anion exchange
coloumn. The purity of F(ab)'^ was over 96% and yield was also impressive.
F(ab)'J fragments were also purified by hydrophobic interaction high performance
liquid chromatography (HPLC) using TSK gel Phenyl - 5PW from pepsin digest of
mouse monoclonal antibodies IgG (Morimoto, 1992). Digests were applied to the gel
equilibrated with phosphate buffered saline containing one molar ammonium sulphate.
F(ab)'j fragments were adsorbed onto the gel and eluted by reducing the ammonium
sulphate concentration to zero molar. This method is useftil for large scale production
of F(ab)'j. Hydrophobic interaction HPLC technique was also used to purify F(ab)'2
from mouse immunoglobulin M (Inouye and Morimoto, 1993). The F(ab)'j derived
from IgM lost the compliment binding activity and the sugar content was also reduced.
Its binding to non specific proteins tumed to be negligible suggesting that the fragments
were usefiil for immunological application. Pascual and Clem (1992) gave an effective
procedure for mouse IgM F(ab)'j production by digesting murine IgM antibodies with
pepsin at low temperature. They reported that digestion performed at 4°C of
immunoglobulin M yields higher ratio of F(ab)'j fragments than those obtained by
digestion performed at 37°C.
Akita and Nakai (1993), reported the formation of F(ab)'j from chicken egg
yolk immunoglobulin Y (IgY). They reported that optimum yield of F(ab)'2 was obtained
after peptic digestion of IgY at pH 4.2 for nine hours at lowNaCl concentration. By
combination of ultra filtration and anion exchange, pure F(ab)\ fragments were easily
obtained in the eluent, as the contaminants bind to the anion exchange column. With
this approach large amount of F(ab)'j were purified using a small column.
F(ab)'j fragments were also obtained by cleaving IgG with immobilized pepsin
preparation (Tomono et al., 1981). Pepsin was immobilized onto glutaraldehyde
activated AH sepharose 4B under acidic conditions and was found to retain 30%
27
activity towards IgG. The immobilized pepsin thus prepared was efficiently used for
the limited cleavage of IgG and gel filtration yielded very pure F(ab)'2.
Benanchi et al (1988) described a two step procedure for purifying F(ab)'^ from
horse immunoglobulins. In the first step, the horse plasma was diluted with ammonium
sulphate and digested with pepsin. In the second step, the perviously dialyzed solution
was chromatographed on DEAE cellulose covalently linked to a synthetic vinyl polymer.
With this assembly, chromatography can be performed at high flow rate with out the
problems related to the use of large columns. The yield and purity of F(ab)'jObtained
was satisfactory.
For the purification of F(ab) fragment papain attached to solid phase CH-
Sepharose 4B was used to digest rabbit IgG. Protein A-Sepharose CL-4B was used
to remove undigested IgG and Fc fragment. The yield reported was 75% (Coulter and
Harris, 1983).
Lihme etal (199 \) reported the binding of almost all serum proteins at high
concentration of ammonium sulphate on affinity matrix prepared by coupling
mercaptoethanol to divinysulfone activated agarose (T-gel). They showed good
recovery of proteins with an excellent separation by gradually lowering the concentration
of salt in the washing buffer. Thiophilic adsorption chromatography (TAG) was also
employed for purification and separation of mouse F(ab) firagment fi-om IgG, monoclonal
antibodies (Yurov et al., 1994). TAG was performed using stepwise elution with
decreasing concentrations of ammonium sulphate. Most contaminating proteins did not
react with the thiophilic adsorbent and chromatography efficiently resolved the F(ab)
and Fc fragments. TAG was also employed for the purification of a recombinant F(ab)
fragment of antibody IN-1 from the periplasmic protein fraction of Escherichia coli
(Fiedler eM/., 1999).
28
1.8 Use of IMAC for purifications of immunoglobulis
Immobilized Metal ion Affinity Chromatography (IMAC) is a highly versatile
separation technique based on interfacial interactions between metal ions immobilized
on a solid support which is usually a hydrophilic cross-linked polymer and basic group
on proteins mainly histidine residues (Fig 1.6). Terminal amino group, tryptophan,
tyrosine, phenylalanine, and cystein residues (Table 1.1) may also be involved in the
binding (Martin, 1979; Sulkowaski, 1985). This concept of using chelated metal for
the selective adsorption of protein was first introduced by Porath (1975) and the
technique renamed as IMAC by Porath and Olin (1983).
Traditional affmity chromatographic methods for immunoglobulins purifications
such as protein A (Fuglistaller, 1989), protein G (Blank and Vetterlein, 1990), k-specific
affinity (Bazin and Cormont, 1975) or anti-Ig specific immuno-adsorbents oflen requires
elution at low pH or with detergents or chaotropic salts. Such elution conditions are
often damaging to the antibody being purified. Further more, chromatographic methods
selective for the N-terminus of IgG will co-purify antibody and degradation products
such as Fab or F(ab)'j. In contrast to aflSnity chromatography, the metal chelate technique
does not damage the antibody or elute metal fi"om the column. The potential use of this
technique for the isolation of human and murine IgG, has been shown by Hale and
Biedler (1994). The near complete removal of albimiin makes IMAC and alternative
to salt precipitation of antibody from ascetic fluids. Further studies by Hale (1995)
showed that the metal chelate column is selective for the Fc portion of the
immunoglobulin. Data base analysis suggest that histidine rich region of C^3 domain is
the specific site of interaction and is well conserved among several immunoglobulin
classes.
Recent application of IMAC range from purification of recombinant growth
hormone (Maisano and Testpro, 1989) and tissue plasminogen activator (Furlong and
Thompson, 1988) to purification of wheat amylases, glycophorins and immunoglobulins
and their subclasses from ascetic fluids (Fuglistaller, 1989).
29
Figure 1.6 A diagrammatic representation of the principle of IMAC.
30
M A T R I X
- A - E
Me = ^
M A T R I X
- A - B
His-protein
EDTA M A T R I X
- A - B
A B Me L1&L2
Linkage Group Chelating Group Metal ion Solvent or buffer molecule
TABLE 1.1
Mechanisms of protein binding to Cu(II), Ni(II) and Zn(II) IDA: Relative contributions to apparent metal affinity from aminoacid side chains and N-terminus
Functional group side chains
His
Cys
Asp,Glu
Lys, Arg
Trp, Tyr, Phe
N - terminus
Contribution
++++
++++*
-
+
+
++
Mechanism
Direct
Direct
Indirect
Indirect**
Indirect
Direct
* An accessible cycteine will bind only in its reduced form. Metals catalyze the oxidation of cystein
** Becomes direct at high pH
Ref : Sulkowski (1985), TIBTECH :3,pp4.
1,9 Objective of the work
Enzyme coupled immunoassays are widely used in revealing antigen-antibody
interactions on solid matrices. Interesting variations involving the use of new and more
stable enzymes, chromogens and strategies like use of biotinylated antibodies and avidin
conjugated enzymes have proved remarkably effective in improving the sensitivity of
the immunoassays. The object of the work was to investigate procedures for improving
the sensitivity of the dot blot, westem blot and ELISA analysis of antigens using multiple
incubation strategy that assembles large amount of the secondary antibody-enzyme
conjugate in quantities proportional to the amount of antigen present on nitrocellulose
strips or in ELISA plates.
Efforts have also been made to develop a simple and rapid procedure for the
isolation of FCab)' fragments from intact IgG. For this purpose the ability of Cu ' -IDA
Sepharose to bind to IgG, Fc fragment but not to F(ab)'2 was made use of. A preparation
of pepsin with attached additional histidyl group was used for the cleavage of IgG,
since it could be quantitatively removed from the reaction mixture inview of its affinity
for Cu^*-IDA Sepharose at neutral pH.
32
v ^ J r\
MateriaCs
MetHods
2.0 Materials
Chemicals and reagents used for the present studies were obtained from the sources
as indicated below. Glass distilled water was used in all the experiments.
B.D.H Poole, England
B.D.H, India
Difco Laboratories Detroit, USA
E.Merck, India
Genei Pvt Ltd, Bangalore
Merck-Schereherdt, Germany
Sigma Cemical Co, USA
Qualigen's Research Laboratories
Bromophenol Blue
Copper sulphate, hydrochloric acid,
trichloroacetic acid
Freund's complete adjuvant, Freund's
incomplete adjuvant
Ammonium sulphate, glycine, methanol,
sodium hydroxide, sodium potassium
tartrate
Molecular weight markers, goat anti-
rabbit-HRP conjugate
(3-mercaptoethanol
Nitrocellulose membrane, pepsin,
sepharose 4B, Comassie brilliant blue
R- 250, sodium dodecyl sulphate, tris
(hydroxymethyl amino-methane),
hemoglobin, HS A
Acetic acid, di-sodium hydrogen
phosphate, glycerol, isopropanol, di-
hydrogen sodium phosphate, sulphuric
acid, sodium chloride, sodium bi
carbonate, zinc chloride, nickel chloride,
cobalt chloride, ferric chloride, copper
Sisco Research laboratories, India
Koch-Light, England
Pharmacia Fine Chemicals, Sweden
Hi-Media, hidia
Greiner-Labor-Technik, Germany
chloride, hydrogen peroxide, ammonium
sulphate, silver nitrate, potassium
dichromate, tween - 20.
N,N'-methylene bisacrylamide,
acrylamide, ammonium persulphate,
bovine serum albumin, cyanogen
bromide, folin's ciocalteu's phenol
reagent, sucrose, di-ethyl amino ethyl
(DEAE) cellulose, ethylene diamine
tetraacetic acid (EDTA).
Glutaraldehyde
Sepharose fast flow (IDA)
Sodium azide
Ninety six welled microtitre plate.
34
3.0 Methods
3.1 Immunological procedures
3.1.1 Preparation of the antigen
For the immunization of rabbits, the antigen (300|ag HSA/0.5ml in 50 mM sodium
phosphate buffer, pH 7.4) was mixed with equal volume of Freund's complete adjuvant
in the ratio of 1:1 .The antigen for booster doses was prepared by mixing solution of
HSA (150^g/0.5ml) with equal volume of Freund's incomplete adjuvant. The oil buffer
mixture was thoroughly emulsified using a 21 gauge syringe.
3.1.2 Immunization of rabbits
Healthy albino rabbits weighing between 2 - 3 Kg were used for immunization.
The rabbits were bled prior to immunization and the sera obtained served as control.
Rabbits were injected subcutaneously with 1.0 ml of the HSA antigen and were rested
for fifteen days. Booster doses were subsequently given weekly for four weeks. After
resting the animal for a week, blood was collected through posterior ear vein. The
blood was allowed to coagulate at 22°C for three hours, centrifuged to collect antiserum
which was then decomplemented at ST C for 30 minutes and stored at -20°C in small
aliquots.
3.1.3 Immunodiffusion
Immunodiffusion was performed essentially by the method of Ouchterlony (1962).
One percent molten agarose in normal saline containing 0.2% sodium azide was poured
on glass plates and allowed to solidify at room temperature. Required number of wells
were cut and the slides were stored at 4°C. Five to ten microliters of suitably diluted
antiserum and required amount of antigen prepared in normal saline were added in the
wells. The reaction was allowed to proceed for 12 - 24 hours in a moist chamber at
room temperature. The Petri-plates were immersed in normal saline to remove non
specific precipitin lines if any.
35
3.1.4 Isolation and purification of immunoglobulin G from the antiserum
Rabbit IgG was isolated by a slight modification of the method of Fahey and
Terry (1979). Fifty ml of antiserum was mixed with equal volume of 20 mM sodium
phosphate buffer, pH 7.2 and 11.4 gm of solid ammonium sulphate added. The mixture
was stirred to dissolve ammonium sulphate and to make it 20% saturated. After 6
hours the sample was centrifuged at 2,000g for 20 minutes and the supernatant was
made 40% saturated with respect to ammonium sulphate by adding additional 12.3gm
of the solid. Precipitation was allowed to proceed for 12 hours at 4°C. The precipitate
obtained was collected by centrifugation at 2,000g for 20 minutes. Most of the gamma
globulin were present only in the 20 - 40 % pellet which was dissolved in minimum
volume of 20mM sodium phosphate buffer, pH 7.2 and extensively dialyzed against
the same buffer with frequent changes and stored at 4°C.
3.1.5 Ion exchange chromatography
Ten grams of DEAE cellulose was suspended in 0.5 N HCl for one hour and
washed in a Buchner funnel with distilled water till the filtrate had neutral pH. The
exchanger was then treated with 0.5 N NaOH for one hour and again washed with
distilled water till the pH of the filtrate was neutral. The DEAE cellulose was then
resuspended in 20 mM sodium phosphate buffer, pH 7.2 to obtain a homogeneous
slurry. Fine particles were removed by decantation and the slurry poured in a clean
vertically mounted column (1.8 cm X 10cm). The flow rate of the column was gradually
increased to 30ml/hr. Buffer was carefully removed from the surface and the 20 - 40 %
ammonium sulphate fraction was applied. Three ml fractions were collected after washing
the column with one bed volume and analyzed for the protein content. The homogeneit}'
of purified protein was assessed by SDS-PAGE.
3.1.6 Preparation of F(ab) monomer
Purified IgG (1 Omg) was dialyzed against 0.1 M sodium phosphate buffer, pH
7.0. Cystein HCl (Img/lOmg IgG), EDTA (O.Smg/lOmg IgG) and papain (1 .Omg/
lOOmg IgG) were added to dialyzed IgG and the mixture incubated for four hours at
37°C. The digest was frozen to inhibit further digestion and was later dialyzed against
0.1 M sodium phosphate buffer, pH 8.0 overnight at 4°C. DEAE cellulose was
regenerated as detailed above and equilibrated with 0.1 M sodium phosphate buffer
pH 8.0. The dialyzed digest was applied to the coliimn and a gradient of phosphate
buffer was run from 0.1 to 0.3 M at pH 8.0. The first peak was F(ab) monomer while
the second peak was predominantly Fc (Catty and Raykundalia, 1988)
3.1.7 Formation of F(ab)\
IgG was digested with pepsin in order to obtain the F(ab)'2 fi^agments as described
by Nisonoff et al (1960). Pepsin (2mg) was dissolved in one ml 0.2 M sodium acetate
buffer, pH 4.5. IgG dialyzed against the same buffer was mixed with the enzyme solution
to give an enzyme: IgG ratio of 1: 100 and the mixture was incubated at 37°C for 24
hours. The reaction was terminated by the addition of solid tris salt that increased the
pH of the reaction mixture to 8.0. The F(ab)', fragments were then characterized by
SDS-PAGE.
3.1.8 Purification of specific IgG
HSA was coupled to CNBr activated sepharose 4B by the procedure of Porath
et a/ (1975). The preparation obtained thus contained 5.0 mg of HSA per 1.0 gm of '
gel. A column of activated sepharose 4B was packed in a 50 ml glass syringe by
carefully adjusting the flow rate. The affinity purification of specific IgG was achieved
by coupling HSA to Sepharose 4B following the procedure of Stults etal (\9S9) with
some modifications. Briefly the HSA specific antiserum was allowed to bind to the
37
HSA 4B affinity column. The column was washed thoroughly with 0.1 M tris HCl, pH
8.9 containing 2.5 M NaCl in order to remove any unbound or non specifically associated
protein. The bound IgG were eluted with 0.1 M glycine-HCl buffer, pH 3.5 and the
eluate immediately neutralized with this buffer.
3.1.9 ELISA
Buffers and substrates:- The following buffers and substrates was used
1. Tris Buffered Saline (TBS):- 20 mM tris, 150 mM sodium chloride, pH 7.4
2. Tris Buffered Saline Tween-20 (TBS-T):- 20 mM tris, 14.3 mM NaCl, 200 mg
KCl and 500 |al tween-20 were dissolved in 800 ml distilled water and pH was
adjusted to 7.4 with IN HCl. The volume was made to one litre.
3. Bicarbonate buffer:- 50 mM, pH 9.5 contained 0.02% sodium azide as
preservative
4. Substrate used is Tetramethylbenzidine/H202 (Commercial preparation diluted
1:4).
Direct ELISA
The titre/generation of human serum specific antibodies was measured in the
sera of HSA immunized rabbits by the ELISA (Autsuka, 1979). Ninty six welled
microtitre plates (Greiner Labor-technik, France) were coated with 100 |il of HSA at
a concentration of 5 mg/ml in 0.05 M carbonate bicarbonate buffer, pH 9.6. the plates
were incubated for 2 hours at room temperature and then overnight at 4°C. The antigen
coated wells were washed three times with TBS-T buffer in order to remove unbound
antigens. Unoccupied sites in the wells were blocked with 150 ml of 1.5% (w/v)
skimmed milk in TBS for three hours at room temperature. The plates were washed
with TBS-T and serially diluted antiserum (100 |j,l/well) in TBS to be tested was added
to each well (1:1000 ^1:102400) and incubated for 2 hours at room temperature.
Bound antibodies were assayed using the commercial secondary antibody-HRP
conjugate at a 1:3000 dilution. After the usual washing steps the peroxidase reaction
was initiated by the addition of the substrate tetramethylbenzidine/H^O,, arrested by
the addition of 4.0 N H,SO. and absorbance of each well was monitored at 450 nm in 2 4
an ELISA reader. Each sample was coated in duplicate and results were expressed as
mean of (A test - A control). A is the absorbance at 450 nm.
3.1.10 Immunoaffinity layering in ELISA
One hundred microliters of serially diluted HSA(l|ig to O.OOlpg) in 0.05 M
sodium bicarbonate buffer pH 9.6 were coated in 96 welled microtitre plates for two
hours at room temperature. The antigen coated wells were washed 3 times with TBS-
T to remove unbound antigens. Unoccupied sites were blocked with 150 |j|l of 1.5 %
(w/v) skimmed milk for three hours at room temperature. The plates were washed with
TBS-T and 1:1000 diluted anti-HS A-IgG in TBS was added to each well and incubated
for one hour at 37°C. After the usual washing steps the wells were again coated with
100 il of 1:3000 diluted goat anti rabbit HRP conjugate and again kept for one hour at
37°C. After the washing steps the peroxidase reaction was initiated by the addition of
the substrate tetramethylbenzidine/ H^O, and arrested by the addition of 4.0 N H^SO .
For improving the sensitivity the plates treated with anti-HS A-IgG and HRP conjugate
were further incubated with anti-HSA-IgG, washed with buffer and again incubated
with the conjugate. The alternate incubation with the anti-HSA-IgG and the conjugate
were performed for the desired number of cycles. Peroxidase activity measured under
standard conditions.
3.1.11 Dot blot/western blot
For immunodetection of HSA dots, appropriate quantities of the protein dissolved
in 0.02 M sodium phosphate buffer pH 7.4 were applied on the nitrocellulose strips
with the help of a micro syringe. The strips were blocked with 2% (w/v) solution of
skimmed milk in the buffer for two hours, washed thoroughly with wash buffer (0.01
M sodium phosphate buffer containing 0.15 M NaCl and 0.05% tween-20) and
incubated in petridishes containing 0. Img/ml anti HSA IgG at 37°C for one hour. The
strips were again washed with the wash buffer and further incubated with the goat anti-
rabbit IgG-HRP for one hour at 37°C. After washing the strips thoroughly once again
with wash buffer, they were stained by incubating with 4-chloro-1 -naphthol reagent
(Hawkes et al, 1982). For the layering steps, the unstained strips were reincubated
with anti-HSA-IgG, washed and incubated once again with the goat anti-rabbit-IgG-
HRP conjugate. The incubation cycles with primary antibody and secondary antibody
enzyme conjugated were repeated as desired prior to staining for the bound HRP.
HSA was subjected to polyacrylamide gel electrophoresis (Laemmli, 1970) and
electrophoretically transferred on to nitrocellulose (Towbin et al, 1979) at 4°C in
Hoeffer Series TE transfer apparatus operating at 100 V for twelve hours. Transfer
buffer contained 20% (v/v) methanol, 192 mM glycine and 25 mM tris. The bands
were visualized by incubating the strips first with the primary antibody followed by
washing and then with the conjugate for the desired number of times as described
before staining with the 4-chloro-1-naphthol reagent.
3.2 Colorimetric Analysis
3.2.1 Determination of protein concentration
Protein was estimated by the method of Lowry etal{\9S\). Aliquots of protein
solution were taken in a set of tubes and final volume made up to 1 ml with 0.01 M
sodium phosphate buffer, pH 8.0. Five ml of alkaline copper reagent (containing one
part of 0.5%) (w/v) copper sulpahte, \% (w/v) sodium potassium tartarte and fifty
parts of 2%) (w/v) sodium hydroxide was added, followed after 10 minutes of incubation
at room temperature with 0.5 ml of 1.0 N Folin - Ciocalteu's phenol reagent. The
tubes were instantly vortexed. The colour developed was read at 660nm after 30
40
minutes against the reagent blank. A standard curve was prepared using BSA as
standard.
3.2.2 Carbohydrate estimation
The procedure described by Dubois (1956) was followed. Two ml aliquots
containing 10 to 70 ig of carbohydrate was pipetted into a test tube and 0.05 ml of 80
% (v/v) phenol added. This was followed by addition of 0.5 ml of concentrated sulphuric
acid, the stream of acid being directed against the liquid surface rather than against the
side of the test tube in order to obtain good mixing. The tubes were allowed to stand
for 10 minutes. Thoroughly mixed and again incubated for 20 minutes at 30°C. The
colour intensity was measured at 490 nm for quantitation of hexose content. Glucose
was used as the standard.
3.2.3 Amino group estimation
The amino groups of protein were determined by the method of Habeeb (1966)
with slight modification. Suitable aliquots of the protein sample were dissolved in 1.0
ml of 0.1 M sodium tetraborate buffer, pH 9.3, 25 \i\ of 0.3 M TNBS added and the
tubes agitated instantly to ensure complete mixing and allowed to stand for 30 minutes
at 25°C. Absorbance of the yellow colour developed was recorded at 420 nm against
a reagent blank. Glycine was used as standard amino acid.
3.2.4 Assay of pepsin
Pepsin activity was assayed according to the protocol of Anson (1932). For the
determination of the proteolytic activity of pepsin, appropriate aliquots of enzyme
(3 mg pepsin in 0.2 M sodium acetate buffer, pH 4.5) were incubated with 1 mi of 2 %
(v/v) hemoglobin in 0.06 N HCl. The reaction was allowed to proceed at 37''C for 20
minutes. The reaction was stopped by the addition of 10 % (w/v) TCA. Acid insoluble
material was removed by centrifligation at 1,000g for 15 minutes. The supernatant was
analyzed for acid soluble peptides with Folins reagent (Lowry et ai, 1951).
41
1 mole of pepsin is the amount that digest 1.0 ^mole of hemoglobin to TCA
soluble peptides/ minute at 37°C.
3.3 Chemical modification of pepsin
A solution of histidine (6mg/ml) in distilled water was mixed with 0.5 % (w/v)
glutaraldehyde and incubated at room temperature for two hours. The glutaraldehyde
linked histidine was loaded on 1 ml of a Cu^^-IDA sepharose. The matrix was washed
with distilled water and the modified histidine was eluted with 5.0 ml of 10 mM EDTA.
The preparation was mixed with 3mg/ml of pepsin in 0.2 M sodium acetate buffer pH
5.5 and incubated for three hours. The sample was dialyzed extensively against the
same buflFer. This pepsin preparation was again loaded on Cu^*-ID A and eluted with
10 mM EDTA to obtain modified pepsin.
3.4 Immobilized metal ion affinity chromatography (IMAC)
Immobilized metal ion affinity chromatography (IMAC) was perfonned by using
the fast flow, Imino Diaacetic Acid (IDA) - Sepharose (Nisonoff e? ai, 1960). IDA-
Sepharose was washed with 50 mM EDTA and 500 mM NaCl (pH 8.0). CuCl solution
(20mM) was used to load the matrix with Cu ^ ions. The matrix was then washed with
the operating buffer (50mM Tris-HCl, 100 mM NaCl, pH 7.5) and mixed with the
dialyzed extract. After incubation for one hour at room temperature, the unbound F(ab)'2
was collected by centrifugation at 1,000g.
3.5 Slab gel electrophoresis
3.5,1 Polyacrylamide gel electrophoresis (PAGE)
Electrophoresis was performed essentially according to the method of Laemmli
(1970) using the slab gel apparatus manufactured by Biotech, India. A stock solution
of 30 % acrylamide containing 0.8 % bisacrylamide was mixed in appropriate proportion
to give the desired percentage of gel. It was then poured into the mould formed by two
42
glass plates (8.5 x 10 cm) separated by 1.5mm thick spacers. Bubbles and leaks were
avoided. A comb providing a template for seven wells was quickly inserted into the gel
film and polymerization allowed to occur. After 15 - 20 minutes, the comb was removed
and the wells were cleaned, overlaid with running buffer. Samples containing 15-35
|ag protein mixed with equal volume of sample buffer (containing 10% (v/v) glycerol,
0.06 M tris HCl, pH 6.8 and traces of bromophenol blue as tracking dye) were applied
to the wells. Electrophoresis was performed at 100 V in the electrophoresis buffer
containing 0.025 M tris and 0.2 M glycine until the tracking dye reached the bottom of
the gel.
3.5.2 Sodium dodecyle sulphate polyacrylamide gel elecrophoresis
(SDS-PAGE)
Sodium dodecyl sulphate (SDS) was performed by the tris-glycine system of
Laemmli (1970) using slab gel electrophoresis apparatus, manufactured by Biotech
India. Concentrated stock solution of 30 % acrylamide containing 0.8 % bisacrylamide,
1.0 M tris (pH 6.8 and 8.8) and 10 % SDS were prepared and mixed in appropriate
proportion to give the final required percentage. It was poured in the mould formed by
two glass plates (8.5 x 10 cm) separated by 1.5mm thick spacers avoiding leakes and
bubbles. A comb providing a template for seven wells was quickly inserted into the gel
before the polymerization began. The comb was removed once the polymerization
was complete and wells were overlaid with the running buffer. Protein samples were
prepared in the sample buffer containing 1 % (w/v) SDS, 10 % (v/v) glycerol, 0.0625
M tris HCl, pH 6.8 and traces of bromophenol blue as tracking dye and where required
5 % f3-mercaptoethanol. The samples were boiled at 100°C for five minutes.
Electrophoresis was performed in electrophoresis buffer containing 0.025 M Tris and
0.2 M glycine at 100 V till the tracking dye reached the bottom of the gel.
3.5.3 Staining procedures
After the electrophoresis was complete the gels were removed and the protein
bands were visualized by staining.
(a) Coomassie brilliant blue staining
Protein bands were detected by staining with 0.1 % coomassie brilliant blue
R-250 in 40 % Isopropanol and 10 % acetic acid. The staining was carried out with
10% glacial acetic acid.
(b) Silver nitrate staining
The procedure described by Merril et al (1982) was followed. After
electrophoresis the protein bands were fixed by rapidly immersing in a mixture of 40 %
(v/v) methanol and 10 % (v/v) acetic acid for one hour with constant shaking. The gel
was washed with 10% methanol and 5 % (v/v) acetic acid twice, each time for 15
minutes to allow the gel to swell to normal size. This was followed by incubation in 3.4
mM potassium dichromate solution containing 3.2 mM nitric acid for 15 minutes and
washing with distilled water. The washed gel was then immersed in 12 mM silver nitrate
solution for 20 minutes and then again washed with distilled water and transferred to
280 mM solution of sodium carbonate containing 0.5% formaldehyde to make the gel
alkaline. The reaction was stopped after 10 minutes by transferring the gel to 3%
acetic acid solution for 5 minutes. The gels were washed 4 to 5 times with distilled
water and finally stored in distilled water.
3.6 Spectral Analysis
3.6.1 UV absorption spectroscopy
The UV absorption spectra of native and modified pepsin was obtained by
measuring the absorption between 200 - 400 nm in a Shimadzu spectrophotometer
using a cuvette of 1cm pathlength. Two hundred micrograms of native and modified
pepsin in a total volume of 1 ml was taken for spectral analysis.
44
3.6.2 Flourescence spectroscopy
Native and modified pepsin was analyzed by measuring intrinsic fluorescence at
25 iO-l^C in Hitachi F 2000 spectroflourometer (Tokyo, Japan). The protein was
excited {X^^ at 280 nm and corrected emission spectra was recorded with extinction
and emission band width of 10 run. Appropriate controls containing the substances
used for the treatment were run and corrections were made wherever necessary.
To study the cumulative effect of tyrosine, tryptophan and phenylalanine the protein
was excited at 280 run. The temperature was maintained at 22°C by thermostating the
cuvette housing during the measurement. The time of mixing was defined as t = 0.
3.6.3 Circular Dichroism spectroscopy
CD of native and modified pepsin were measured in a Jasco 720
spectropolarimeter equipped with a temperature controlled sample cell holder. Spectra
were recorded w^th a scan speed of 20 nm/minutes and with a response time of one
second. Each spectrum was the average of two scans. Measurement in the far UV
(200 - 250 nm) were taken using pepsin concentration of about 500 nM (ie about
0.35 gm /litre) with a 1 mm path length cell. Whereas cells with 10 mm path length and
pepsin concentrations of about 1.5 i M (ie about 1.2g/litre) was used in the near UV
(200 - 300 nm). For each sample the buffer was used as blank.
The results were expressed as mean residual ellipticity (MRE) in deg.cmldmol-1.
Which is defined as
MRE = e ^ (m deg)/( 10 x n x 1 x Cp)
Where 9 ^ is the CD in millidegree, n is the number of amino acid residues, 1 is
the path length of the cell and Cp is the mole fraction.
45
3.7 Size exclusion high performance liquid chromatography (HPLC)
Analysis of the FCab)' formed was carried out using an HPLC apparatus
equipped with a gel filtration column (Protein Pak® Diol (OH)), 10|im, (7.5 x 300nm),
equilibration, calibration and fractionation were carried out at room temperature with a
0.4ml/min flow rate of 50 mM sodium phosphate and 150 mM NaCl (pH 7) buffer.
The protein concentration in the eluate was monitored by its absorbance at 280 nm.
46
4.0 Results
4.1 Immunogenicity of HSA and cross reactivity of anti HSA antibodies
Human serum albumin used for the study was quite immunogenic and readily
induced antibody formation in rabbits. Pre-immune and immunized antisera from 100
to 100,000 dilution was coated in the wells for titre determination. After blocking of
the wells with skimmed milk, they were again incubated with the enzyme conjugate.
The plates were then developed for the peroxidase activity. HSA was found to be
highly immimogenic, as it gives a titre of 31,622 (Fig 4.1). Sharp precipitin lines were
obtained on immimodiffusion of anti-HSA antiserum and the DEAE fraction against
HSA (Fig 4.2 A). In Fig 4.2(B) the precipitin lines obtained show that antisera raised
against HSA readily cross reacted with human serum but not with bovine serum.
4.2 Immunoaffinity layering
4.2.1 Immunodot analysis of HSA by immunoafHnity layering
Earlier studies from others (Alvarez-Kaza, 1993; Vanderberg, 1994) and from
this laboratory (Farooqi et al., 1999) have shown that amounts of enzyme immobilized
on a solid surface can be raised several folds by alternate incubations with the enzyme
and anti-enzyme antibody. The strategy was described as immunoafFmity layering. It
was envisaged that the strategy could be used to enhance the binding of secondary
antibody-enzyme conjugate to the enzyme fixed on nitrocellulose and incubated with
primary antibody. To enhance the sensitivity of the standard immunodot analysis
procedure, immunoaffinity layering was performed with anti-HSA antibodies and the
goat anti-rabbit IgG-HRP conjugate. HSA dissolved in 10 mM sodium phosphate
buffer pH 7.4 was applied as spots on the nitrocellulose strips. A set of four strips
prepared thus contained spots bearing 22.6 pg to 22.6 [ig of HSA . The strips were
blocked with skimmed milk in order to prevent non specific binding. They were then
thoroughly washed with PBS-T buffer and incubated with anti-HSA-IgG for one hour,
47
Figure 4.1 Titre determination of the raised antisera by ELISA
Ninty six well microtitre plate was loaded with serially diluted antisera.
Ajfter blocking with skimmed milk and washing with the wash buffer the
plate was again incubated with the enzyme conjugate. The enzyme activity
was read at 405 nm. For details see the text.
48
o c (0
o (0 <
• ••
-•— Preimmune sera • Immunized sera
-r-3
—r-5
-Log serum dilution
Figure 4.2 (A) Ouchterlony double diffusion of HSA against anti-HSA
antiserum and IgG fractions isolated thereof.
Rabbits were immunized with HSA. The antiserum obtained was
fractionated to isolate the P-globulin fraction as detailed in the text. The
central well contained 30|i)g of HSA. Well a, 30^g of immunize serum;
well b, 30|Xg of 20-40% ammonium sulphate cut and well c, BOp-g of
DEAE fraction.
(B) Ouchterlony double diffusion of anti-HSA-IgG against purified
HSA, Human serum and BSA
Rabbits were immunized with HSA. The antiserum obtained was
fractionated to isolate y-globulin fraction as detailed in the text. The
central well contained 50)J,1 of antiserum. Well 1 and 2 had 30ng of BSA
and HSA respectively and well 3 contained 50|il of human serum.
49
^
\
B
followed after thorough washing with the conjugate for the same duration. Strip I was
stained for the peroxidase activity while strips II, III and IV were again incubated,
after washing with bufifer, with anti-HSA-IgG followed by the conjugate, prior to staining.
After an additional incubation with the primar)' antibody and the conjugate, strip II was
stained for the peroxidase activity. Strip III and IV were carried through additional one
and two incubation cycles respectively prior to staining for peroxidase activity.
Fig 4.3 shows the quantity of HSA readily detectable under standard conditions
used was 22,600 pg, although the sample bearing 2,260 pg of HSA was also visible as
a very faint spot. The intensity of the HSA spots increased remarkably and step-by-
step on further incubations with anti-HSA-IgG and the conjugate, and those bearing
far lower concentrations of HSA, not detectable under standard conditions, also became
visible. As evident from Fig 4.3, the HSA spot bearing 22.6 pg of the protein was also
readily detectable after four incubation cycles with anti-HSA-IgG and the conjugate.
While it was possible to further increase the sensitivity of the procedure by increasing
the number of the incubation cycles with anti-HSA-IgG and the enzyme conjugated
secondary antibody, this was accompanied by a marked increase in background staining.
4.2.2 Effectof time of incubation
Attempts were made to ascertain if the enhancement in the observed sensitivity
of detection of HSA with multiple incubation assays described in the previous experiment
were not merely related to the extended durations for which HSA was exposed to the
primary and secondary antibodies. For this purpose eight nitrocellulose strips each
bearing 22,600 pg, 2,260 pg, 226 pg and 22.6 pg of protein applied as spots a, b, c
and d were incubated for var>'ing intervals with primary antibody followed by incubation
with the conjugate under standard conditions.
As evident from Fig 4.4 in the strip I incubated for 10 minutes with primary
antibody, only the spot bearing 22,600 pg of antigen was barely visible while the other
50
Figure 4.3 Immunodot analysis of HSA using the anti HSA antibody and HRP
conjugate by multiple incubation assay.
Nitrocellulose strips bearing different HSA concentration were incubated
with primary antibody and enzyme conjugate for layering process. Dot
a, 22.6 pg; b, 226 pg; c, 2,260 pg; d, 22,600 pg of HSA. I, II, III and IV
indicates the number of layers the strip is subjected to.
51
IV
III
II
a b e d
Figure 4,4 Effect of incubation time on immunodot assay. j
Nitrocellulose strips bearing different antigen (HSA) concentrations were
subjected to dot blot analysis. Dot a, b, c and d contained 22,600 pg,
2,260 pg, 226 pg and 22.6 pg of HSA respectively. Strips I, II III IV V
VI and VII were subjected for 10 min, 15 min, 30 min, 1 hr, 4 hr, 8 hr
and 16 hr respectively for each primary and secondary antibody
incubation.
VIII
c d
three spots containing 2,260 pg, 226 pg and 22.6 pg of HSA were not detectable. In
strip II the intensity of the dots bearing 22,600 pg was somewhat increased, while strip
III incubated for 30 minutes and then stained for peroxidase activity showed clear
intensification of the spot with 22,600 pg of HSA and that containing 2,260 pg of HSA
became clearly visible. In case of strip IV which was subjected to 1 hour incubation,
the 22,600 pg spot of HSA became more intense accompanied by significant increase
in the intensity of spot containing 2,260 pg of the antigen. Strip V, VI and VII that were
incubated with the antibody for 4, 8 and 16 hours respectively showed no further
improvement in the intensity of the spots. Longer incubations were also ineffective in
rendering the 226 pg and 22.6 pg of HSA visible. Incubations beyond 8 hours infact
resulted in decrease in the intensity of the spots supporting the observation of Gershoni
(1984) that antigen-antibody complex may dissociate after long incubations.
4.2.3 Immunodot analysis of HSA with intact anti HSA-IgG and F(ab)'j derived
thereof
In view of the probability of secondary antibody component of the conjugate
recognizing the non-Fc regions of the primary antibody, it was envisaged that removal
of the Fc portion of the latter may improve the binding process by decreasing the steric
hindrance. Experiments were therefore undertaken to investigate the usefulness of the
replacement of anti-HSA-IgG with the F(ab)', prepared thereof on the sensitivity of
HSA detection by the multiple incubation procedure. Purified HSA was spotted on
nitrocellulose strips and five spots of varying HSA concentrations were applied on two
strips. While one strip was taken through four incubation cycles with anti-HSA-IgG
followed by goat anti-rabbit IgG-HRP conjugate as described earlier, the other strip
was subjected to the alternate incubations with anti-HSA- ^{ah)\ and the conjugate.
Fig 4.5, shows a comparison of immunodot analysis of HSA using anti-HSA-IgG and
F(ab)', derived thereof after four incubation cycles. The improvement in sensitivity as a
result of substitution of F(ab)'2 (strip A) for IgG (strip B) was not very high but significant.
53
Figure 4.5 Immunodot analysis of HSA with intact anti-HSA-IgG and F(ab)'
derived thereof.
Nitrocellulose strips bearing dififerent HSA concentrations were subjected
to immunoaffinity layering with intact anti-HSA-IgG (B) or F(ab)'2
derived thereof (A) and goat anti-rabbit-HRP conjugate after four
incubation cycles. Dot a, 11.3 pg; b, 22.6 pg; c, 226 pg; d, 2,260 pg and
e, 22,600 pg of HSA.
54
B
• • -^y
The spot bearing even 11.3 pg of HSA was clearly visible after four incubation cycle
involving F(ab)'2 and the intensities of the spots bearing higher concentration of the
proteins increased when compared with those stained after multiple incubations using
intact anti-HSA-IgG. Removal of the Fc region thus appears to facilitate a moderate
enhancement in binding of secondary antibody conjugate, presumably by making antigen
binding domains on FCab)' region more accessible.
4.2.4 Immunoaffinity layering using native and glutaraldehyde crosslinked anti
HSA-IgG
An attempt to improve the sensitivity of detection was also made using anti-
HSA-IgG crosslinked with glutaraldehyde. As shown in Fig 4.6, anti-HSA-IgG migrates
as two bands corresponding to light and heavy chain and the IgG was transformed to
high molecular weight adduct on treatment with gluteraldehyde. The preparation cross
linked with 0.5% glutaraldehyde did not enter the gel on electrophoresis indicating the
very high molecular weight nature of the adduct. Eight strips spotted with 15.6 pg,
156 pg, 1,560 pg and 15,600 pg of HSA as dots a, b, c and d respectively. Four were
incubated with anti-HSA-IgG (Fig 4.7, Panel A) and four processed with glutaraldehyde
crosslinked anti-HSA-IgG, (Fig 4.7, Panel B). In strip I, Panel (A) the dot bearing
15,600 pg of HSA is very clear while that with 1,560 pg is visible as a very faint spot.
In contrast in strip I, Panel (B) even the dot bearing the 156 pg of antigen is clearly
detectable. While the dots bearing 15,600 pg and 1,560 pg were also more intense as
compared to Panel (A).
4.2.5 Immunodot analysis of HSA using affinity purified anti-HSA specific
antibody
Experiments performed thus far were performed using the IgG fraction isolated
from the immune sera. Specific antibodies against HSA were isolated by affinity
purification using HSA coupled to CNBr activated Sepharose 4B following the published
55
Figure 4.6 Gel electrophoresis of glutaraldehyde treated anti-HSA-IgG
Anti-HS A-IgG was treated with glutaraldehyde and characterized using
10% acrylamide gel in presence of P-mercaptoethanol. The gel was
stained and de-stained according to the procedure given in the text.
Lane a and b contained 0.5% and 0.05% glutaraldehyde treated
preparations. Lane c contained native anti-HSA-IgG.
56
a b
Figure 4.7 Immunodot analysis of HSA by immunoaffinity layering using
native anti HSA-IgG (A) and gluteraldehyde linked anti-HSA-IgG
(B).
Strips of nitrocellulose membrane bearing 15.6 pg, 156 pg, 1,560 pg
and 15,600 pg in dots a; b; c and d respectively. The strips were carried
through One (I), two (II), three (III) and four (IV) incubation cycles as
described in the text.
57
IV
III
II
a b c d
B 1
^
1' 1
• •
#
o
o
#
•
. •
e
IV
III
II
a b c d
procedure (Stults, 1989). The preparation obtained thus contained 5mg/10ml of HSA.
HSA specific antiserum was allowed to bind to the HSA-Sepharose 4B affinity column.
The column was washed to remove any unbound IgG and the specific IgG eluted with
0.1 M glycine HCl buffer pH 3.5. These immunoaffinity purified anti-HSA antibodies
were used to perform immunodot analysis of HSA. Four nitrocellulose strips were
spotted with 8.2 pg, 82 pg, 820 pg and 8,200 pg of HSA as dots a, b, c and d
respectively. The strips were first blocked with skimmed milk and then incubated with
affinity purified anti-HSA-IgG for 1 hour and with the conjugate also for 1 hour at
37°C. Strip I was stained for the peroxidase activity, and strip II, III ans IV were
taken through further incubation cycles (Fig 4.8). The dot bearing the 82 pg of antigen
was very faint but detectable by the standard assay procedure. In the strip subjected
to two cycles of incubation 82 pg spot could be seen clearly. The 8.2 pg spot was also
visible in strip III as a faint spot. After four incubation cycles, 8.2 pg of HSA spot
could also be clearly seen. The intensity of the spots also increased with each successive
incubation cycle. The sensitivity of detection however appeared to improve only
moderately when affinity purified antibodies were used instead of the IgG fraction.
4.2.6 Immunodot analysis of HSA in human serum using the multiple incubation
assay
Four nitrocellulose strips were spotted with varying amounts of human serum as
five dots. The strips were then taken through one, two, three and four incubation
cycles, as described earlier in experiments, and Fig 4.9 shows the result of the study.
As evident, HSA in the human serum sample bearing 3,944 pg of protein was barely
detectable by the standard procedure. Successive alternate incubations as described
earlier caused the appearance of new spots and marked intensification of those already
visible. At the end of four incubation cycles, the spot of the human serum sample
bearing 32 pg of protein was also detectable as a faint spot while that containing 39 pg
protein turned more intense. Considering that albumin constitutes about half of protein
58
Figure 4.8 Immunodot analysis of HSA using affinity purified anti HSA-IgG
on nitrocellulose strips by multiple incubation assay.
Nitrocellulose strips bearing various concentration of HSA were carried
through One (I), two (II), three (III) and four (IV) incubation cycles as
described in the text. Dot a, 8.20 pg; b, 82 pg; c, 820 pg; d, 8,200 pg of
HSA
59
IV
III
II
a b e d
Figure 4.9 Immunodot analysis of human serum by immunoaffmity layering.
Nitrocellulose strips were spotted with whole human serum of different
dilutions and subjected for four incubation cycles with anti-HSA-IgG
and goat anti-rabbit-HRP conjugate. Dot a, 32 pg; b, 39 pg; c, 394 pg;
d, 3,944 pg; e, 39,440 pg; of protein. I, II, III and IV indicates the
number of layers the strips are subjected to.
60
o e IV
III
II
a b c d e
in plasma, the sensitivity of its detection in plasma samples appears to be comparable
with that of purified HSA.
4.2.7 Studies of albumin aggregation using multiple incubation assay
The applicability of the multiple incubation procedure to detect HSA aggregation
at low concentrations was also investigated. HSA was subjected to 7.5% gel
electrophoresis under non denaturing conditions (Fig 4.10). The lanes a, b, and c
contained 5.6 ng, 56 ng, and 560 ng of albumin respectively. Subsequent to
electrophoresis the protein bands were transferred on to nitrocellulose membrane
according to the Towbin's protocol (Towbin et a/., 1979). Strips of panel 1 - IV were
subjected to single, two, three or four incubation cycles respectively with primary
antibody and the conjugate as described earlier in the text. As evident from Fig 4.10,
panel I (a), aggregation is barely visible in the sample containing about 5.6 ng of HSA
that migrated as a fast moving major band and a very faint band migrating about 75%
of its distance, presumably representing the aggregated form of the protein. The slow
migrating band was visible somewhat more prominently in the sample bearing 56 ng
but was very clearly visible in that with 560 ng of protein, which also showed the
presence of an additional far slower moving bands. Further incubations with primaiy
antibody and secondary antibody conjugate however resulted in visualization of
additional bands and at the end of the four incubation cycles, even the sample bearing
5.6 ng showed additional slow migrating bands. Evidently even at the lowest
concentration of HSA used, formation of aggregates takes place, but the aggregates
were apparently present at concentration below the limits of detection of the standard
staining procedure. The aggregation could however be clearly visualized by the multiple
incubation procedure and additional bands of various mobilities could be seen even in
samples containing 5.6 ng of protein.
61
Figure 4.10 Multiple incubation assay showing albumin aggregation
HSA was subjected to polyacrylamide gel electrophoresis and transferred
to nitrocellulose membrane. Strips of panel I-IV were subjected
respectively to single two, three and four incubation cycles with anti-
HSA-IgG and conjugate. Lanes a; b and c contains 5.6 ng, 56 ng and
560 ng of protein.
62
II III IV
a b c a b c a b c a b c
4.2.8 Multiple incubation assay in ELISA
Wells of the polystyrene microtitre plates were coated with various HSA
concentrations ranging between 0.0001 pg to 1 |ig. They were blocked with 2% (w/v)
skimmed milk, incubated with anti HSA-IgG for 1 hour at 37°C. This was followed by
the incubation with goat anti-rabbit-HRP conjugate and estimation of the peroxidase
activity colorimetrically. After four incubation cycles even 1 pg of antigen was detectable.
Sensitivity appears to increase nearly 10 fold after four incubation cycles (Fig 4.11).
4.2.9 Multiple incubation assay in ELISA using F(ab)'2
The experiments described in section 4.2.8 was also performed using anti-HSA-
F(ab)'2 instead of intact IgG. Plates were coated with varying amounts of HSA ranging
from 0.0001 pg to 1 fig. Following the same standard procedure for layering, the
plates were developed for peroxidase activity. Fig 4.12, shows the result after four
incubation cycles. With F(ab)\ 0.1 pg of antigen could be detected after four incubation
cycles. This suggested significant improvement in sensitivity over the procedure that
employed intact IgG.
63
Figure 4.11 Multiple incubation assay on ELISA plate
Ninty six well microtitre plate bearing HSA (0.0001pg to lug) were
incubated with anti-HSA-lgG and enzyme conjugate through One (I),
two (II), three (HI) and four (FV) incubation cycles as described in the
text.
64
0.7
0.6
0.5 -
^ 0.4
o « 0.3
<
—•-
o
^
- Layer 1 Layer II
- Layer III Layer IV
0.2
0.1
0.0
^
^
10-10 10-9 10-8 I 1 1 1 1 1 1 1 —
10-7 10-6 10-5 10-4 10-3 10-2 10-1 100
HSA (^g)
Figure 4.12 Multiple incubation assay on ELISA plate using F(ab)'2.
Ninty six well microtitre plate bearing HSA (0.000 Ipg to l|ag) were
incubated with anti-HSA-F(ab)'2 and enzyme conjugate for multiple
incubation process. Results shown are of plates taken through four
incubation cycles.
65
0.7
0.6
0.5 -
g 0.4 c CO
€ I 0.3 <
0.2
0.1
0.0
-— • -
o
~
1
• Intact IgG F(ab)'2
1 1
d
1
o
1
o
1
o
1
o '
1
. o
1
.o •
1
• O
1
10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 100
HSA (ng)
5.0 Modification of pepsin
5.1 Preparation of pepsin modified with histidine
Porcine pepsin contains 328 aminoacid residues, including two arginines, one
histidine and one lysine. Our preliminary experiments indicated that affinity of pepsin
for IDA Sepharose was only moderate, presumably due to the presence of the single
histidine residue. The lysine residue protrudes firom the surface behind the active site
cleft on the C-terminal domain of porcine pepsin (Cottrell et al ,1995). It was therefore
envisaged that additional histidine could be coupled to the side chain of lysine residue
of the enzyme to facilitate stronger binding of the enzyme to the immobilized metal ion
support.
In order to achieve covalent attachment of histidine, 6 mg of the aminoacid was
mixed with 20-fold molar excess of glutaraldehyde in a total volume of one ml 0.02 M
phosphate buffer pH 7.4 and incubated at room temperature for two hours. Amino
group content of the glutaraldehyde treated histidine was determined with the help of
TNBS method (Synder and Sobosinsta, 1975) and at the end of the incubation it was
observed that greater than 95% of the amino groups were modified. In order to remove
unreacted glutaraldehyde, the mixture was loaded on one ml column of Cu-' -IDA
Sepharose. The matrix was washed with distilled water and the bound histidine eluted
with five ml of 20 mM EDTA. The eluted preparation of modified histidine was mixed
with 3 mg of pepsin in sodium acetate buffer pH 5.5 in a total volume of 6.0 ml and the
mixture incubated for three hours at 4°C. This mixture was again loaded on the Cu-"-
IDA Sepharose and the modified pepsin eluted with 20 mM EDTA. The eluted sample
was extensively dialyzed against 200 mM sodium acetate buffer pH 3.5. The pepsin
preparation retained 85% of enzyme activity after its treatment with glutaradehyde
treated histidine.
66
5.2 Properties of modified pepsin
5.2.1 Kinetic behaviour
5.2.1.1 Effect of substrate concentration
When pepsin catalyzed haemoglobin digestion was measured as a function of
substrate concentration at 37°C in 0.2 M sodium acetate buffer pH 3.5, a typical
Mechalis Menton behaviour was observed in both the cases of native and modified
pepsin. The Line Weaver Burk plot indicated K^ values for native and modified pepsin
preparations as 200 mM and 222 mM, indicating only a minor alteration in the affinity
of the enzyme for protein substrate as a result of the chemical modification. The V^^ of
the modified pepsin remained nearly unchanged (Fig 5.1).
5.2.1.2 Effect of temperature
Temperature activity profiles for native and modified preparations were
investigated in 0.2 M sodium acetate buffer, pH 3.5 and shown in Fig 5.2. No alteration
in temperature maximum was observed and both native and modified pepsin showed
maximum activity at 40°C. However the modified pepsin retained a relatively higher
fraction of maximum enzyme activity at temperatures above 40°C
5.2.1.3 Effect of pH
Activity profiles of native and modified pepsin were investigated in 0.2 M
sodium acetate or sodium phosphate buffer at various pH at 40°C (Fig 5.3). The
optimum pH value of the modified preparation was identical with that of the native
enzyme.
5.2.2 Electrophoretic behaviour
The porcine pepsin modified as described above was dialyzed thoroughly against
0.2 M sodium acetate buffer, pH 3.5 and subjected to electrophoresis. Fig 5.4 (.A.)
67
Figure 5.1 Line Weaver Burk Plot for haemoglobin digestion by pepsin and
modified pepsin preparations.
30 ^g of pepsin and modified pepsin preparations were incubated in a
series of tubes with the standard assay mixture containing varying
concentration of substrate under standard conditions, and the activity
was determined.
68
^ ?
-0.01
34
" • Native Pepsin A IModified pepsin
/
X '
/ y / * X * A /
X * V * -X ^
/ ^ A k\ 1 1 >
(1 0.01 0.02 0.03
*
0.04 0.05
1/S (mlM)
Figure 5.2 Temperatute activity profiles of native and modified pepsin
Aliquots of pepsin and modified pepsin were incubated at indicated
temperatures for 15 minutes and assayed for enzyme activity under
standard conditions of pH and substrate concentration.
Figure 5.3 pH activity profiles of native and modified pepsin preparations.
Aliquots of native and modified pepsin preparation were incubated in
0.2 M sodium acetate (pH 3.0 - 5.0) or 0.2 M sodium phosphate
(6.0 - 8.0) for 15 minutes and the activity determined under standard
assay condition.
69
> (0
E 3
E X (0
E
120
100
80
60
40
20
-•— Native pepsin
• Modified pepsin
— I —
40
— I —
80 20 60 100
Temp (°C)
120
100
• >
(0
E
X m
E
80 -
60
40
20 -
-•— Native pepsin • Modified pepsin
2 4 6
PH
8 10 12
Figure 5. 4 (A) Gel electrophoresis of native and modified pepsin.
Native and modified preparations were subjected to gel electrophoresis
in 12.5% polyacrylamide in the presence of P-mercaptoethanol and SDS.
Lane a, molecular weight markers; lane b, native pepsin (35^g); lane c,
modified pepsin (35ng).
(B) Molecular weight determination of modifled pepsin by Weber
and Osborn procedure.
The relative mobility of the standard marker proteins from the SDS gel
(Figure 5.4 A) were plotted against logarithm of molecular weight using
least square analysis. Arrows indicate the position of the native and
modified pepsin.
70
98Kda
68Kda
43Kda
29Kda
20Kda
14Kda
a
5.2
5.0 -
4.8 -
§. 4.6
4.4
4.2 -
4.0
A Modified pepsin •^ Native pepsin
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Relative mobility (cm)
0.9
shows the electrophoretic behaviour of pepsin in reducing SDS PAGE. As evident
from the Fig 5.4 (A) there was a very slight difference in the mobility of modified and
native pepsin, with modified pepsin migrating slighlty slower than native enzyme. The
molecular weight of modified pepsin as calculated was 37.2 KDa and the native pepsin
had a molecular weight of 35.8 KDa (Fig 5.4, B).
5.2.3 Autolysis of pepsin and modified pepsin
In order to compare the susceptibility to autolysis, native and modified pepsin in
0.2 M sodium acetate buffer, pH 3.5 were incubated for 18 and 24 hrs at 40''C. SDS-
PAGE under reducing condition shows one major band for modified pepsin, and three
in native preparation incubated for 18 hrs (Fig 5.5, lane d and e). Even after 24 hours
the modified preparation showed only two major bands while native pepsin was cleaved
to several fragments (Fig 5.5, lane b and c). A small band migating slower than pepsin
was observed in the modified pepsin preparation incubated for 24 hours.
5.2.4 Spectral studies
5.2.4.1 Ultra violet spectroscopy
The UV spectrum of native pepsin shows maximum absorbance at 277 nm,
while modification resulted in slight alteration in the spectrum with a shift in the absorption
maximum from 271 to 274 nm. There was observed a 3 nm blue shift with a decrease
in the magnitude of absorbance (Fig 5.6).
5.2.4.2 Fluorescence spectroscopy
The histidine modified pepsin was also characterised for its fluorescence using
an excitation wavelength of 280 nm over the range of 300 - 400 nm. As shown in Fig
5.7 the emission spectra obtained gave maximum intensity at 368 nm, both for native
and modified pepsin preparation, but showed a decrease in the magnitude of
fluorescence intensity in case of the modified preparation.
71
Figure 5.5 Autolysis of native and modified pepsin
Native or modified pepsin were subjected to SDS-polyacrylamide gel
electrophoresis after their incubation for 18 or 24 hours at 40°C at pH 2.
Lane a, molecular weight markers (98,68,43,29,20 and 14 KDa); lane b,
modified pepsin after 24 hours incubation; lane c, native pepsin after 24
hours; lane d, native pepsin after 18 hours; lane e, modified pepsin after
18 hours; lane f, native pepsin at zero hour.
72
a b c d e f
Figure 5.6 Ultravoilet spectra of native and modified pepsin
UV spectra of native and modified preparations in 0.2 M sodium acetate
buffer were taken at pH 3.5. The protein concentration was 200 |ig/ml
and the pathlength was 1 cm.
Figure 5.7 Fluorescence spectra of native and modifled pepsin
Fluorescence spectra of pepsin and modified pepsin in 0.2M sodium
acetate buffer at pH 3.5. The protein concentration was 200 ng/ml.
Samples were excited at 280 nm and band width was 10 nm.
73
o oo CM
(Q
0) O C (0
o en
<
Native pqjsin Modified pepsin
240 260 280 300
wavelength (nm)
320 340
400
350
300
250 H
Native pepsin Modified pepsin
300 320 340 360
Wavelength (nm)
380 400
5.2.4.3 Circular dichroism of native and modified pepsin
The far UV spectrum for native pepsin is characterized by the presence of a
minima at 216 run (Fig 5.8). Modification of pepsin with glutaraldehyde Unked liistidine
resuked in only a marginal alterations in the spectrum leading to a slight dip at 219 nm.
The CD spectra in the near UV region reflects the contribution of aromatic side
chains, disulphide bonds, and of prosthetic groups (Kuwajima, 1989; Dryden and
Weir, 1991). Fig 5.9 shows the near UV-CD spectrum of pepsin in 250 - 300 nm
range. Near UV-CD spectrum of native pepsin is characterized by a band at 258 nm
and the modified pepsin also showed nearly similar spectrum. The small decrease in
the MRE value indicates a slightly less ordered structure in case of the modified pepsin.
5.2.5 Binding of native and modified pepsin on Cu^MDA Sepharose
Native or modified pepsin (2mg/ml) were mixed and incubated with Cu^ -IDA
Sepharose at increasing pH (2 - 8) for one hour. The matrix was centrifuged and the
amount of protein in the supernatant was used to calculate percent binding. Fig 5.10
shows that at pH 2, both native and modified pepsin showed only about 19 % binding
to IDA Sepharose. At pH 4, there was a slight increase in the binding of modified
pepsin to IDA over native pepsin which shows only 20% binding. At pH 6 and 8
modified pepsin showed a marked increase in binding to the support over the native
enzyme.
5.2.6 Elution of pepsin and modified pepsin bound to Cu^*-IDA with EDTA
Elution profile of native and modified pepsin was studied at varying EDTA
concentration. Native and modified pepsins were eluted with 5 ml of (4 - 24 niM)
EDTA. Fig 5.11 shows that native pepsin elutes completely at 16 mM EDTA while
modified pepsin elutes only 80 % under the condition. At 20 mM EDTA however both
the preparations were eluted completely from the support.
74
Figure 5.8 Far UV-CD spectra of native and modified pepsin
Experiments were carried out at 0.2 M sodium acetate buffer pH 3.5
using 1 ml (0.35 mg/ml) of native ( ) or modified pepsin
( ) solution in a 1 cm path-length cuvette after two scans from
200 to 250 nm at room temperature under continuous nitrogen flush.
75
m I O
o
e •D
CM
e •
<b <b t . CT <b X3 n a> u
200 210 220 230
X(n m)
240 250
Figure 5.9 Near UV-CD spectra of native and modifled pepsin
Spectra were generated using 1 ml (1.2 mg/ml) solution of native
( ) and modified pepsin ( ) in a 1 cm path length cuvette
after two scans from 250 to 300 nm at room temperature under
continuous nitrogen flush.
76
en I O
o
e CM
6 < j
<u a> i _
o>
n CD u
250 260 270 280
^ (nm)
290 300
Figure 5.10 Binding of native and modified pepsin to Cu'MDA Sepharose
Native or modified pepsin was allowed to bind to Cu^^-IDA at a pH of
2, 4 ,6 and 8
77
120
100
I Native pqjsin
I Modified pq}sin
80
60
.S
.S 60 X!
40
20
pH
Figure 5.11 Elution profile of native and modified pepsin from Cu ' -IDA
Sepharose
Native and modified pepsin bound to Cu -IDA was eluted with increasing
EDTA concentration.
78
G O
3
w
I ^ U
100 -
80 -
60 -
40 -
20 -
0 -
••-• Modified pepsin
1 1
' •
• " '
1
— - * — — — • . • • • "
1 1
10 15 20
EDTA (mM)
25 30
5.3 Purification and pepsinization of IgG and F(ab)', formation.
5.3.1 Purification of IgG from anti-HSA antiserum
Commercial HSA (Sigma) is quite immunogenic in rabbits and readily elicited
the formation of precipitating antibodies. Its higher titre generation is shown in
Fig 4.10.
For the purification of anti-HSA antibodies, the procedure described by Catty
and Raykundalia (1988) was followed. The antisera were precipitated between 20 -
40 % ammonium sulphate saturation, and the precipitate dissolved in 20 mM sodium
phosphate buffer pH 7.2, was subjected to ion exchange chromatography on a DEAE
cellulose column (1.8x10 cm) equilibrated with 20 mM sodium phosphate buffer, pH
7.2. Purification of IgG was followed by polyacrylamide gel electrophoresis performed
in presence of SDS. Whole antiserum and ammonium sulphate fractions show bands
corresponding to IgG and several other polypeptides (Fig 5.12, lane a and b). Nearly
all other bands were removed after DEAE cellulose chromatography and the purified
IgG preparation gave a single band (Fig 5.12, lane c) on electrophoresis.
5.3.2 Molecular weight of IgG
In order to determine the molecular weight of the isolated IgG preparation, SDS-
PAGE was run along bovine serum albumin (68 KDa), ovalbumin (43 KDa), carbonic
anhydrase (29 KDa), soyabean trypsin inhibitor (20 KDa) and lysozyme (14.2 KDa)
as markers (Fig 5.13 A). The molecular weight of the polypeptides of IgG was calculated
as per the procedure of Weber and Osborn (1969) by plotting the mobility of marker
proteins Vs the logarithim of their molecular weights (Fig 5.13 B). The position of the
migration of IgG polypeptides corresponded to their apparent molecular weights of 48
KDa and 25 KDa.
79
Figure 5.12 Purification of IgG
IgG was purified by salt fractionation and DEAE cellulose
chromatography, and subjected to SDS-PAGE. Lane a, rabbit serum;
Lane b, 20 - 40% ammonium sulphate fration; Lane c, DEAE fraction
80
a b
Figure 5.13 (A) SDS-PAGE of IgG during various stages of purification
Antiserum, 20 - 40% ammonium sulphate fraction and fraction from
DEAE cellulose and markers were incubated with sample buffer at 100°C
for five minutes and subjected to 12.5% polyacrylamide gel
electrophoresis in presence of SDS. Lane a, DEAE cellulose fraction;
lane b, 20 - 40% ammonium sulphate fraction; lane c, antiserum; lane d,
molecular weight markers.
(B) Molecular weight determination by Weber and Osborn
procedure
The relative mobility of the standard marker proteins from SDS gel
(5.13 A) was plotted against logarithm of molecular weight using least
square analysis. Arrows indicate the positions of the large and small
molecular weight peptides from the DEAE fraction.
81
66Kda
45Kda
31 Kda
21.5 Kda
14.4 Kda
0.0 0.2 0.4 0.6
Relative mobility (cm)
1 .» -
4.8-
4.7 -
4.6 -
S w 4.5 -o _ i
4.4 -
4 . 3 -
4.2 -
4.1 -
i
^
<—
^ •
A L N .
1 1 1
I T 1 •
Heavy chain Light chain
\ »
1
0.8 1.0
5.3.3 Cleavage of IgG with modified pepsin and purification of F(ab)' by IMAC
The IgG fraction obtained by ammonium sulphate fractionation and DEAE
cellulose chromatography was subjected to proteolysis with the modified pepsin
preparation to obtain F(ab)'2 fragments. Initially, the procedure described by Nisonoff
et al (1960) for IgG cleavage by pepsin was followed. Two mg/ml of IgG solution was
incubated with modified pepsin in a ratio of (1:100) at 40°C for 24 hours at pH of 4.5.
The reaction was terminated by adding tris salt that increased the pH to 8.
Fragmentation of the IgG was studied by SDS PAGE under reducing conditions.
The gel shows the disappearance of the band corresponding to IgG with a significant
increase in the band of higher mobility (Fig 5.14, lane b) suggesting the cleavage of
IgG into VidbyjYc fragments. The faster moving protein bands presumably correspond
to degraded Fc fragment.
With a view to achieve the separation of F(ab)'2 generated from intact IgG/Fc,
IMAC was used. Fig 5.14, lane a shows a single band with mobility corresponding to
IgG inferring the complete binding of intact IgG, Fc and fragmented Fc to Cu-"-IDA
Sepharose.
Optimum conditions of IgG cleavage by modified pepsin, were evaluated by
studying the dependence of the process on temperature , pH and time of incubation.
5.3.3.1 pH dependent cleavage of IgG
IgG (2 mg/ml) cleavage with modified pepsin preparation was investigated
after 12 hours incubation at 40°C at various pH values between (3.5 to 5.0). The
reaction was stopped by the addition of 0.2 MNaOH. Fig 5.15 shows that IgG was
best cleaved at pH 3.5 while at pH 5.0 there was very little cleavage of IgG . At pH
4.0, most of IgG was cleaved leaving only a faint band corresponding to heavy chain.
82
Figure 5.14 Pepsinization of IgG
2 mg/ml IgG was exposed to pepsin at pH 4.5 at 37°C. The reaction was
terminated after 24 hours by the addition of tris salt. Fragmented IgG
and F(ab)'2 obtained was subjected to non reducing PAGE. Lane a,
supernatant obtained after binding of the pepsin digested IgG on Cu^ -
IDA Sepharose Intact IgG; Lane b, IgG treated with pepsin; Lane c,
intact IgG.
a b c
Figure 5.15 pH dependent cleavage of IgG
Pepsin digestion of the IgG was carried out at various pH values and the
digest subjected to SDS-PAGE. Lane a, intact IgG; lane b; c; d and e,
pepsin digests prepared at pH 5.0,4.5,4,0,3.5 respectively.
84
a b o d e
5.3.3.2 Temperature dependent cleavage of IgG
IgG was cleaved with the modified pepsin preparation at pH 3.5, for 12 hours
at various temperatures between 4°C to 40°C. Fig 5.16, lane b shows that the
fragmentation of IgG was barely detectable at 4°C, while at 20°C cleavage was quite
significant (Fig 5.16, lane c). Presence of a single band in the preparation exposed to
protease at 40°C shows complete fragmentation of IgG (Fig 5.16, lane d).
5.3.3.3 Time dependent cleavage of IgG
IgG was incubated with modified pepsin preparation at pH 3.5 at various time
intervals at 40°C, the reaction was stopped by adjusting the pH to 8 with 0.2 M NaOH
and the reaction products characterized by SDS-PAGE. Fig 5.17, lane d shows that
after one hour incubation nearly all the IgG was cleaved.
5.4 Binding of F(ab) monomer to Cu-'-IDA Sepharose
Experiments were performed to determine if the F(ab) monomers bind to IDA
Sepharose. This was considered important since small amounts of the monomer may
be formed during the proteolysis of IgG to F(ab)',. For the purpose 50 |ig of each
F(ab)'2 and F(ab) obtained as described in methods were rhixed and loaded on Cu-'-
IDA Sepharose, and the unbound fraction was characterized by PAGE. Fig 5.18, lane
b shows 2 bands corresponding to F(ab)', and Fab, lane a had only one band
corresponding to F(ab)', indicating essentially complete binding of F(ab) monomer to
the Cu-*-IDA Sepharose.
5.5 Isolation of F(ab)\ from unfractionated serum
Five hundered microlitres of serum was adjusted to pH 3.5 with the help of
0.06 N HCl, digested with 500 |ig of modified pepsin at 40°C. After one hour
incubation, the reaction was terminated with 0.2 N NaOH. One hundered \i\ of digested
serum was loaded on 300 |il of IDA Sepharose loaded with Cu-', Fe-" , Ni-*, Co-* or
85
Figure 5.16 Temperature dependent cleavage of IgG
SDS-PAGE of the pepsin digest of IgG carried out at various
temperatures, assessed under reducing conditions. Lane a, Intact IgG;
Lane b; c and d has IgG digested at 4°C, 20°C, 40''C respectively. Lane
e, marker proteins (98,68,43,29,20 and 14 KDa).
86
a b c d e
Figure 5.17 Time dependent cleavage of IgG
IgG digested with pepsin for various time intervals was subjected to
SDS-PAGE. Lane a, marker proteins (98,68,43,29,20 and 14 KDa);
lane b, intact IgG; lane c; d; e; f and g pepsin digests obtained after 30
minutes, 1 hour, 4 hours, 8 hours and 12 hours respectively.
87
a b c d e f g
Figure 5.18 Binding of F(ab) monomer to Cu^MDA.
A mixture of F(ab)'2 and F(ab) prepared as described in methods was
incubated with Cu^"'-IDA-Sepharose. The unbound fracure was subjected
to 7.5% non-reducing PAGE. Lane a, IDA supernatant; Lane b, mixture
of F(ab)'2 dimerand F(ab) monomer.
Figure 5.19 SDS-PAGE of serum digested with modified pepsin and binding to
IDA-Sepharose loaded with various metal ions
The serum digest as described in the text was allowed to bind to Cu " -,
Fe^*-, Ni-^- and Zn^ - IDA Sepharose. Unbound fraction was subjected
to PAGE in absence of |i-mercaptoethanol (Panel A) or in presence of
P-mercaptoethanol (Panel B).
Panel A, lane a, pepsin digest, lane b,c,d,e, and f had the supernatant of
Fe^ -, Cu^"-, Co^^-, Ni - and Zn^^-chelated IDA Sepharose.
Panel B, lane a, pepsin digest, lane b,c,d,e and f contained the supernatant
of Zn^*-, Ni-"-, Fe^^-, Co^ - and Cu-"- chelated IDA Sepharose.
90
a b c d e f
B
a b c d e f
Figure 5.20 High performance gel filtration chromatography of IgG and its
pepsin digest.
Rabbit IgG was digested with pepsin and the digest incubated with Cu ""-
IDA Sepharose. The samples were analyzed using a Protein Pak ®
column. (A) Intact IgG, (B) Pepsin digest of IgG, (C) supernatant of
IgG digest incubated with Cu " - IDA Sepharose.
91
m 5.00 10.00 m \m im m HinuUs
10.00 1100 18.00 i m 25,111) 3 0 1
Hintites
m 10.00 ] m
Figure 5.21 High performance gel filtration chromatography of serum and its
pepsin digest.
Rabbit serum was digested with pepsin and the digest incubated with
Cu^*- IDA Sepharose. The samples were analyzed using a Protein Pak*
column. (A) serum, (B) pepsin digested serum proteins, (C) supernatant
of serum digest incubated with Cu^*-IDA
92
2.00 e.oo 10.00 n.oo it.DO R O D 2i.oo )m HinullS
M,00 30.00
HiouKs
tenninated by adjusting the pH of the reaction mixture to 8 with the help of 20 fil of 0.2
M NaOH. The sample was centrifuged and the supernatant analyzed both by reducing
and non-reducing SDS-PAGE. Fig 5.22 panel (A), lane b, c and Fig 5.22 panel (B),
lane a, b shows a single band indicating the formation of F(ab)' .
5.7.1 Isolation of F(ab)'j in a single tube from various mammalian species
IgG was isolated from the sera of sheep, goat, buffalo, rabbit and rat. Prior to
incubation with the reaction mixture, the pH of the IgG was adjusted to 3.5. The IgG
solution (1 mg/ml) from each species was added to the tube, 0.2 M NaOH was added
to stop the reaction after one hour incubation at 40°C. Supernatant collected was run
on SDS-PAGE. IgG from all the species showed the presence of only F(ab)'2 in the
unbound fraction (Fig 5.23). All the lanes give a single band confirming their
homogeneity.
5.8 Antigenicity of F(ab)'2 isolated from IgG
Immunodiffusion was also performed to check the antigen binding affinity of
F(ab)'2 preparation. Sharp precipitin lines were obtained on immunodiffusion of anti
HSA- F(ab)'2, and affinity purified anti-HSA-F(ab)'2 prepared from the above
procedure indicating the homogeneity of the preparation (Fig 5.24).
Figure 5.22 Production of purified F(ab)'j from IgG in single tube.
1 mg/ml IgG was incubated with modified pepsin, IDA-Sepharose and
CuClj at pH 3.5 for 1 hour unbound fraction was subjected to SDS-
PAGE under reducing condition (Panel A), non reducing condition
(Panel B)
Panel A, lane a, marker proteins (98,68,43,29,20 and 14 KDa); lane b
and c contains 40 and 80 |il of the unbound fraction. Panel B, lane a and
b had 80 and 40 |J.l of unbound fraction respectively.
94
«4
a b
B
Figure 5.23 Preparation of F(ab)'2 from IgG derived from various mammals
IgG derived from various mammalian sera were digested with modified
pepsin and the reaction mixture purified on Cu(II)-IDA Sepharose. The
supernatant was subjected to SDS-PAGE along with ^-mercaptoethanol
as described in the text. Lane a; b; c; d and e had 50 ^\ of fraction that
did not bind to IDA Sepharose from sheep, goat, buffalo, rabbit and rat
respectively.
95
a b c d e
Figure 5.24 Ouchterlony double diffusion of anti-HSA- IgG and anti-HSA-IgG-
F(ab)'j against HSA.
Rabbits were immunized with HSA antiserum obtained was fractionated
to isolate the whole y-globulin and anti HSA IgG fraction (affinity
purified) as detailed in the text. IgG obtained was treated with modified
pepsin preparation to yield F(ab)'2 in the single tube incubation procedure.
The central well contained 30|ig of HSA; well 1, anti-HSA antiserum;
well 2, anti-HSA-IgG; well 3, F(ab)'2 derived thereof; well 4 and 5 had
anti-HSA specific IgG and F(ab)'2 derived thereof respectively.
96
HJ •Mm
(Discussion
6.0 Discussion
Enzyme coupled immunoassays are widely used in revealing antigen
antibody interactions on solid matrices such as nitrocellulose membranes, in dot
blot and western blot procedures, tissue sections, cell smear preparations as well
as in ELISA. Immunoassays and analogous assay systems are of increasing
importance both in basic and applied research as well as for routine diagnostic
purposes. Sensitivity in most cases is the key problem in immunological detection
systems. Until recently, there has been an explosive increase in the application of
the technique in detection and quantitation of antigens/antibodies as well as in
efforts to improve them (Towbin et al., 1984; Chu et al., 1989). Interesting
variations involving the use of biotinylated antibodies and avidin conjugated
enzymes have proved remarkably effective in improving the detection of dot blot
analysis (Guedson et al, 1979; Ogata et al, 1983).
In efforts to raise the sensitivity of Clark electrode based glucosensors, large
amount of the enzymes have been assembled on Sepharose support as alternate
glucose oxidase/polyclonal antiglucose oxidase IgG layers and the technique was
described as immunoaffmity layering (Farooqui et al., 1999).
The present work employs a similar strategy which is remarkably effective
in increasing the limits of detection of protein antigens on nitrocellulose in dot
analyses as well as after transfer from polyacrylamide gels and in ELISA. Studies
described in chapter 4 shows that sensitivity of immunodetection of HSA on
nitrocellulose strips can be raised upto 100-fold by repeated incubations with the
primary antibody and the secondary antibody HRP conjugate prior to staining
them with the substrate 4-chloro-1 -naphthol/H^Oj. The conjugate being oligomeric,
is expected to have multiple binding sites for the primary antibody. On incubation
of the conjugate with the nitrocellulose strips bearing HSA with bound primary
antibody, binding occurs. A number of binding sites of the oligomeric conjugate
97
for primary antibody are however expected to remain free due to non availability
or inaccessibility of the sites on the primary antibody. Consequently, when the
strips are incubated further with anti HSA-IgG (primary antibody), additional
binding of the later occurs. Some of the binding sites of the primary antibody thus
bound to the complex may also remain free, similarly facilitating additional binding
of the conjugate during subsequent incubations with the later. Continuation of the
alternate incubations with the primary antibody and the conjugate may therefore
result in the formation of multiple layers of primary antibody and the conjugate
resulting in the association of large amount of the enzyme (HRP) with the antigen
(Fig 6.1). This results in remarkable increase in the sensitivity of detection of the
antigen. Fig 4.3 shows that quantity of HSA readily detectable by dot analysis
using the standard procedure is 22,600 pg but a faint spot of the sample bearing
2,260 pg of HSA was also visible. The intensity of the HSA spots increased
remarkably on taking the strips through further incubation cycles and as evident
from Fig 4.3 the spot bearing as low as 22.6 pg of HSA was also detectable after
four incubation cycles.
In order to eliminate the possibility that the longer duration of the incubations
for which the strips are exposed to primary antibody and the conjugate during
multiple enzyme assay, are responsible for the observed increased sensitivity, effect
of extending the duration of incubation was studied with primary/secondary
antibody. It is clear from the Fig 4.4 that there is no increase in the intensity of
spots after one hour incubation and the intensity of spots of strips incubated for 4,
6 and 8 hours was not higher than that incubated for one hour. Instead the intensity
of spots actually decreased slightly after 8 hours of incubation time (Fig 4.4, strip
VIII), supporting the observation of Gershoni (1984) that antigen-antibody complex
may dissociate on long incubations. This study thus rules out that observed increase
in the intensities of the spots after multiple incubations is not the result of more
longer exposure to the primary antibody or the conjugate.
98
Figure 6.1 Diagrammatic representation of the multiple incubation procedure.
99
^
One incubation cycle Antigen
Two incubation cyck
Y Primary antibody
Three incubation cycles
X Enzyme conjugate
Four incubation cycles Niti'ocellulose membrane
With a view to achieve improvement in the sensitivity F(ab)'2 fragments
derived from anti HSA-IgG were used instead of whole IgG in the multiple
incubation assay.The removal of the Fc portion from primary antibody decreases
the steric hindrance for the binding of the conjugate and lowers non-specific binding
(Ishikawa, 1983). Fig 4.5 shows that the spot bearing 11.3 pgofHSA was readily
detectable after four incubation cycles when ¥(aby^ but not intact IgG was used.
The observed improvement in sensitivity was however only moderate with an
apparent 2-fold increase in the detection limit.
In an attempt to further increase the sensitivity of detection of the antigen,
usefulness of using crosslinked primary antibody vvas investigated. It was envisaged
that oligomeric/poiymeric antibodies will be left with greater number of unoccupied
antigen sites after binding to the antigen immobilized on the nitrocellulose. This
would result in enhancement in the binding of the conjugate molecules and hence
improved detection in the sensitivity. For this purpose anti-HSA-IgG crosslinked
with the bifunctional crosslinking agent glutaraldehyde was used in place of the
IgG in the multiple incubation assay. Glutaraldehyde treatment of the IgG resulted
in the formation of large aggregates that did not enter the gel (Fig 4.6). Fig 4.7
shows that the multiple incubation assay with glutaraldehyde linked primary
antibody slightly improved the sensitivity of antigen detection and it was possible
to detect 15.6 pg of HSA. There was approximately only 1.5-fold increase in
sensitivity as compared to the standard multiple incubation procedure with
crosslinked anti-HSA-IgG. The improvement in sensitivity achieved by using the
crosslinked antibodies is not remarkable, presumably due to masking inactivation
of some of the antigen binding sites as a result of cross linking.
Use of affinity purified anti-HSA-IgG instead of IgG also resulted in only
modest improvement in the sensitivity of the multiple incubation procedure. The
sensitivity increase was nearly 2.7-fold as compared to the standard procedure
(Fig 4.8). 100
The applicability of the multiple incubation assay for the detection of HSA
in the unfractionated serum samples was also studied. HSA spot in the human
serum sample was detectable by standard procedure only when an aliquot of serum
bearing a minimum of 3.9 ng of protein was applied on the strip. The multiple
layering procedure could however lower the detection limit in the serum sample
to 32 pg, there by increasing the sensitivity about to 100-fold. This is in accordance
with the observed increase in sensitivity while using pure HSA (Fig 4.9).
Applicability of the multiple incubation assay for increasing the sensitivity
of detection of HSA after electrophoresis and western blotting on nitrocellulose
strips was also investigated. Albumin has been shown to aggregate at neutral pH
especially when the concentration of the samples was high (Jordan et al., 1994;
Katakam and Banga, 1995; Ortega-Vinuesa, 1998). It is usually inferred that
aggregation is not significant at low protein concentration. As shown in Fig 4.10
aggregation was barely visible with the standard procedure in 5.6 ng of HSA
sample, but was clear after four inbubation with primary antibody and the enzyme
conjugate.
The multiple incubation strategy with the primary antibody and the conjugate
also worked quite well in ELISA, and the sensitivity of the assy could be increased
significantly (Fig 4.11). One pg of HSA could be quantitated after four incubation
cycles, while the limit of the standard ELISA procedure was only 10 pg. Multiple
incubations was also performed in ELISA using F(ab)'2, and it is evident from Fig
4.12 that after four incubations 0.1 pg of HSA could be conventionally quantitated.
The detection limit of all the procedures used are summarised in Table 6.1.
The repetitive incubation with primary antibody and the conjugate as well
as the process of washing between successive incubations makes the procedure
time consuming. Taking into consideration the remarkable improvement in
101
Table 6.1
Sensitivity of HSA detection/quantitation by the multiple incubation assay'
Technique used
Immunodot
analysis
Western blotting
(HSA aggregation)
ELISA
Primary antibody
Anti-HSA-IgG
Anti-HSA-F(ab)'2
Glutaraldehyde linked anti-HSA-IgO
Affinity purified Anti-HSA-IgG
Anti-HSA-IgG
Anti-HSA-IgG
Anti-HSA- F(ab)'2
Detection limit
After single
incubation
cycle (pg)
2,260
1,700
1,560
820
56,000
10
1
After four
incubation
cycles (pg)
22.6
11.3
15.6
8.2
5,600
1
0.1
* The assays were performed using the secondary antibody-HRP conjugate and
the standard procedure as described under methods
102
sensitivity achieved, the efforts may however be considered worthwhile. In addition
considering that the antigen antibody interactions involved in the entire multiple
incubation procedure are not different from those of the standard procedure, the
specificity of the assay is not compromised.
The multiple incubation approach with the moderate improvement in
sensitivity of detection of the cells/tissue bound antigens/antibodies (Halveson et
al., 1981; Lansdorp et al., 1984) and in the construction of biosensors (Farooqui
et al., 1999) have been described earlier. To our knowledge amplification of
sensitivity of detection on nitrocellulose membrane of the magnitude described in
this study has not been achieved thus far. Naser et al (1990) described a single
incubation multiple layer immune technique in which the immobilized antigen is
incubated simultaneously with primary antibody and secondary antibody enzyme
conjugate. While the author claims 4-20 fold improvement in sensitivity, this could
not be substantiated in our laboratory due to precipitation of the immune complex
from the solution during the incubation.
In most instances of infection, parasite specific antibodies are more easy to
detect and hence used as diagnostic tools. However diagnosis based on antigen
may be more reliable although antigen concentrations present in blood even at the
peak of infection period may be quite low. This together with the difficulty of
obtaining adequate human biological samples for the assays, necessitates highly
sensitive antigen detection procedures. The multiple incubation procedure described
in this study may be very help ful in this respect. Another remarkably attractive
feature of the procedure is its potential in increasing the sensitivity of antigen
detection using commercial kits.
The growing use of immunological assays requires rapid, reproducible
protocols for preparation of fragments from polyclonal and monoclonal antibodies.
102
Preparation of FCab)' fragments by pepsinolysis is a well established procedure
for producing antibody fragments that retain full binding activity but lack the
effector functions conferred by the Fc domain (Wisonoffet al., 1960; Andrew and
Titus, 1997). Removal of the Fc portion of the IgG also restricts the steric hindrance
in the binding of antigen. Such FCab)' fragments have also proven very useful in
experiments designed to analyze the role of the Fc segment in various biological
functions (Turner et al., 1970). Enzyme cleaved antibodies are also widely used in
the treatment of snake envenoming (Meyer et al., 1997; Moran et al., 1998).
The classical method of preparation of F(ab)'2 involves the isolation of the
y-globulin class of serum proteins, digestion with pepsin (Nisonoff er al., 1960)
and final recovery of F(ab)'2 fragments by gel filtration (Jaquet and Cebra, 1965)
or ion exchange chromatography (Clezardin et al, 1985). Such procedures as
well as the simplified method of Madson and Rhodkey (1976), using digestion of
whole globulin fraction are effective but quite laborious, time consuming and
expensive.
In the present study efforts have been made to develop a single step procedure
for the production of F(ab)'2 from purified IgG as well as serum fractions. The
metal chelate affinity binding technique described by Hale and Biedler, (1994)
was exploited for the purification of the F(ab)'2 fragments. Hale (1995) has shown
that metal chelate (Cu^*-IDA Sepharose) column is selective for the Fc portion of
Immunoglobulin and the well conserved histidine rich region of C^3 domain is the
principal specific site of interaction. The site is apparently well conserved among
several immunoglobulin classes. Thus selective affinity for immobilized metals for
the Fc portion of antibodies provides an effective technique for purification of
F(ab)'2 which is devoid of Cj 3 domain and thus does not bind to the support.
104
The objective of the technique described here is to isolate F(ab)'2, free of
pepsin used for the proteolytic cleavage of IgG, Fc fragment or the fragment
thereof or any remaining intact IgG. In order to achieve the goal, it was necessary
to ensure to have pepsin preparation that possessed strong affinity for the
immobilized metal ions and this was achieved by attaching an additional histidine
to the enzyme (Fig 6.2). The covalent attachment of a histidyl residue was
accomplished by treating pepsin with glutaraldehyde treated histidine. Porcine
pepsin contains a single lysine residue (Cottrell et al., 1995). The imadazole group
is expected to be attached mainly at position 319 although the aldehyde treated
histidine may react with other amino acid side chains as well (Habeeb et al., 1968;
Fraenkel-Conrat and Olcott, 1968). The modified pepsin obtained exhibited
remarkable higher affinity towards Cu ' -IDA Sepharose as compared to the native
enzyme and could be purified by binding onto and elution from the support.
Various kinetic and spectral parameters studied to investigate the behaviour
of the modified pepsin. Fig 5.1 shows that there was only a marginal increase in
the K^ value of modified pepsin, which suggest that the modification does not
cause major alterations in conformation of the active site and hence the affinity
for substrate. The modified preparation was almost equally active as native enzyme
with identical V^ ^ and exhibited unaltered temperature optimum. The temperature-
activity profile of the modified pepsin was also comparable with the native pepsin
(Fig 5.2). The pH activity profiles however suggested that the modified enzyme is
less resistant to extremes of pH as compared to native pepsin although both
preparations have same pH optima (Fig 5.3). The result is in accordance with that
of Cottrell et al (1995), who showed a decrease in the inactivation pH for the
pepsin in which lysine was modified with methionine with no appreciable shift in
the pH optimum.
105
Figure 6.2 Diagrammatic representation of the procedure of coupling
histidine to lysine residue of pepsin with the help of glutaraldehyde
106
8
I I I + 9i
3 t S 9
o
o o-
-^ o
+ 2 s O
^
g
o a O I o—o-
I o
• t^
u—X
o 2 o u—u—n: O
.S B.
a
o a
I
In SDS-PAGE the modified pepsin migrated signifantly slowly as compared
to the native pepsin indicating an increase in molecular weight. The molecular
weight of modified pepsin comes out to be 1.4 KDa more than the native pepsin,
which is higher than what is expected if the attachment occurs of a single
glutaraldehyde linked histidine to the lone lysine of the enzyme. Evidently the
modified histidine may have reacted with residues other than lysine as described
earlier. Alterations in the rigidity of the polypeptide due to the chemical modification
may also be responsible for the slower mobility of the modified pepsin in SDS-
PAGE (Fig 5.4).
The decrease in the activity of pepsin at higher temperature may be related
both to the unfolding of the polypeptide and autolysis. One can therefore speculate
that modified pepsin being more resistant to autolysis (Fig 5.5) may retain greater
fraction of enzyme activity at higher temperature. While the side chain residues of
aminoacids that undergo modification as a result of glutaraldehyde treatment may
not contribute to the pepsin susceptible bonds (Fruton and Bergmann, 1939;
Konigsberg et al., 1963), the observed lower autolysis of modified pepsin may be
related to a minor alteration in conformation. It is also difficult to explain the
presence of the band migrating slower than pepsin in the modified preparation
incubated for 24 hours (Fig 5.5). It has been reported that chemical modifications
of several enzymes including proteases results in improving resistance to autolysis
and inactivation (Bogacheva et al., 1977; Nakajima et al., 2002). The improvement
in stability at high teniperature is certainly a significant advantage in their utilization
in the degradation of proteins, since higher temperatures in addition to contributing
towards enhanced reaction rates also result in unfolding of the substrate proteins
(Inadaera/., 1995).
The UV absorption spectrum of modified pepsin showed a 3nm blue shift
(Fig 5.6) indicating that hydrophobic groups of the modified enzyme may be slightly
107
more buried as compared to native enzyme. However the fluorescence spectrum
depicted a decrease in the intrinsic fluorescence (Fig 5.7) obtained for modified
pepsin indicating some environmental changes around the chromophoric groups
as a consequence of the attachments of histidine to the lysine residue of porcine
pepsin. The quenching can be attributed to the shifting of some tryptophan residues
to a more polar environment. (Friedfelder, 1982).
Circular dichroism (CD) is widely used for studying the conformational
changes of proteins in solutions (Proven-Cheer and Glockner, 1981). Fig 5.8 shows
the far UV CD spectrum for pepsin and modified pepsin with the characterstic
minimum at 215 run for acid proteases (Tello-Sollis et al., 1995; Allen et al.,
1990). In the spectral region of 200 - 250 nm, the CD signal of proteins is mainly
due to its secondary structure (Manavalan, 1983; Hennessay et al., 1981). The
CD spectra obtained for modified pepsin and native pepsin have no significant
difference, indicating no major change in the secondary structure of modified pepsin.
Although the spectrum for modified pepsin also shows a slight dip at 219 nm,
inferring a stable ^-sheet structure for modified preparation (Yada et al., 1986;
James et al., 1986). The similar observations was also found when polyhistidine
tag was attached to ovine growth hormone (Khan et al, 1998).
Differences in ellipticity within the spectral region of 250-300 nm reflects
modifications in the immediate structural and electronic environment of aromatic
amino acid residues (Jirgensons, 1970; Creighton, 1991). The mean residue
ellipticity of modified pepsin is decreased at 260 nm indicating the presence of
slightly less ordered tertiary structure as compared to native pepsin (Fig 5.9).
Modified pepsin showed a remarkable increase in binding to the chelating
matrix. Its binding capacity was 49% higher than that of the native pepsin at pH 8
(Fig 5.10). The elution profile also shows the high affinity of the modified pepsin
108
for Cu ' -IDA Sepharose as modified pepsin gets eluted at a higher salt concentration
than native pepsin (Fig 5.11). While it has been shown that single accessible surface
histidine may be adequate to retain a protein strongly on metal chelate support
(Arnold, 1991), attachement of additional histidine residue may increase the affinity
of the enzyme for the support. Thus Chaga (1994) have coupled histidine residues
to glycosyl chains of glycoproteins subsequent to their oxidation by periodate.
Thus it was possible to remarkably increase the affinity of P. chrysogenum glucose
oxidase for metal ion chelated support and confer affinity to HRP that does not
bind to metal chelate support in the native form.
For the purification of F(ab)'2 fragment, IgG was incubated with the modified
pepsin preparation at pH 4.5 for 24 hours at 37°C (Nisonoff ef al., 1960; Parham,
1986) and F(ab)'2 was obtained by loading the modified pepsin digest to Cu^ -
IDA Sepharose (Fig 5.14). The single band in lane a, Fig 5.14 shows that any
fragmented Fc or intact IgG present in the digested preparation were bound to the
matrix while F(ab)'2 remained in the unbound fraction.
Experiments performed to study the cleavage of IgG with modified pepsin
shows that IgG is best cleaved at pH 3.5 (Fig 5.15). It is also evident from Fig
5.16 and 5.17 that IgG is cleaved in one hour incubation with modified pepsin at
40°C.
Interestingly even the monomeric F(ab) is selectively separated from F(ab)'2
during the procedure, when F(ab) and F(ab)'2 were mixed and loaded on
Cu^*-IDA. The supernatant collected after centrifugation of the Cu' ^-IDA-
Sepharose revealed the presence of F(ab)'2 only (Fig 5.18 lane a). Possibility of
the formation of F(ab) monomers exists during treatment of IgG with proteases
like pepsin (Hale and Biedler, 1994).
109
At pH 3.5 there is little binding of Cu-* to the IDA-Sepharose hence there is
poor binding of modified pepsin to the support (Fig 5.10). At pH 3.5 therefore
most of the modified pepsin remains in solution and acts actively on IgG. When
the pH is adjusted to 8.0 most of the Cu ^ binds to IDA-Sepharose and in turn
bind all pepsin. The Cu ' -IDA Sepharose also binds any intact IgG, F(ab) fragment
or Fc fragment containing the histidine rich binding site leaving in solution only
F(ab)'2 and as shown earlier (Fig 5.14) any F(ab) monomer formed is also likely to
be removed from the solution leaving only F(ab)'2 (Fig 5.18).
Significantly the procedure for the preparation of F(ab)'2 could also be
applied directly to serum. Incubation of the serum with the modified pepsin resulted
in degradation of majority but not all the proteins. It is evident from the Fig 5.19
that Cu^*-IDA Sepharose binds nearly all the other proteins present in the digest
of the serum leaving F(ab)'2 in the supernatant. The high affinity of the support
for the serum proteins is effective in the complete removal of all the protein other
than F(ab)'2. High binding affinity of Cu ' -IDA Sepharose for albumin was earlier
reported by Hanson and Kagedal (1981).
The fragmentation of IgG and purification of F(ab)'2 was also substantiated
by HPLC. On fragmentation of IgG with modified pepsin and subsequent binding
of the mixture on Cu^*-IDA Sepharose, a single peak with retention time of 13
minutes was obtained that corresponded with that of F(ab)'2 (Fig 5.20). The
chromatogram with the digested but not incubated with Cu ' -IDA Sepharose shows
a small peak at the retention time of 11 minutes which corresponds v/ith the peak
for native IgG. This sugests that the metal ion support completely binds any intact
IgG left in the preparation. The chromatogram obtained from size exclusion HPLC
using the whole antiserum processed similarly also shows a single peak at the
retention time of 13 minutes (Fig 5.21).
10
In order to make the technique of F(ahy^ formation more simple and
applicable to small sample volumes, attempts were made to isolate F(ab)'j from
IgG in a single tube .A diagrammatic representation of single tube incubation
system was shown in Fig 6.3. CuClj, IDA-Sepharose and modified pepsin were
taken in sodium acetate buffer pH 3.5 to which was added IgG. After one hour of
incubation at 40''C adequate NaOH was added to change the pH to 8 and terminate
the reaction. Single band in PAGE and reducing PAGE confirms that all the proteins
and the peptides except F(ab)'2 binds to the matrix (Fig 5.22). While the modified
pepsin is expected to completely bind to the IDA-Sepharose under the conditions.
The procedure appears to be applicable for the preparation of F(ab)'2
fragment from IgG derived from various mammalian sources (Fig 5.23). Also the
F(ab)'2 seems to retain their ability to bind the antigen effectively as observed by
immunodiffusion (Fig 5.24).
Figure 6.3 Diagrammatic representation ofthe preparation of F(ab)'2 from IgG
and its separation from uncleaved IgG, pepsin and Fc fragment
112
Cu YV Cu
Cu
i l l Cu
Cu Y
pH 8
f Cu
Cu-|
C i r^
' Cu—I
• Cu-^
' C u - |
. C u H
V V
V V
V Ill Cu
F(ab)'2 Modified Fragmented Copper pepsin Fc ion
Y Intact antibody
IDA matrix
Test eppendorf
7.0 Summarj'
Recent years have witnessed a remarkable interest in the improvement of
sensitivities of enzyme based immunoassays for detection and quantification of
various anaiytes. Immunoassays combine the remarkable specificity of antibodies
with that of enzymes to catalytically generate a chromogen and can be applied
virtually to any substance of interest. The first part of the thesis deals with the use
of a multiple incubation assay with primary antibody and the secondary antibody
HRP conjugate for enhancement in the sensitivity of the HRP-based immunoassays
using human serum albumin (HSA) as the model antigen.
The multiple incubation assay is based on the observation that when a
polyvalent antibody binds to an antigen immobilized on solid support, all the binding
sites do not have the opportunity to combine with the antigen for steric reasons
and some of these remain free. Similarly when the secondary antibody-enzyme
conjugate is incubated with the nitrocellulose strips bearing antigen with bound
primary antibody some of the binding sites on the conjugate also remain unoccupied.
When the strips are again incubated with primary antibody, significant binding of
the later therefore occurs and the unoccupied sites of the later have the potential
to bind additional conjugate molecules. Thus "layers" of primary antibody and the
conjugate can be assembled that contain enzyme conjugate in quantities
proportional to the concentration of the antigen. Using anti-HSA-IgG and HRP-
anti rabbit-IgG conjugate it was possible to remarkably improve the sensitivity of
antigen detection on the nitrocellulose strips. The sensitivity of detection increased
over 100-fold after four incubation cycles with primary antibody and the conjugate
as compared to that of the standard procedure. While it was possible to further
increase the sensitivity of detection by increasing the number of incubation cycles,
the background staining also increased significantly.
113
The sensitivity of detection of HSA by multiple incubation procedure could
be increased, but only moderately, by replacing the intact primary anti-HSA-IgG
with the F(ab)'2 fragment derived thereof as well as by crosslinking the antibody
with glutaraldehyde. The assay appeared suitable for the micro-detection of HSA
in human serum samples.
The multiple incubation assay could also be used to demonstrate the
aggregation of HSA at very small concentrations of the protein. For this purpose
HSA was subjected to electrophoresis and transferred onto nitrocellulose
membrane, and the nitrocellulose strips taken through the multiple incubation assays
prior to staining for HRP activity. While only the sample bearing 560 ng of protein
exhibited the aggregation when visualized by the standard procedure, aggregation
was evident even in samples containing 5.6 ng of protein after four incubation
cycles
The multiple incubation assay was also useful in increasing the sensitivity of
HSA by ELISA and unlike the standard procedure with the detection limit of 10
pg, even 1 pg of HSA could be quantitated conveniently after four incubation
cycles.
F(ab)'2 fragments of antibodies are currently of great interests both as
diagnostic and therapeutic agents, as they retain full binding activity but lack the
effector functions conferred by the Fc domain. Generally the procedures available
for the purification of ¥{ab)\ are time consuming, requiring several steps for
digestion of IgG as well as separation and purification of the fragments.
Contamination of the F(ab)'2 preparation with the proteolytic enzyme used for the
cleavage of IgG, F(ab) or Fc poses are quite problematic. Therefore attempts
have been made to develop a simple procedure for obtaining pure F(ab)', using
immobilized metal ion affinity chromatography (IMAC). As shown in second part
of the thesis, pepsin was chemically modified for the purpose by attaching histidine
14
to the lysine residue of the enzyme. The modification resuhed in 49% enhancement
in the binding affinity of the modified pepsin for Cu^*-IDA Sepharose. Kinetic and
spectral parameters studied for porcine pepsin after modification showed no
significant change in the K^ and V^ ^ values. UV, fluorescence and CD spectra
also showed that there is no major change in the structure of modified pepsin.
Investigations of the dependence of IgG cleavage on temperature, pH,
incubation time with modified pepsin were studied and it was found that IgG
(Img/ml) was best cleaved at pH 3.5 in one hour incubation time at 40°C.
The modified pepsin was effective in converting IgG to F(ab)'2. When the
pepsin IgG digest was allowed to bind to Cu^'^-IDA-Sepharose at pH 8, the modified
pepsin gets bound to the matrix along with any F(ab) and Fc fragment present in
the digest leaving F(ab)'2 in the solution. Purity of F(ab)'2 obtained thus was
confirmed by size exclusion HPLC and showed the presence of a single peak with
the retention time of 13 minutes that corresponded with that of F(ab)'2. The
procedure was also effective in purification of F(ab)'2 directly from the serum
without the need for IgG purification.
The complete procedure of F(ab)'2 purification was achieved in a single
tube incubation system to make it more convenient and effective for small sample
volumes. For this purpose IgG was transfered to small tubes containing CuCl^,
IDA-Sepharose and modified pepsin in sodium acetate buffer pH 3.5 and incubated
at 40°C for one hour. Subsequently the pH of the reaction mixture was adjusted to
8 by adding NaOH solution. At pH 8 the modified pepsin and other major
polypeptides were bound to the support, and electrophoresis of the unbound
fraction showed only a single band in SDS-PAGE corresponding to the of F(ab)',.
The procedure was useful in the preparation of F(ab)'2 from the IgG of
various mammalian species. The F(ab)', prepared thus also retained their ability to
combine with the antigen as revealed by immunodiffusion.
115
7.0 Bibliography
Abad-Zapatero, C , Rydel, T. J. and Erickson, J. (1990) Proteins. 8, 62
Adair, S. W. (1982) Anal Biochem. 125, 299
Akita, E. M. and Nakai, S. (1993) J immunol Methods. 162, (2), 155
Allen, B., Blum, M., Cunnigham, A., Chou T. G. and Hoffman, T. J. (1990) Biol Chem. 265, 5060
Alvarez-Kaza, M. and Bilitewski, W. (1993) Anal Chem. 65, 525 A
Alwine, J .C, Kemp, D.J. and Stark, G.R. (1977) ProcNatl Acad Sci. USA.74, 5350
Alwine, J .C, Kemp, D.J., Parker, B.A., Reiser, J., Renart, J., Stark, G.R. and Wahl, G.M. (1979) Methods in Enzymology (Wu, R .ed) Vol. 68, pp 220, Academic Press New York
Ancell. (1839) Lancet.l, 222
Andrew, S. M. and Titus, J. A. (1997) Current Protocols in Immunology, Wiley, Newyork, pp 2.7.1
Anel, A., Calvo, M., Naval, J., Iturraalde, M., Alava, M.A. and Pineiro, A. (1989)FEBSLett. 250, 22
Anson, M. L. and Mirsky, A.E. (1932) J Gen Physiol. 16, 59
Arnold RH. (1991) Biotechnology. 9, 151.
Autsuka, S., Okawa, M., Ikebe, K. and Yakahari, R. (1979) J Immunol Methods. 28, 149
Avrameas, S. (1969a). Immunochemistry. 6, 43
Avrameas, S. (1969b). Immunochemistry. 6, 825
Bala, S., Seth, S. and Seth, RK. (1987). Acta Paediatr Hung. 28, 187
Balbin, M., Vizoso, F. and Sanchez, L.M. (1991) Clin Chem. 37, 547
Barnes, D. and Sato G. (1980) Anal Biochem. 102, 255
Bates, D.L. (1987) Trends Biotechnol. 5, 204
Bazin, H., Cormont, F and Dc Clereq, L. (1984) J Immunol Methods. 71, 9.
Beeker, J.M. and Wilchek, M. (1972) Biochem Biophys Acta. 264, 165
Bell, M. L. and Engvall, E. (1982) Anl Biochem. 123, 329
Benanchi, PL., Gazzei, G. and Giannozzi, A (1988) J chromatogr. 450, 133.
Bennich, H. and Johansson, S. G. O. (1971) Adv Immunol. 13, I
Bisgaard, K. and Pluzek, K.J. (1996) 10th International Congress of Histochemistry and Cytochemistry, Kypto, Japan
116
Bisgaard, K., Lihme, A. Rolsted, H. and Pluzek, KJ . (1993) Scandinavian Society for Immunology XXIVth Annual meeting, Arrhus, Denmark
Bittner, M., Kupferer, P. and Morris, C.F. (1980) Anal Biochem. 102, 459
Blake, D.A., Skarstedt, M.T., Shultz, J. L. and Wilson, D.P. (1984) Clin Chem. 30, 1452
Blank, G.S. and Vetterlein D (1990) Anal Biochem. 190, 317.
Bleeker, W.K., Agterverg, I., Rigter, G., de Vries-Van., Rossen A. and Bakker, J. (1987) Vox Sang. 52, 281
Bleeker, W.K., Agterverg, I., Rigter, G., Feeling, J. L., Tool, A. J., Koenderman, A.H., Kuijpers, T. W. and Hack, C. E. (2000) Blood. 95, 1836
Bleeker, W.K., Agterverg, I., Rigter, G., Van Rooijen, N., Rossen A. and Bakker, J. (1988) Clin Exp Immunol. 77, 338
Blumsohn, T.A., Proce, A., Morris, B.W., Griffiths, H. and Gray, T.A. (1991) Ann clin Biochem. 28,187
Bobrow, M.N., Harris, T.D., Shaughnessy, K.J. and Litt, G.J. (1989) J. Immunol Methods. 125, 279
Bogacheva, T.F., Mirgorodskaia, O.A. and Moskvichev, B.(1977) Biokhimiia. 4, 609.
Bondi, A., Chieregatti, G., Eusebi, V, Fulcherie, E. and Bussolati, G. (1982) Histochemistry. 76,153
Bowen, B., Steinberg, J., Laemmli, U.K. and Weintraub, H., (1980) Nucl Acids Res. 8, 1
Bradford, M.M. (1976) Analytical Biochem. 72, 248.
Brandtzaeg, R (1998) J. Immunol Methods. 216, 49
Brandtzaeg, R and Rognum, T.O. (1983) Histochem. J. 15, 655
Brandtzaeg, R, Halstensen, T.S., Huitfeldt, H.S. and Valnes, K.N. (1997) Immunochemistry 2-A. Practical Approach (A.P. John Stone and M. W. Turner eds) pp 71, Oxford University Press, Oxford, U.K
Brodersen, R., Vorum, H., Krukow, N. and Pederson, A.O. (1991) Eur J Biochem. 197,461
Burnette, T. C. and Schmidt, C. L. A. (1921) J Immunol. 6, 255
Burnette, W.M. (1981) Anal Biochem. 112, 195
Butcher, H., Kenntte, W., Collins, O., Demoor, J. and Koropatnick, J. (2003) J. Immunol Methods. (1-2), 247
Callister, S.M., Case, K.L., Aggar, W.A., Schell, R.F., Johnson, R.C. and Ellingson, J.L. (1990) J Clin Microbiol. 28, 363
17
Capra, J.D. and Edmundson, A.B. (1977) Scientific American 236, 50.
Cartlidge, P.H. and Rutter, N. (1986) Arch Dis Child. 61, 657
Catty, D. and Raykundalia, C. (1988) Antibodies- A Practical Approach. Catty, D (edO IRL Press. 1, 50
Chaga, C (1994) Biotechnol Appl Biochem 20, 43.
Chu, N., Janckila, A. J., Wallace, J. H. and Yam, L. T. (1989) J Histochem Cytochem. 17,257
Cistola, D.R and Small, D.M. (1991) J. Clin Invest. 87, 1431
Clark, C.A., Downs, E.C. and Primus, F.J. (1982) J Histochem Cytochem. 30, 27
Clark, L., Hitzeman, R. and Carbon, J. (1979) Methods in Enzymology (Wu, R. ed) Vol 68, pp 436, Academic Press, New York
Clezardin, P., Mc Gregor, J. L., Manch, m., Boukerche, H. and Dechavanne, M. (1985) J Chromatogr. 319, 67
Coons, A.H. and Kaplan, M.H. (1950) J. Exp. Med .91,1
Coons, A.H., Creech, H.J. and Jones, R.N. (1941) Proc. Soc. Exp. Biol. Med. 47, 200
Cooper, J.K. and Gardner, C. (1989) J. Am Geriatr Soc. 37, 1039
Cottrell, T.G., Harris, L.J., Tanaka, T. and Yada, R. Y. (1995) J Biol Chem. 271,19974
Coulter, A. and Harris, R. (1983) J Immunol Methods. 59, 199.
Creighton, T. E. (1991) in Protein Structure ( A Practical Approach, 2nd edn) pp 261. IRL pRess, Oxford University Press. UK.
Danscher, G. (1981) Histochemistry, 71,81.
de Alwis, W. U. and Wilson, G. S. (1989) Talanta. 112, 249
De Waele, M., Renmans, W., Segers, E., Jochmans, K. and Van Kamp, B. (1988) J. Histochem Cytochem. 36, 679
Deliconstantions, G., Tsopanakis, C , Karayjannakos, P. and Sklkeas, G. (1986) Atherosclerosis. 61, 67
Derrer, M.M., Miescher, S., Johansson, B.. Frost, H. and Gordon, J. (1984) Allergy Clin Immunol. 24, 621
Dieter, R, Krause, H. and Schulze, S.A. (1990) Eicosanoids. 3, 45
Dryden, D. and Weir M.R (1991) Biochim Biophys Acta. 94, 1087.
Dubois, M.K., Gilles, A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956) Anal Biochem. 28, 350
118
Edelman, G. M. (1969) Biochem. 9, 3197
Edelman, G. M. and Gall, W. E. (1970) Ann Rev Biochem . 38, 415
Ehle, H. and Horn, A. (1990) Bioseparations. 1, 97
Engvall, E. and Perlmann, P (1971), Immunochemistry 8, 87.
Erickson, P. R, Minier, L.N. and Lasher, R.S. (1982) J Immunol Methods. 51, 241
Ernst-Carbrera, K. and Wilchek, M. (1988) Trends Anal Chem. 7, 58
Fahey, J.L. and Terry, E.W. (1979) In Handbook of Experimental Immunology (Weired, D.M, ed) pp 8.1, Blackwell Scientific Publications, Oxford
Farooqui, M., Sosnitza, P., Saleemuddin, M., Ulber, R. and Scheper, T. (1999) Appl Microbiol Biotechnol. 52, 373
Faulk, W. and Taylor, G. (1971) Immunochemistry. 8,1081
Fernandez-Pol, J. A. (1982) FEBS Lett. 143, 86.
Fiedler M. and Skerra, A (1999) Protein Expression and purification 17,421.
Figge, J., Rossin, T.H. and Fencl, V. (1991) J Lab Clin Med. 117, 453
Fleminger, G., Itada, E., Wolf, T and Solomon, B. (1990) Appl Biochem Biotechnol. 23, 123.
Fonda, M.L Trauss, C. and Guempel, U.M.(1991) Arch Biochem Biophys. 288, 79
Fraenkel - Conrat, H and Olcott, H.H. (1968) J Am Chem Soc. 70, 2673.
Friedfelder, D. (1982) Physical Biochemistry, 2nd ed. Freeman and Company, New York.
Fruton J.S. and Bergmann. (1939) J Biol Chem. 127, 627.
Fuglistaller, R (1989) J Immunol Methods. 24, 171
Furlong, A.M., Thomson, T. R., Marotti, K. R., Post, L. E. and Sharma, S. K. (1988) Biotech & Appl Biochem. 10, 454
Gershoni, J.M. and Palade, J.E. (1984) Anal Biochem. 131, 1
Gershoni, J.M., Palade, J.E., Hawrot, E., Klimowicz, D.W. and Lentz, T.L. (1982) J. Cell Biol. 95, 422a (21046)
Gersten, D.M. and Marchalonis, J.J. (1978) J Immunol Methods. 24, 305.
Gitlin, D and Edelhoch, H. (1951) J Immunol Methods. 66, 67.
Glenney, J.R. and Weber, K. (1980) J. Biol Chem. 225, 10551
Coding, J. M. (1985) Monoclonal: Principles and Practices, Academic Press, New York, pp 9
19
Coding, J.R. (1983) Monoclonal : Principles and Practices, Academic Press, New York, pp 188
Gordon, J. and Rosenthal, M. (1984b) J. Rheumathol. 28, 231
Gordon, J., Hawkes, R., Niday, E., Rordorf, C , Rosenthal, M. and Towbin, H. (1983) Immunoenzymatic Techniques, edn. Avrameas, S., Druet, P., Massayeff, R., Feldmann, G., (Elsevier, Amsterdam) pp 303
Gorini, G., Medgyesi, G. A. and Doria, G. (1969) J Immunol. 103, 1132
Gornall, A.G., Broadwill, G.J. and David, M.M. (1949) J Biol Chem. 177, 751.
Green,N.M. (1963) Biochem. J. 89, 585
Gronman, E. and Wilchek, M. (1987) Trends Biotechnol. 5, 220.
Guedson, J.L., Ternynck, T. and Avrameas, S. (1979) J. Histochem Cytochem. 27, 1131
Haba, S. andNisonoff, A. (1990) J Immunol Methods. 138,15
Habeeb, A. F. S. A. (1966) Anl Biochem. 14, 328
Habeeb, A.F.S.A. and Hiramoto, R. (1968) Archs Biochem Biophys. 126,16.
Haeggstrom, J., Fitzpatrick, F., Radmark, O. and Samuelsson, B. (1983) FEBS Lett. 164, 181
Hale, J. E. (1995) Anal Biochem. 231, 46
Hale, J. E. and Beidler, D.E. (1994) Anal Biochem. 222, 29
Hall, J.E. and Hargreaves, W.R. (1984) Europeon patent application No 84104371.4 (EP 123265)
Halveson, C. A., Falini, B., Taylor, C. R. and Parker, J. W. (1981) Am J Pathols. 105,254
Hanker, J.S., Yates, P.E., Metzeb, C.B. and Rustioni, A. (1977) Histochem. J. 9,789
Hansen, R.S. and Beavo, J. A., (1982) Proc Natl Acad Sci. USA, 79, 2788
Hanson, H. and Kagedal, L. (1981) J Chromatogr. 215, 333
Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Harris, C.C. (1984) US patent No 4,463,090
Hawkes, R. (1982) Anal Biochem. 123, 143
Hawkes, R., Niday, E. and Gordon, J. (1982) Anal Biochem. 119, 142
Hayman, E. G., Engvall, E. A., Hearn, E., Barnes, D., Piersch-bacher, M. and Ruoslahti, E. (1982) J Cell Biol. 95, 20
120
Heggeness, M.H. and Ash, J.F. (1977) J. Cell Biol. 73, 783
Heinsohn. C , Polgar, H., Fishman, J. and Taylor, L. (1987) Arch Biochem Biophysics. 257, 251
Hennessey, J. P. and Johnson, W. C. (1981) Biochemistry. 20, 1085
Herbrink, P., Van Bussels, F.J. and Warnaar, S.O. (1982) J. Immunol Methods. 48, 293
Holgate, C.S., Jackson, R, Cowen, RN. and Berd, C.C. (1983) J. Histochem Cytochem. 31,938
Hood, L. and Talmage, D. (1970) Science. 168, 325
Hristova, G. (1968b) AUer Clin Immunol. 135, 439
Hristova, G. (1970b) Proc Imunobiol Stand. 4, 99
Hudson, S.M., Stinson, R C , Shiman, R. and Jefferson, L.S. (1987) Am J Physiol. 252,291
Huet, J., Sentenac, A. and Fromageot, P. (1982) J. Biol Chem. 257, 2613
Inada, Y., Koelera, Y., Matuschima, A., Yaseukochi, T. and Kraut, J. (1995) Ann Rev Biochem. 46, 331
Inouye, K. and Morimoto, K. (1993) J Biochem Biophys Methods. 1, 27
Ishikawa, E., Imagawa, M., Hashida, S., Yoshitake, S., Hamaguchi, V. and Veno, T. (1983) J Immunoassay. 4, 209
Jafri, F., Hussain, F. and Saleemuddin, M. (1993) Biotechnol Appl Biochem. 18,401
James, M. N. G. and Sielecki, A. R. (1986) Nature. 319, 33
Jaquet, H. and Cebra, J. (1965) Biochem. 4, 954
Jasiewicz, M.L., Schoenber, D.R. and Mueller, G.C. (1976). Exp Cel Res. 100,213
Jirgensons, B. (1970) Biochim Biophys Acta. 200, 9
Johannsson, A., Ellis, D.H., Bates, D.L., Plumb, A. and Mandstanely, C.J. (1986) J. Immunol Methods. 87, 7
Johannsson, A., Stanley, C.J. and Self, C.H. (1985) Clin Chem Acta. 148,119
Jolley, M.E., Wang, C.H., Ekenberg, S.J., Zuelke, M.S. and Kelso, D.M. (1984) J. Immunol Methods. 1,21
Jones, R. G. A. and Landon , J. (2002) J Immunol Methods. 57, 263,
Jones, R. G. A. and London, J. (2002) J Immunol Methods. 263, 57
Jones, R. G. A., Corteling, R. 1. and Landon , J. (1999) Am J Med Hyg. 61, 361
121
Jordan, G.M., Yoshioka, S. and Teraro, T. (1994) J Pharm Pharmacol. 46, 182
Joshi, U.M., Rao, K.S. and Mehendaie, H.M. (1987) Int J biochem. 19, 1029
Kabat, E. A., Wu, T. T. and Bilofsky, H. (1970) J Biol Chem. 252, 6609
Kabat, E.A. amd Mayer, M.M. (1961) Experimental Immunochemistry. Charles C. Thomas, Springfield, IRL.
Kakita, K., 6 Connel, K. and Permutt, M.A. (1982) Diabetes. 31, 648
Karlson-Stiber. C. and Persson, H. (1994) J Inter Med. 235, 57
Katakam, M. and Banga, A.K. (1995) J Pharm Pharmacol. 47, 103
Kaysen, G.A., Jones, H., Jr., Martin, V. and Hutchison, F.N. (1989) J. Clin Invest. 83, 1623
Kekwick, R. A. (1940) Biochem. J. 34, 1248
Kemeny, D.M. and Chantler, S. (1988) An Introduction to ELISA. In: D.M. Kemeny and S. Challamcombe (Eds), ELISA and other solid phase immunoassays. Wiley, NY, pp. 1.
Kerstens, H.MJ., Poddighe, P.J. and Hanselaaar, A.G.J.M. (1995) J. Histochem Cytochem. 43, 347
Khan, R.H., Appa Rao, K.B.C., Eshwari, A.N.S.,Totay, S.M. and Panda, A.K. (1998) Biotechnol Prog 14, 722.
Kohler, G. and Milstein, C. (1975). Nature. 256, 495
Konigsberg W and Hill R.J. (1963) J Biol Chem. 237, 3157.
Krystoffersen, E. K., Matre, R., Ulvested, E. and Veddler, C. A. (1994) J Immunol Methods. 167(1-2), 15.
Kuwajima, K. (1989) Proteins. 6, 87.
Laemmli, U.K. (1970) Nature. 227, 680
Lamoyi, E. and Nissonoff, A. (1983) J Immunol Methods. 56, 235
Lansdorp, P. M., Vander-Kwast, T. H., De Boer, T. and Zeijlemaker, W.P. (1984) J Histochem Cytochem. 32, 172
Lauritzen, E., Manon, M., Rubin, I. and Holm, A. (1990) J Immunol Methods. 131(2), 257
Levy, H. B. and Sober H. A. (1960) Proc Soc Exp Biol Med. 103, 250
Li, C.Y., Zeismer, S.C, Yam, L.T., English, M.C. and Janckila, A.J. (1984) Am J Clin Pathol. 81, 204
Li, X., Jeffers, L.J., Shao, L., Reddy, K.R., Demedina, M., Scheffel, J., Moore, B. and Schiffer, E.R. (1995) J. Virol Hepatitis. 2, 227
Lihme, A. and Heegaard, RM. (1991) Anal Biochem 192, 64.
122
Lin, W. and Kasamatsu, H. (1983) Anal Biochem. 128, 302
Lin, X., Loy, J. A., Sussman, F. and Tang, J. (1993) Protein Science. 2, 1383
Lindup, W.E. (1987) Prog Drug Metab. 10, 141
Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randell, R.J. (1951) J. Biol Chem. 193, 265
Lumb, R., Smith, P.S., Davies, R., 6 Donoghue, P.J., Atkinson, H.M. and Lanser, J.A. (1989) Immunol Cell Biol. 67, 267
Lundsgaard, H.P. (1986) Curr Stud Hematol Blood Transfusion. 1, 17
Madson, L. H. and Rhodkey, L.S. (1976) J Immunol Methods. 9, 355
Maisano, F. and Testpro, S. A. (1989) J Chromatogr. 472, 422
Malavoiti, M., Cervak, S. and Shehan, K.L. (1989) Lipids. 24, 273
Manavalan, P. and Johnson, W. C. (1983) Nature. 305, 831
Mancini, G., Vaerman, J.P., Carbonara, A.D. and Heremans, J.F. (1964) In: T. Peeters (Ed), Protides of the biological fluid, 11th Colloquium, Elsevier, Amsterdam.
Mangold, B.J., Cook, R. A., Cranfiels, M.R., Huygen, K.and Godfrey, H. P. (1999) J Wildl Med. 23,200
Manning, J., Pellegrini, M. and Davidson, N. (1977) Biochemistry. 16, 1364
Mariani, M., Cauragra, M., Tarditi, L. and Seccariani, E. (1991) Mol Immunol. 28,69
Martin, R. B. (1979) In Metal Ions in Biological Systems (Sigel, H. ed) Vol 9, pp 2, Marcel Dekker
Mason, D.Y. and Sammons, R. (1978) J. Clin Pathol. 31, 454
Mason, D.Y., Abdulaziz, Z., Falini, B. and Stein, H. (1983) Immunochemistry: In Practical Application in Pathology and Biology (J.M. Polak and S. Van Noorden eds) 1st edn, pp 113, Wright, Bristol, U.K
Matyash, L., Ogloblina, O. and Stepanov, V. (1973) 35, 540
Mc Donald, V, Mc Crosson, M.V. and Petery, F. (1995) Parasitology. 110, 259
Mendel, CM., Miller, M.B., Siiteri, RK. and Murag, J.T. (1990) J Steroid BiochemMolBiol. 37, 245
Merril, G. R., Goldman, D. and Keuren, M.I.V., (1982) Electrophoresis. 3, 17
Mesa-Tejada, R., Pascal, R. R. and Fenoglio, CM. (1977) Human Pathol. 8, 313
Meyer, W. P., Habib, A. G., Onayade, A. A., Yakubu, A., Smith, D. C , Nasidi, A., Daudu, I . J., Warrell, D. A. and Theakston, R. D. J. (1997) Am J Trop MedHyg. 56, 291
Milstein, C. and Pink, J.R.L. (1970) Progs Biophys Mol Biol. 21, 204
Mistrello, G., Gentili, M., Roncarolo, D., Antoniotti, P., Ottoboni, F. and Falagiani, R (1998) J Med Entmol. 35(2), 143
Moran, N. P., Newman, W. J., Theakston, R. D. J., Warrell, D. A. and Wilkinsin, D. (1998) Trans See Trop Med Hyg. 92, 69
Morimoto, K. and Inouye, K. (1992) J Biochem Biophys Methods (1-2), 107
Morrill, W.E., Barbaree, J.M., Fields, B.S., Sanden, G.N. and Martin, W.T. (1990) J Clin Microbiol. 28, 616
Muilerman, H.G., Terhart, H.G.J, and Van Dijk, W. (1982) Anal Biochem. 120, 46
Naiem, M., Gardes, J., Abdulaziz, Z., Sunderland, C.A., Stein, H. and Mason, D.Y. (1982) J. immunol Methods. 50, 145
Nakajima, N. Ishihara, K. Sugimoto, M. Nakhara, T. and Tsuji.(2002) T Biosci Biotechnol Biochem. 12, 2739.
Nakane, RK. (1968) J. Histochem Cytochem. 16, 557
Nakane, RK. and Pierce, G.B. (1967). J. Histochem Cytochem. 14. 929
Naser, W. L. (1990) J Immunol Methods. 129, 151
Ngu, J.L., Nkenfou, C , Capuli, E., Mc Moli, T.E., Perler, F., Mbwagbor, J., Tume, C , Nlatte, O. B., Donfack, J. and Asonganyi, T. (1998) Trop Med Int Health. 3(5), 339
Nissonoff, A., Wissler, F.C., Lipman, L. and Worenley, N.D.L. (1960) Arch Biochem. 89, 230
Northrop, J, H. (1930) J Gen Physiol. 13, 739
Ogata, K., Arakawa, M., Kasahara. J. and Shiori-Nakano, K. (1983) J Immunol Methods. 65, 75
Olmsted, J. B. (1981) J Biol Chem. 256, 11955
Olufemi, O.S., Humes, R, Whittaker, RG., Read, M.A., Lind, T. and Halliday, D. (1990) Eur J Clin Nutr. 44, 351
Ortega-Vinuesa, J.L., Tengvall, P. and Lundstrom, I.I. (1998), J Colloid Interface Sci. 207, 228
Padridge, W.M. (1988) Ann N.Y. Acad Sci. 538, 173.
Pappas, M.G., Hajkowski, T. and Hockmeyer, W.T. (1983) J Immunol Methods. 64, 205
124
Parham, P. (1986) In Weir, C. D. M (ed) Handbook of Experimental Immunology, Vol 1, Blackwell Scientific Publishers, Oxford, pp 14.1
Pascual. D.W. and Clem, L. W. (1992) J Immunol Methods. 146. (2), 249
Pepper, D. S. (1992) Methods Mol Biol. 11, 173
Pereira, L.G., Torrance, L., Roberts, I.M. and Harrison, B.D. (1994) Virology. 203, 277.
Peters, T. Jr. (1970) Adv Clin Chem. 13, 37
Peters, T. Jr. (1985) Adv Protein Chem. 37, 161
Phillipy, B.O. and McCarthy, R.D. (1979) Biochem Biophys Acta. 584, 298
Pitts, J. E., Dhanraj, V., Dealwis, C. G., Mantafounis, D., Nugenr, P., Orprayoon, P., Cooper, J.D., Newman, M. and Blundell, T. L. (1992) Scand J clin Lab Invest. 52, suppl. 210, 39
Podzorski, E and Wells, F.E. (1989) Ann. Clin. Biochem. 26, 556.
Porath, J. and Olin, B. (1983) Biochem. 22, 1621
Porath, J., Carrlsson, J., Olsson, I. and Belfrage, G. (1975) Nature. 258, 598
Porter, R. R. (1959) Biochem J. 73, 119
Portsman, B., Portsman, T., Neugel, E. and Eers, U. (1985) J. Immunol Methods. 29, 27
Posner, I. and De Sanctic, J. (1987) Comp Biochem Physiol B. 87, 137
Poulsen, F. and Hjort, T. (1980)Acta Pathol Microbiol Scand. Sect C. 88, 241
Presswood, W.G. (1981) Membrane Filteration , Applications, Techniques, Problems (Dutka, B.J., ed) Dekker, New York/ Basel
Provencher, S. W. and Glockner, J. (1981) Biochemistry. 20, 33
Pryzwanski, K.B. (1982) Techniques in Immunochemistry (G.R. Bullock and P. Petrusz eds). Vol 1, pp 77, Academic Press, London
Quash, G., Roch, A.M., Nivlean, T., Kevlouangkhot, T. and Huppert, J (1978) J Immunol Methods. 22, 165.
Raap, A.K., Van de Corput, M.RC, Vervenne, R.A.W., Van Gijlswijk, R.RM., Tanke, H.J. and Wiegant, J. (1995) Hum Mol Genetics. 4, 529
Rabilloud, T., Asselineau, D. and Darmon, M. (1988) Mol Biol Rep. 13, 213
Raymond, S. and Weintraub, L. (1959) Science. 130, 711
Reading, R.F., Ellis, R. and Fleetwood, A. (1990) Early Human Dev. 22, 81
Reinhart, M. R and Malamud, D. (1982). Fed Proc. 41, 873
Reiser, J. and Wardale, J. (1981) Eur J Biochem. 114, 569
Renart, J., Reiser, J. and Stark, G.R. (1979) Proc Nat Acad Sci. USA, 76, 3116
^ 125
Richards, E.W., Hamm, M.W. and Otto, D.A. (1991) Biochem Biophys Acta. 1076,23
Richards, E.W., Hamm, M.W., Fletcher, J.E. and Otto, D.A. (1990) Biochem Biophys Acta. 1044,361
Robinson, B.S., Baisted, D.J. and Vance, D.E. (1989) Biochem J. 264, 125
Sabattini, E., Bisgaard, K., Ascani, S., Piccioli, M., Ceccarelli, C , Pierl, F., Fraternali - Ocioni, G. and Pileri, S.A. (1998) J. Clin Pathol. 51, 506
Schaltman, K. and Pongs, O. (1980) Hoppe-Seyler's, Z. Physiol Chem. 361, 207
Schneider, Z. (1980) Anal Biochem. 108, 96
Schramm, W. and Pack, S. (1992) Anal Biochem. 205, 47
Self, C.H. (1985) J. Immunol Methods. 76, 389
Self, C.H. (1986) U.S patent No 4,598,042
Seto, Y., Iba, T. and Abe, K. (2001) Luminescence. 4, 285
Shami, E. Y., Ramjeesingh, M., Rothstein, A. and Zywulko, M. (1989) Trends Biotechnol. 7, 86
Siddiqui, F. A. and Francis, J.L. (2002) Blood Coagul Fibrinolysis. 13(3), 173
Solomon, B., Koppel, R., Pires, G. and Katchalski - Katzir, E (1986) Biotechnol Bioeng. 28, 1213.
Soukka, T., Pankkunen, J., Harma, H., Lonnberg, S., Lindroos, H. and Lovgren, T. (2001) Clin Chem. 7, 1269
Stanworth, D. R. and Turner, M. W. (1986) in "Handbook of Experimental Immunology" Vol I, Immunochemistry (D. M. Weired) Blackwell Scientific Publications, Alden Press, Oxford, pp 12.1
Steinitz, M., Rosen, A. and Klein, G. (1991) J. Immunol Methods. 136, 119
Sternberger, L.A., Hardy, RH., Cuculis, J.J. and Meyer, H.G. (1970) J. Histochem Cytochem. 18, 315
Stults, N.L. and Asta, L.M. (1989) Anal Biochem. 180, 114
Sulkowski, E. (1985) Trends biotechnol. 3, 1
Sumi, M.G., Annamma, M., Sarada, C. and Rdhakrishnan, V.V., (2000) Acta Neurol Scand. 101(1), 61
Sunderberg, L. and Porath, J. (1974) J Chromatogr. 90, 87
Suzuki, J., Sasaki, H., Saso, Y., Hama, A., Kawasaki, H., Nishiyama, M., Horinouchi, S. and Beppu, T. (1989) Protein Engg. 2, 563
Symington, J., Green, M. and Brackmann, K. (1981) Proc Nat Acad Sci. USA. 78, 117
126
Synder, S. and Sobocinsta, Y. (1975) Anal Biochem. 64, 284.
Szabolcs, M. and Francia, I. (1989) Acta Biochem Biophys. Hung. 24, 245
Taylor, C.R. (1978) Arch Pathol Lab Med. 102, 113
Taylor, C.R. (1980) J Histochem Cytochem. 28, 777
Tello-Sollis, S. R., Romero, A. R. and Arana, A. H., (1995) Biochem Mol Biol Int. 33, 759
Ternynck, T. and Avrameas, S. (1976) Ann Immunol. 127C, 197
Theakston, R. D. G. and Warrell, D.A. (2000) Lancet. 365, 2104
Tomono, T., Sizuki, T. and Tokunaga, E. (1981) Biochem Biophys Acta. 660 (2), 186
Towbin, H. and Gordan, J. (1984) J Immunol Methods. 72, 313
Towbin, H., Ramjone, H. R, Kuster, H., Liverani, D. and Gordon, J. (1982) J Biol chem. 257,12709
Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc Nat Acad Sci. USA, 76, 4350
Turner, M. W. and Bennich, H. H. (1970) Clin Exp Immunology. 7, 603
Valnes, K. and Brandtzaeg, P. (1982) J. Histochem Cytochem. 30, 518
Vander Loos, CM. and Becker, A. E. (1994) J Histochem Cytochem. 42, 289
Vanderberg, E. T., Brown, R. S. and Krull, U. J. (1994) Immobilized Biosystems: Theory and Practices, Velicky, J. A., Mc Lean, R. J. C. L (eds) Blackie Academic and Professional, pp 129
Vesey, G., Slade, J.S., Byrne, N. Spheherd, K and Dennis, RJ (1990) J Appl Bacter. 75, 87.
Vesey, G.D. Deere G.J., Weir N., Ashbolt K.L. and Veal D.A. (1997) Lett Appl Micribiol. 25, 316.
Vyberg, M. and Nielsen, S. (1998) Applied Immunohistochem. 6, 3
Walsh, B.J., Wrigley, C.W. and Baldo, B.A. (1984) J Immunol Methods. 66, 99.
Watanabe, S., Tani, T. and Seno, M. (1991) Biochim Biophys Acta 1073, 275.
Wilson, D. S., Wu, J., Peluso, R and Nock, S. (2002) J Immunol Methods. 260, 29
Wold, R.T., Young, RE., Tarn, E.M. and Farr, R.S. (1968) Science 161, 806.
Yada, R. Y. and Nakai, S. (1986) J Food Biochem. 10, 155
Yen, T.S. and Webster, R.E. (1982) Cell. 29, 337
Yurov, G.K., Neugodova, G.L., Verkhovsky, O.A. and Naroditsky, B.S. (1994) 177, 89.
127