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Benha University Faculty of Veterinary Medicine, Moshtohor Department of Bacteriology, Immunology and Mycology ---------------------
A Thesis Presented By
WAFAA RAGAB ABD EL-AZIZ EL-SAYED B.V.SC., Zagazig University(Benha branch) 2000
M.V.Sc., Benha University(Bacteriology,Immunology and Mycology) 2005
Under The supervision of
Prof. Dr. ALI MOHAMED SOLIMAN EL-GED Prof. of Microbiology and Chairman of the
Dept. of Bacteriology, Immunology and Mycology Faculty of Veterinary Medicine, Moshtohor
Benha University
Prof. Dr. AHMED AHMED EL-BASIOUNY Chairman of Dept. of Hygiene and Preventive Medicine
Faculty of Veterinary Medicine, Kafr El-Sheikh University
Prof. Dr.FAYKA KAMAL AHMED
Chief Researcher of Microbiology Veterinary Serum and Vaccine Research Institute,
Abbassia, Cairo
For The Drgree of Ph.D in Veterinary Science
(Bacteriology, Immunology and Mycology)
(2011)
٣٢األيه : سورة البقرة
LISTS
i
Acknowledgement
First of all, many thanks to the All Merciful God, who gave me every
thing I have.
I wish to express my sincere appreciation and gratitude to Dr. Ali
Mohamed Soliman El-Ged, Professor of Microbiology and
Chairman of the Department of Bacteriology, Immunology and Mycology.
Faculty of Veterinary Medicine, Moshtohor, Benha University ,who has given
me so much of his valuable time, experience and scientific knowledge.
I wish to express my great gratitude to Dr. Ahmed Ahmed El-
Basiouny, Chairman of Hygiene and Preventive Medicine, Kafr El-
Sheikh, Tanta University for his stimulating supervision, encouragement and
interest in this work.
I am deeply indebted to Dr. Fayka Kamal Ahmed, Chief
Researchers of Microbiology, Veterinary Serum and Vaccine Research
Institute, Abbassia, Cairo for scientific advice and facilitation of requirements
for achieving this work.
LISTS
ii
LIST OF CONTENTS page
1. Introduction .............. 1
2. Review of literature .............. 3
- Nature of tetanus toxin .............. 3
- Preparation of tetanus toxin ……….. 11
- Tetanus toxin fragment C ……….. 18
- Preparation of tetanus antitoxin ……….. 28
- Purification of tetanus antitoxin ……….. 29
3. Material and methods .............. 45
3.1. Materials ……….. 45
3.1.1. Strain ……….. 45
3.1.2. Media ……….. 45
3.1.3. Antisera and toxoids ……….. 48
3.1.4. Experimental animals ……….. 48
3.1.5. Apparatuses and equipments ……….. 48
3.1.6. Buffers and solutions ……….. 49
3.2. Methods ……….. 54
3.2.1. Preparation of tetanus toxin ……….. 54
3.2.2. Evaluation of the prepared tetanus toxin … 54
3.2.2.1. Determination of minimal lethal
LISTS
iii
dose of tetanus toxin(MLD) ..... 54
3.2.2.2. Determination of Lf (Limits of flocculation)
value of toxin ……….. 55
3.2.3. Purification of tetanus toxin ….……. 56
3.2.4. Measuring protein by Bradford method …. 57
3.2.5. Digestion of tetanus toxin ………. 58
3.2.6. Evaluation of fragment C ………. 60
3.2.7. Production of antitetanic serum ………. 63
3.2.8. Purification of antitetanic serum ………. 63
3.2.9.1. Preparation of IgG by ammonium sulphate ... 63
3.2.9.2. Preparation of IgG by caprylic acid ....... 64
3.2.8.3. Preparation of F(ab)2 …….... 64
3.2.8.4. Preparation of F(ab)2 (pepsin+caprylic)…….. 65
3.2.8.5. Preparation of F(ab) fragment ………. 66
3.2.9. Evaluation of the prepared fragments ………. 66
4. Results ………… 70
5. Discussion ………… 100
6. Summary .………... 113
7. References ..……….. 116
Arabic summary
LISTS
iv
LIST OF TABLES
Table Title Page
1 Purification of tetanus toxin from culture filtrate using
ammonium sulphate.
71
2 Chromatography of tetanus toxin 73
3 Chromatography of papain digested tetanus toxin 76
4 Toxicity test of tetanus toxin and fragment c 80
5 Preparation of IgG using different concentrations of
Caprylic acid
82
6 Preparation of IgG using caprylic acid at different pH values. 84
7 Preparation of F(ab)2 using pepsin enzyme at different pH
values
86
8 analysis of SDS-PAGE results of IgG and F(ab)2 (lanes 1-13) 89
9 Analysis of SDS-PAGE results of IgG and F(ab)2 (lanes 14- 91
10 Preparation of F(ab)2 using pepsin enzyme and caprylic acid. 93
11 Preparation of F(ab) fragment by papain digestion at different
digestion time.
95
12 Analysis of Fig.(5) 97
13 Percentage of survival of mice treated with IgG, F(ab)2 or
F(ab) 24 hr after the inoculation of tetanus toxin.
99
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v
LIST OF FIGURES
Figure Title Page 1 Bradford bovine serum albumin standard curve. 58
2 Chromatography of tetanus toxin 74
3 SDS-PAGE of tetanus toxin digestion fragments 78
4 SDS-PAGE of F(ab)2 and IgG products of antitetanic
serum(lanes 1-13)
88
5 SDS of F(ab)2 and IgG products of antitetanic serum.
(lanes 14-25)
90
6 SDS-PAGE for papain digested serum
96
LISTS
vi
List of abbreviations
BoNT Botulinum neurotoxin CNS Central Nervous System ELISA Enzyme Linked Immunosorbent Assay
FPLC Fast Protein Liquid Chromatography
GLn-Lys bond Glycine – Lysine bond
HPLC High performance Liquid Chromatography
kDa Kilo Dalton
MLD Minimal Lethal Dose
TeNT Tetanus Neurotoxin
TIG Human anti-tetanus immunoglobulin
VAMP Vesicle associated membrane protein
INTRODUCTION
﴿ ﴾
1
1. Introduction Tetanus is a devastating disease worldwide, and although the
true incidence is unknown, it has been estimated that up to 1 million
cases occur annually. It remains endemic in many countries of the
developing world. The global fatality rate of tetanus has been
estimated as 30-50 percent. At least 50 percent of these deaths
tragically occur in neonates, who become infected with Clostridium
tetani shortly following birth and lack maternal antibodies for
protection. Neonatal tetanus is the second leading cause of death
from vaccine-preventable diseases in children world wide. Tetanus a
tragic and regrettable disease, could be prevented by vaccination
which is the most cost-effective public health intervention.(Jhonson,
2005)
Tetanus toxin is comprised of heavy (H, 100 kDa) and a light
chain (L, 50 kDa) linked by a disulphide bond and non-covalent
interactions. The carboxy-terminus of the heavy chain (HC) binds
with extraordinary affinity and specificity to nerve terminals (Caleo
and Schiavo ,2009).
Tetanus toxin may be degraded by papain. This enzyme splits a
polypeptide bond approximately in the middle of the heavy chain subunit,
yielding fragment C, corresponding to the carboxy terminal portion of the
heavy chain, with a molecular weight of about 47,000 dalton and
fragment B, comprising the N-terminal part of the heavy chain and the
entire light chain polypeptide, with a molecular weight of 95,000. Peptide
C can bind to ganglioside and show retrograde transport in axons, is
INTRODUCTION
﴿ ﴾
2
atoxic, and a good imunogen producing antibodies which neutralize
tetanus toxin (Burns, 2002).
Passive immunization with hyperimmune serum obtained from
actively immunized sheep or horse confers effective protection against
tetanus in unimmunized animals and humans. It’s effective within hours
of administration but its protection doesn’t persist for longer than three
weeks. It’s used either for the protection of animals in which the possible
development of tetanus can be anticipated, or for the treatment of animals
suffering from tetanus (Odendaal and Kriek, 1994).
Today enzyme cleaved antibodies are used widely throughout the
world for the treatment of, for example, drug overdose (digoxin),
bacterial toxins (diphtheria, tetanus and botulinum), viral infections
(rabies) and envenoming (snake, spider and scorpion). The production of
polyclonal antibody fragments for therapeutic use usually involves
numerous steps designed to retain their effectiveness while reducing the
incidence and severity of side-effects (Jones and Landon,2003).
Therefore the present study was carried out to investigate the
following items:
Production of highly potent tetanus toxin.-1
2- Digestion of tetanus toxin to produce peptide C (fragment C).
3-Sudying the immunogenicity of peptide C.
4- Evaluating different methods for obtaining purified antitetanic serum.
5-Comparing the protective capacity of antitetanic whole IgG, F(ab')2,
F(ab), against tetanus.
Review of literature
﴾ 3 ﴿
2. REVIEW OF LITERATURE
NNAATTUURREE OOFF TTEETTAANNUUSS TTOOXXIINN::
Bizzini et al. (1973) proposed that tetanus toxin might consist of
two identical subunits with molecular weight of 75,000. Each subunit
being formed from two non identical chains with molecular weights of
50,000 and 25,000 respectively, held together by disulphide links.
Matsuda and Yoneda (1974) analyzed the neurotoxin protein of
Clostridium tetani by polyacrylamide gel electrophoresis and showed
that the toxin as purified from culture filtrates (extracellular toxin,
molecular weight 160,000) could be dissociated into two polypeptide
chains of molecular weight 53,000 (fragment α) and 107,000 (fragment
β) by treatment with dithiothreitol and sodium dodecyl sulfate. The
toxin as purified from bacterial extracts (intracellular toxin) was found
to consist of a single 160,000 KDa (kilo Dalton) polypeptide chain,
which is undissociable by such treatment but, when pretreated with
trypsin, becomes dissociable into two fragments apparently identical
with α and β.
Helting et al. (1978) pointed out that tetanus toxin is a protein
composed of two polypeptide chains, the heavy chain with a molecular
weight of 93,000 KDa, and the light chain at 48,000 KDa. Neither
chain appears to possess any toxicity. They reported the degradation of
tetanus toxin to yield two polypeptide fragments, B and C. Whereas
Review of literature
﴾ 4 ﴿
fragment C was derived from one portion of the heavy chain
polypeptide, fragment B was shown to contain the remainder of the
heavy chain as well as the light chain of the tetanus toxin.
Finn et al. (1984) prepared a pool of synthetic oligonucleotides
based on the amino terminal amino acid sequence of tetanus toxin. This
probe hybridized to plasmid DNA isolated from three toxigenic strains
of Clostridium tetani but not to plasmid DNA from a nontoxigenic
strain. These results show that the structural gene for the toxin is on the
plasmid. The p CL1 plasmid from one of the toxigenic strains
spontaneously deleted 22 kilobase pairs of DNA to form p CL2. Strains
harboring this deleted plasmid are nontoxigenic. However, the probe
mixture hybridized to p CL2. indicating that the DNA encoding the
amino terminus of the toxin had not been deleted.
Krieglestein et al. (1990) stated that tetanus toxin is a 151-kD
protein. The complete amino acid sequence is known. The mature toxin
is made of two peptide and contains 10 half-cystine residues. Treatment
with 4-vinylpyridine in the presence of 6M guanidine converted six of
them into s-pyridylethyl cysteine residues are determines by amino acid
analysis. When alkylation was preceded by mercaptolysis, all 10 half-
cystine residues were recovered in the s-pyridylethylated form. It was
therefore concluded that the toxin contains six sulfhydryl groups and
two disulfide bonds.
Bartels and Bigalke (1992) reported that tetanus toxin inhibits
transmitter release in vivo and in vitro. The clinical symptoms of the
Review of literature
﴾ 5 ﴿
tetanus disease result mainly from a block of exocytosis in spinal
glycinergic intraneurons. Recovery of the physiological functions is a
slow process and cannot be prompted by intravenous or intrathecal
application of specific antitetanus antibodies because antibodies cannot
pass through the plasma membrane of nerve cells in which the toxin
performs its crucial action. In spinal cord cultures as well as in vivo,
antibodies cannot mitigate the course of intoxication once the
electrophysiological disorder has set in.
Poulain et al. (1992) Examined the temperature dependencies of
binding, internalization and intracellular action of both botulinum type
A (BoNT) and tetanus (TeTx) neurotoxins to gain further information
on their neuroselectivity. The respective neuroselective actions of
botulinum type A (BoNT) and tetanus (TeTx) neurotoxins on
cholinergic and non-cholinergic synapses of Aplysia are mainly due to
differences in their extracellular neuronal targetting. After reduction of
temperature from 22 to 10 ºC, the binding of neither BoNT nor TeTx
was significantly altered. Although TeTx internalization could be
detected at the low temperature, its intracellular activity was greatly
attenuated compared to that of BoNT. It is inferred that the uptake
mechanisms are different for these two related but distinct toxins.
Schiavo et al. (1994) stated that tetanus and botulinum
neurotoxins block the fusion of neurotransmitters or peptides
containing vesicles with the plasma membrane. The authors have
shown that the light chains of these Clostridial neurotoxins are
intracellular enzymes .They are zinc-endoproteinases, whose activity is
Review of literature
﴾ 6 ﴿
set free upon nicking of the single-chain toxin and reduction of the
single interaction disulfide bond. Tetanus and botulinum B and F
neurotoxins act specifically on VAMP/synaptobrevin, a membrane
protein of the vesicles, which is cleaved at a single site. The single Gln-
Phe peptide of VAMP is specifically cleaved by tetanus and botulinum
B neurotoxin, while serotype F cleaves the single Gln-Lys bond of the
sequence.
Williamson and Neale (1994) tested the way by which tetanus
toxin internalize to neurons before it can block calcium-dependant
exocytosis and indicate that tetanus toxin requires an acid environment
to penetrate cellular membranes . The authors proposed that the toxin
enters neurons by receptor-mediated endocytosis and that upon
physiologic acidification of the endosome, the toxin forms a channel in
the endosomal membrane through which it translocates to the cell
cytoplasm to exert its action.
Williamson et al. (1999) examined toxin binding and action in
spinal cord cell cultures grown in the presence of fumonisin B1 , an
inhibitor of ganglioside synthesis. Mouse spinal cord neurons grown for
3 weeks in culture in 20 µM fumonisin B1 developed dendrites, axons,
and synaptic terminals similar to untreated neurons, even though thin
layer chromatography shows a greater than 90% inhibition of
ganglioside synthesis. Absence of tetanus and cholera toxin binding by
toxin-horseradish peroxidase conjugates or immunofluorescence further
indicates loss of mono- and polysialogangliosides. In contrast to control
cultures, tetanus toxin added to fumonisine B1 –treated cultures, does
Review of literature
﴾ 7 ﴿
not block potassium-stimulated glycine release, or abolish
immunoreactivity for vesicle-associated membrane protein, the toxin
substrate .These data demonstrate that fumonisine B1 protects against
toxin-induced synaptic blockade and that gangliosides are a necessary
component of the receptor mechanism for tetanus toxin.
Rossetto et al. (2001) reported that the neuroparalytic syndromes
of tetanus and botulism are caused by neurotoxins produced by bacteria
of the genus Clostridium. They are 150 kDa proteins consisting of
three-domains, endowed with different functions: neurospecific
binding, membrane translocation and specific proteolysis of three key
components of the neuroexocytosis apparatus. After binding to the
presynaptic membrane of motoneurons, tetanus neurotoxin (TeNT) is
internalized and transported retroaxonally to the spinal cord, where it
blocks neurotransmitter release from spinal inhibitory interneurons.
TeNT and BoNT-B, -D, -F and -G cleave specifically at single but
different peptide bonds, VAMP/synaptobrevin, a membrane protein of
small synaptic vesicles. BoNTs are increasingly used in medicine for
the treatment of human diseases characterized by hyperfunction of
cholinergic terminals
Kegel et al. (2002) stated that tetanus neurotoxin is a 150 kDa
protein produced by Clostridium tetani. The 50 kDa light chain of this
neurotoxin belongs to the family of zinc metalloproteases. It cleaves
synaptobrevin, a small synaptic vesicle protein, which is involved in
neuroexocytosis, at the single Q76-F77 peptide bond.
Review of literature
﴾ 8 ﴿
Chin et al. (2003) generated 600 human hybridoma cell lines by
the fusion of lymphocytes from hyperimmunized people with
heteromyeloma cells, Even though seven cell lines produced antibodies
against tetanus toxoid, only two antibodies from hybrid CH8 and CH5
neutralized the tetanus toxin and completely protected the mice that had
been challenged with the toxin even at the level of 90 mean lethal dose.
The cDNA of light (L) chain and heavy (H) chain variable region was
isolated, and then inserted into expression vectors containing human
IgG constant regions. After transfection of the recombinant human IgG
gene into Chinese Hamster Ovary (CHO) cells, transformants secreting
the complete human antibody were selected. The recombinant human
antibodies produced from CHO cells possessed neutralizing activity
against tetanus toxin just like the original human antibodies produced
from human hybridoma cell lines.
Teng et al. (2005) pointed out that Clostridial neurotoxins light
chain component (LC) inhibits synaptic transmission by digesting
vesicle-docking proteins without directly altering neuronal health. An
adenoviral vector containing the LC of tetanus toxin (AdLC) was
constructed. LC expressed in differentiated neuronal PC12 cells was
shown to induce time- and concentration-dependent digestion of mouse
brain synaptobrevin in vitro as compared to control transgene products.
Spontaneous functional recovery was observed to parallel the cessation
of LC gene expression. These results suggest that light chain gene
delivery within the nervous system may provide a nondestructive
means for focused neural inhibition to treat a variety of disorders
Review of literature
﴾ 9 ﴿
related to excessive synaptic activity, and prove useful for the study of
neural circuity.
Kegel et al. (2007) pointed out that tetanus neurotoxin (TeNT1)
is a bacterial protease which specifically cleaves the vesicle protein
synaptobrevin-2 (vesicle associated membrane protein-2, VAMP-2).
This proteolytic feature of the toxin has been used to develop a
sensitive endopeptidase assay for the detection of TeNT activity as an
alternative to the in vivo assay for TeNT toxicity. Recombinant
synaptobrevin-2 (rSyb2) is immobilized onto a microtiter plate, and the
cleavage of immobilized rSyb2 by TeNT is detected with a polyclonal
antibody directed against the newly generated C-terminus of the
cleavage product. This antibody is shown to be a highly specific tool
for detecting rSyb2 proteolysis by TeNT. The method reaches a
detection limit of less than 1 pg TeNT/ml. In the future, the assay may
also serve as a basis for the replacement of the in vivo safety control of
tetanus vaccines
Foster (2009) Stated that TeNT enters the body via wounds and
initially binds and internalizes into the peripheral terminals of
motorneurons. it is transported by retrograde axonal transport to the
motorneuron soma in the spinal cord. Once in the somatodendritic
region TeNT appears to be transported to somatodendritic postsynaptic
sites, from where it is released into the synaptic cleft. At the spinal cord
level, having been released into the synaptic cleft, TeNT undergoes
receptor mediated uptake into an acidic vesicular compartment in the
Review of literature
﴾ 10 ﴿
presynaptic termini of the inhibitory interneurons, from where it
translocates into the cytosol and inhibits neurotransmitter release.
Nakajima et al. (2009) reported two cases of severe tetanus
infection. Case 1: A 73-year-old non-vaccinated man who developed a
wound on the left little finger. The wound was debrided and a tetanus
toxin shot given on day 4 following the injury. He developed trismus
on day 6 requiring deep sedation and mechanical ventilation in the
intensive care unit (ICU), with human anti-tetanus immune globulin
(TIG) and antibiotics administered. Recovered and was discharged
mobile after 2 months of rehabilitation. Case 2: A 37-year-old woman
fully vaccinated against tetanus in her childhood had apparently had
booster vaccine for at least 20 years. She sustained two lacerations on
the fingers. Diagnosed clinically as having tetanus and underwent a
shot of tetanus toxin, TIG, and antibiotics. She died the next day due to
endotoxin shock caused by other bacteria. These cases underscore the
importance of maintaining adequate tetanus antibody levels through
booster administration every 10 years in immune adults and appropriate
post-exposure treatment with tetanus toxin and/or prophylactic TIG
administration.
Hatamabadi et al. (2010) investigated the sensitivity,
specificity, and the positive and negative predictive values and cost-
effectiveness of TQS (Tetanus Quick Stick), an
immunochromatographic dipstick test, developed to determine the
tetanus immunity of the patients. Tetanus vaccine and immunoglobulin
administration are challenging decisions mostly because of the fact that
Review of literature
﴾ 11 ﴿
the current protocol for immunization against tetanus is based on 2
variables: the vaccination status of the patient and the nature of wound
and its exposure. This study revealed TQS test to be appropriate and
cost-effective for ED (emergency department) use especially in
evaluating patients who do not remember or cannot give their tetanus
immunization history.
PREPARATION OF TETANUS TOXIN:
Mueller and Miller (1954) investigated various factors
influencing tetanus toxin production. Most important, is the variation
among different batches of pancreatic digest of casein. They contain
some components which seem to inhibit toxin formation. Some of these
inhibitory factors in the digest can be removed by charcoal treatment.
The necessity for the inclusion of beef heart infusion introduces another
series of variables. The influence of iron is largely unexplained. Best
results are obtained with Merck s "Iron by hydrogen" added to the
completed medium and allowed to settle. Other variables such as size
and method of inoculum and exact age of seed culture seem not to be
critical.
Fisek et al. (1954) studied the part played by growth
accessories, present in the infusion of beef heart, in toxin formation.
The study provided certain amount of information on the qualitative
and quantitative requirements of a few vitamins and growth accessories
which are essential for maximal toxin formation and it has been
possible gradually to modify the composition of the original toxin
Review of literature
﴾ 12 ﴿
medium by decreasing the quantity of beef heart infusion and
substituting certain vitamins in pure form. Pantothenic acid and uracil
seemed to be the two substances having the most striking effect. The
other accessories obviously are supplied at almost the optimal levels by
the small amount of heart infusion and by the pancreatic digest of
casein.
Thomson (1957) reported large-scale semi-continuous
production method. A 80 liter open tank of aluminum with an outlet at
the bottom .The sterile tank was filled with approximately 70 liter .of
autoclaved medium at 35c˚.After incubation the contents stirred
continuously at 60 rpm for 2 days.70 liter of culture was produced
every 48 hour. Approximately 1 liter. of culture was left in the tank as
an inoculum for the next batch of medium.
Latham et al. (1962) modified the formula of Mueller and
Miller (1954), their formula did not contain beef heart infusion. They
removed phosphate salts as they are inhibitory to toxin production and
cause blackening of the culture. No tyrosine was added as considerable
amount is present in the casein digest. Nicotinic acid and vitamine B12
were added, reduced iron was replaced by ferric chloride salt. They
indicated that adequate heating appeared to be an indispensable
physical requirement, aside from its sterilizing function. They indicated
an interesting interaction between iron and cystine, increasing both of
them together cause a sharp drop in toxin yield associated with the
blackening of the culture. Their medium gave an average toxin titer of
80 Lf/ml and 600,000 MLD/ml in mice.
Review of literature
﴾ 13 ﴿
Nielsen (1967) used Latham medium (a protein-free medium)
for tetanus toxin production in a 100-gal, steam –jacketed, glass-lined
fermentor. Either nitrogen or air was introduced to the culture surface
within the tank through a sterile filter .Toxin titers of 10-40 Lf /ml
were reported .They concluded that satisfactory toxoid can be produced
in this manner. In addition, the Massachusetts medium would seem to
be the medium of choice, since all ingredients are commercially
available and the absence of protein in the medium may result in a
more refined product.
Hepple (1968) earlier methods of cultivation of the Harvard
strain of Clostridium tetani in glass containers have been replaced by
the use of 1100 liter batches of medium in closed, stirred, stainless steel
fermentor vessels, equipped for the forced removal of waste gases
inhibitory to toxin formation .A slight modification of the medium was
required as reduced iron powder was replaced by soluble iron salts and
the phosphate salts were omitted. This method achieves a two-fold
increase in the titer of toxin as well as permitting at least a two-fold
increase in batch size.
Mellanby (1968) confirmed the specific effect of glutamic acid
on toxin production. a culture grown on a variant of the Muller-Miller
medium supplemented with 1 % sodium L-glutamate grew more
rapidly than a culture without added glutamate for the first 24 hours
after inoculation and then started to autolyze ,whereas the ordinary
culture continued to grew for a further 30 hours and produced 30%
more dry weight of organisms. At 36 hours, a glutamate culture
Review of literature
﴾ 14 ﴿
(176,000 LD50 /ml) had twice the toxin content of an ordinary culture
(77,000 LD50 /ml) and most of the toxin is extracellular, probably due
to autolysis; at 89 hours the glutamate culture (522,000 LD50 /ml) had
1/5 of the toxin of the ordinary culture (2,650,000 LD50 /ml),which by
now had also autolyzed).
Zacharias and Bjőrklund (1968) carried out continuous toxin
production in a one liter stirred culture vessel for as long as 65 days.
Toxin production of approximately 120 flocculating units per ml was
maintained with a dilution rate of 0.125 hr ¹־ , a temperature of 34 ˚C, a
pH of 7.4 , and the addition to the medium of 0.1gm of potassium
chloride per liter. The average minimal lethal intraperitoneal dose of
the toxin in mice was approximately 106
per ml.
Vinet and Fredette (1970) demonstrated the influence that the
mode of cultivation has on toxin yield. Clostridium tetani produced
fewer toxin when cultivated in ordinary flasks than when it was
cultivated in jars. It was established that growth came to an end about
24 hours earlier in jars than in flasks. Since anaerobiosis is less strict in
jars than in flasks, contact of the culture surface in jars with oxygen is
likely to promote lysis and release of the toxin from the bacterial cells
at a time when most of the toxin has already been synthesized.
Matsuda and Yoneda (1975) used a substrain (Biken) of the
Harvard A47 strain of Clostridium tetani for toxin production in a
modified Latham medium. The Latham medium was modified by
Review of literature
﴾ 15 ﴿
replacing 25gm of casein digest by 20gm of polypeptone and 10gm of
whale heart extract and by adding 100 µg of folic acid per liter.
Watt and Brown (1975) show that the commercially available
defined medium “109” supplemented with cysteine and ascorbic acid
and prepared in a liquid or solid form, can support the growth of
Clostridium tetani strain. Two of three toxigenic strains passaged in the
liquid medium (CA 109-L) retained their ability to produce toxin when
supernates of cultures in this medium were injected into mice.
Ikbal et al. (1988) Tested the growth of eight local and two
foreign strains of Clostridium tetani on different medium . Papain
digested beef was the medium of choice followed by thioglycolate,
cocked meat and peptone water. They found that when the curve of
tetanus toxin was followed during 12 days of growth on Papain
digested beef; lethal toxin reached the peak at the sixth day of growth.
Optimal temperature was 35-37 ˚C, pH 7.5 and the best carbohydrate
for toxin production was glucose.
Vrany et al. (1988) provided a cultivation technique employing
dialyzed cultures of microorganisms , this technique is suitable for both
research and production of Clostridial toxins. A 10-fold increase of the
antigen concentration in filtrates of dialyzed cultures is found in
comparison with normal cultures. A dialyzed culture ensures a well-
balanced production of toxic filtrates that contain highly concentrated,
relatively pure and strongly immunogenic antigens.
Review of literature
﴾ 16 ﴿
Maria et al. (1997) investigated the effect of exposing cultures
of Clostridium tetani to nitrogen (N2) gas on the recovery of tetanus
toxin to be processed for the preparation of its toxoid .Nitrogen was
bubbled through nine 10-liter culture during the growth of the bacteria,
while nine parallel control incubations were maintained without
bubbling. They found that treatment of the Clostridium tetani anaerobes
with an inert gas in this manner during cultivation produced a highly
significant increase (about two fold) in the yield of tetanus toxin from
them in comparison with the standard procedure.
Prado et al. (1999) purified tetanus anatoxin on a small scale
using Sephacryl S-200 High Resolution (gel filtration) and obtained
successful high-yield purification. On the basis of these results, by
combining conventional tangential flow filtration (TFF) at 50,000
N.M.W.L. (Nominal Molecular Weight Limit) ultrafiltration membrane
with gel filtration on Sephacryl S-200 High Resolution, they have been
able to purify 14 lots of tetanus anatoxin using the Bioprocess System
to a large scale operation.
Flu et al. (2002) developed Bakstim, a new biostimulating
preparation obtained from the organs of the immune system of animals.
The impact of Bakstim on the growth and toxigenic function of
Clostridium tetani production strain Copenhagen-471 was evaluated.
The addition of the preparation to Gluzman commercial medium for
obtaining tetanus toxoid led to an increase in the yield of bacterial
biomass from 1.9 to 4-fold and an increase in the toxoid production
from 2 to 2.8-fold. The optimum concentration of this biostimulant
Review of literature
﴾ 17 ﴿
ensuring the maximum yield of tetanus toxin from the production
culture was determined (1,000 mg/l). Bakstim will supposedly be used
as additive to nutrient media for the production of tetanus toxoid.
Fratelli et al. (2005) Investigated the simultaneous effects of
the starting levels of glucose (G0) and pancreatic digest of casein; N-Z
Case TT (NZ0) as carbon and nitrogen sources, respectively, on the
production of tetanus toxin in static cultivations by means of a five-
level star-shaped experimental design and evaluated by response
surface methodology (RSM) for optimization purposes. The highest
final average yield of tetanus toxin (72 Lf/mL), achieved at G0= 9.7 g/L
and NZ0= 43.5 g/L, was 80% higher than that obtained with standard
cultivations (G0= 8.0 g/L and NZ0= 25.0 g/L).
Demain et al. (2006) revealed that tetanus toxin was made by
fermentation with Clostridium tetani, the traditional source of iron is
an insoluble preparation called reduced iron powder. This material
removes oxygen from the system by forming FeO2 (rust). When
inoculated in a newly developed medium lacking animal and dairy
products and containing glucose, soy-peptone, and inorganic salts,
growth and toxin production were poor without reduced iron powder.
The optimum concentration of reduced iron powder for toxin
production was found to be 0.5 g/L. Inorganic iron sources failed to
replace reduced iron powder for growth or toxin formation. The iron
source that came closest was ferrous ammonium sulfate. The organic
iron sources ferric citrate and ferrous gluconate were more active than
the inorganic compounds but could not replace reduced iron powder.
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﴾ 18 ﴿
Insoluble iron sources, such as iron wire, iron foil, and activated
charcoal, were surprisingly active. It thus appears that the traditional
iron source, reduced iron powder, plays a double role in supporting
tetanus toxin formation, i.e., releasing soluble sources of iron and
providing an insoluble surface.
Fratelli et al. (2010) Studied the effects of the initial nitrogen
source (NZ Case TT) level and the protocol of glucose addition during
the fed-batch production of tetanus toxin by Clostridium tetani. An
increase in the initial concentration of NZ Case TT (NZ0) accelerated
cell growth, increased the consumption of the nitrogen source as well as
the final yield of tetanus toxin, which achieved the highest values (50-
60 Lf/mL) for (NZ 0) ≥ 50 g/L. The addition of glucose at fixed times
(16, 56, and 88 h) ensured a toxin yield ( approximately 60 Lf/mL)
about 33% higher than those of fed-batch runs with addition at fixed
concentration ( approximately 45 Lf/mL) and about 300% higher than
those obtained in reference batch runs nowadays used at industrial
scale. The results of this work promise to substantially improve the
present production of tetanus toxin and may be adopted for human
vaccine production after detoxification and purification.
TETANUS TOXIN FRAGMENT C:
Latham et al. (1965) demonstrated the value of gel filtration for
the analysis of the tetanus toxoid as well as for its purification. The
simplicity of the technique as well as the low cost of materials are
additional advantages of the system. Filtration of partially purified
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﴾ 19 ﴿
tetanus toxoid through the dextran gel Sephadex G-100 has yielded at
least four clearly identifiable fractions .The first two fractions,
containing 55 to 65% of the nondialyzable nitrogen in the starting
material ,were antigenically the same as the parent toxoid .The third
fraction was poorly antigenic. The fourth fraction was inactive both in
vitro and in vivo.
Helting and Zwisler (1974) treated tetanus toxin with papain at
55˚c which resulted in breakdown of the molecule to yield an atoxic
fraction with a molecular weight of approximately 40,000. The highly
purified material exhibited partial immunological identity with the
parent toxin, showed no toxicity and elicited the formation of
neutralizing antibodies against tetanus.
Goretzki and Habermann (1985) characterized enzymatic
fragments of tetanus toxin by immunoblotting using a set of previously
characterized antibodies and a set of novel antibodies. The selected
antibodis recognized the light chain, fragment C (β1) and the
complementary piece (β2) of the heavy chain when blotted on
nitrocellulose. All toxin preparations contained intrinsic esteroprotease
activity which became manifest in the presence of urea. The main
product of papain hydrolysis is fragment C, which appears as a double
band under non reducing conditions but is homogeneous when reduced.
Chymotryptic digestion hydrolyses the heavy chain well but leaves the
light chain largely intact. Tetanus toxin is very resistant against trypsin
as compared with other proteases, although this enzyme splits
numerous different links.
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﴾ 20 ﴿
Ozutsumi et al. (1985) described a rapid, simplified method for
production of tetanus toxin from bacterial extracts. The extracts were
prepared by stirring young cells (about45 hr culture) of Clostridium
tetani in 1M NaCl-0.1M Sodium citrate, PH 7.5, over night at 0 to 4˚C.
The toxin was purified by a combination of (i) ammonium sulfate
fractionation(0 to 40% saturation),(ii)ultracentrifugation for removal of
particulate materials ,and (iii) gel filtration by high liquid
chromatography(HPLC) on a TSK G-3000 SW-type column. So this
method required 6 days. The minimum lethal doses of the purified toxin
preparations for mice was 1.4 x107 to 1.5 x107 per mg of protein and
they showed 360 to 390Lf (flocculating activity) per mg protein and a
280/260 absorbance ratio of 2.0 to 2.1. The final recovery of the toxin
from bacterial extracts was 90 to 93%. The purified preparations gave a
single band of toxin protein with a molecular weight of 150,000 on
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed
immunoelectrophoresis, the purified toxin preparations gave a single
precipitation arc against anti-crude toxin serum.
Ozutsumi et al. (1989) prepared a highly purified fragment [A-
B] of tetanus toxin by combination of gel permeation chromatography,
adsorption chromatography in an HPLC and immunoadsorption
chromatography using anti-Fragment [C] as a ligand. The purified
Fragment [A-B] (200 micrograms) elicited a peculiar toxicity,
'hypoactivity' or 'weakness', and killed the mice in ca. 73 hr and 88 hr
when it was injected intravenously and intramuscularly, respectively.
However, contamination by the whole toxin was not detectable, in the
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﴾ 21 ﴿
purified fragment preparation, when up to 600 micrograms was tested
by the mouse toxicity assay.
Fishman et al. (1992) Pointed out that the non-toxic binding
fragment of tetanus toxin (fragment C) binds avidly to neural tissue and
has a growing number of neurobiological uses. Its current utility is
limited by both its high commercial cost and the complex procedure for
its preparation requiring highly purified tetanus toxin. A short
procedure was developed which prepares fragments of tetanus toxin
from crude C. tetani extracts. The resultant proteins are atoxic with
molecular sizes and immunological properties closely resembling
fragment C. These proteins undergo retrograde axonal and apparent
transneuronal transport in a fashion similar to fragment C.
Fischer and Howden (1994) Studied the immunochemical
structure of the heavy chain polypeptide from tetanus toxin. Numerous
antigenic determinants were identified by probing a set of overlapping
peptides derived from the amino acid sequence of tetanus toxin with
polyclonal anti-toxoid antibody preparations. Synthetic antigens
representing continuous epitopes were prepared and used to immunize
mice. The capacity of the resulting anti-peptide antibodies to react with
tetanus toxin in vitro and in vivo was determined. The majority of
antibodies bound to tetanus toxin and three epitopes capable of eliciting
neutralizing antibodies were identified.
Ledoux et al. (1994) Indicated that botulinum and tetanus
neurotoxins are water-soluble proteins (mol. wt 150,000) produced by
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﴾ 22 ﴿
Clostridium botulinum and Clostridium tetani, respectively. It is
believed that these neurotoxins, once internalized via receptor-mediated
endocytosis, form membrane channels in order to traverse the
endosomal membrane and enter the cytoplasm of the nerve terminal.
That is, an association between neurotoxin monomers could result in an
oligomeric form of the neurotoxin necessary for assembly of a channel
through the hydrophobic interior of the endosomal membrane, thereby
allowing passage of the neurotoxin or its active fragment through the
resulting pore.
Clare et al. (1998) Demonstrated that the yield of fragment C
was only modestly affected by Mut phenotype, and the site and type of
integration event. Fragment C accumulation was closely correlated with
gene dosage and maximal expression levels required high gene copy
number.Yields were greatly increased in controlled fermenters,
compared to shake-flasks, owing to the high cell density achieved and
to an increased efficiency of induction (2.5- to 10-fold).In fermenter
inductions of a 14-copy strain, fragment C accumulated to 27% of total
protein, giving an estimated yield of 12 g/L. Considerable clonal
variation in the level of expression occurred with transplacement
transformants, and this was owing to a diversity of different integration
events and to differences in gene copy number. These multicopy
transplacement events occur by in vivo circularization of transforming
DNA fragments followed by repeated single-crossover integration.
Umland et al. (1998) Obtained two crystal forms of recombinant
tetanus neurotoxin C fragment. The C fragment corresponds to the C-
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﴾ 23 ﴿
terminal 451 amino-acid residues of tetanus neurotoxin and is the
subunit responsible for receptor binding by the toxin. Both forms
belong to space group P212121. Form I has unit-cell dimensions of a =
71.3, b = 79.7, c = 94.0 Å, and produces thin plate crystals. Form II has
unit-cell dimensions of a = 67.4, b = 79.7, c = 91.1 Å and produces
thick rod-shaped crystals. Diffraction data to 2.6 Å, have been collected
from form II.
He et al. (2000) reported that the fragment C of tetanus
toxin(TTC) was amplified from Clostridium tetani DNA by
PCR(polymerase chain reaction). This fragment was cloned into
expression vector pET-28a (+), under the control of the T7 promoter.
Expression of this plasmid in E.coli resulted in the production of a
protein consisting of 6xHis of the vector fused to the N-terminal 451
amino acids of tetanus toxin. The protein product accounted for 8.2%
of the bacteria total protein in soluble form. Immunization of mice with
rTTC resulted in the production of antibodies that were able to protect
mice against a challenge with tetanus toxin furthermore; rTTC in vivo
appeared to be able to undergo retrograde axonal transport.
Herrers et al. (2000) stated that tetanus neurotoxin (TeNT) is a
powerful bacterial protein toxin that cleaves VAMP/synaptobrevin, and
consequently blocks neurotransmission. The extreme neurospecificity
of TeNT is determined by the binding of its C-terminal domain
(fragment C or H(C)) to neuronal receptors. Its C-and N-terminal
halves was expressed as recombinant proteins and analysed their
binding abilities to rat phaeochromocytoma (PC12) cells differentiated
Review of literature
﴾ 24 ﴿
with nerve growth factor. It was found that the C-terminal subdomain
of the fragment C of TeNT is necessary and sufficient for cell binding
and for the interaction with the 15 kDa putative receptor. In contrast,
the N-terminal half showed a very poor interaction with the cell
surface. These results restrict the binding domain of TeNT to the C-
terminal half of the fragment C and highlight the importance of this
domain for the neurospecific interaction of the toxin with the synapse.
Furthermore, these findings support the use of this portion of TeNT as a
neurospecific targeting device.
Le et al. (2003) Evaluated bacterial spores as vaccine vehicles.
Bacillus subtilis spores displaying the tetanus toxin fragment C (TTFC)
antigen were used for oral and intranasal immunization and were shown
to generate mucosal and systemic responses in a murine model. TTFC-
specific immunoglobulin G titers in serum (determined by ELISA)
reached significant levels 33 days after oral dosing, while responses
against the spore coat proteins were relatively low. Tetanus antitoxin
levels were sufficient to protect against an otherwise lethal challenge of
tetanus toxin (20 LD50). The robustness and long-term storage
properties of bacterial spores, coupled with simplified genetic
manipulation and cost-effective manufacturing, make them particularly
attractive vehicles for oral and intranasal vaccination.
Miana-Mena et al. (2003) analysed the non-toxic C fragment of
tetanus toxin fused to the beta-galactosidase enzyme as a
neuroanatomical tracer. After intramuscular injection in rat tongue, its
location in the hypoglossal network was compared with other classic
Review of literature
﴾ 25 ﴿
tracers such as neurotropic viruses. The hybrid protein reached second
and higher-order neurons after crossing several synapses. It appears to
be a powerful tool to map neuronal circuits since the protein is easy to
handle and detect and its trans-synaptic transport is potential activity-
dependent.
Francis et al. (2004) used the non-toxic neuronal binding
domain of tetanus toxin (tetanus toxin fragment C, TTC) as a vector to
enhance delivery of potentially therapeutic proteins to motor neurons
from the periphery following an intramuscular injection. The unique
binding and transport properties of this 50-kDa polypeptide suggest that
it might also enhance delivery of proteins to neurons after direct
injection into the CNS. Using quantitative fluorimetry, they found that
labeled TTC showed vastly superior retention within brain tissue after
intracerebral injection compared to a control protein (bovine serum
albumin).Concluded that TTC may be a useful vector to enhance
neuronal delivery of potentially therapeutic enzymes or trophic factors
following direct injection into the brain.
Tregoning et al. (2005) reported that Plant-expressed vaccines
may provide a unique opportunity for generating anti-pathogen
immunity, especially in countries where cold storage is lacking. In the
following study, they show that soluble protein from tobacco leaves
expressing fragment C of tetanus toxin protected mice against a lethal
tetanus toxin challenge. More importantly, they show that a single
intranasal (i.n.) vaccination was as efficient as oral delivery, inducing
high levels of activated CD4(+) T cells and anti-toxin antibody. Unlike
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﴾ 26 ﴿
the oral route, i.n. delivery did not require the presence of adjuvant
(cholera toxin). Indeed, addition of cholera toxin induced bystander
immune responses to plant proteins as well. Plant-based vaccines are
promising because they are more heat stable, are easy to produce, cheap
and do not require needles.
Qazi et al. (2006) compared the immunogenicities of the
nontoxic HC fragment of tetanus toxin and derivatives lacking
ganglioside binding activity with that of tetanus toxoid after
subcutaneous immunization of mice. Wild-type HC (HCWT) protein
and tetanus toxoid both elicited strong antibody responses against
toxoid and HC antigens and provided complete protection against toxin
challenge. Mutants of HC containing deletions essential for ganglioside
binding elicited lower responses than HC WT. Concluded that the
presence of the ganglioside binding site within HC may be essential for
induction of a fully protective anti-tetanus response comparable to that
induced by tetanus toxoid by subcutaneous injection.
Slade et al. (2006) reported herein the results of an in situ
scanning probe microscopy study of the interaction of tetanus toxin C-
fragment (Tet C) with supported planar lipid bilayers containing the
ganglioside receptor GT1b. results show that Tet C preferentially binds
to the surface of fluid phase domains within biphasic membranes
containing GT1b and that with an extended incubation period these
interactions lead to dramatic changes in the morphology of the lipid
bilayer, including the formation of 40–80 nm diameter circular cavities.
Combined atomic force microscopy/total internal reflection
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﴾ 27 ﴿
fluorescence microscopy experiments confirmed the presence of Tet C
in the membrane after extended incubation. These morphological
changes were found to be dependent upon the presence of GT1b and
the solution pH.
Caleo and Schiavo (2009) stated that Clostridium tetani is
comprised of heavy (H, 100 kDa) and a light chain (L, 50 kDa) linked
by a disulphide bond and non-covalent interactions. The carboxy-
terminus of the heavy chain (HC) binds with extraordinary affinity and
specificity to nerve terminals. Following internalization, the amino-
terminal portion of the heavy chain (HN) inserts into the membrane of
the endosome at acidic pH and assists the translocation of the L chain
into the cytosol. Finally, the L chain is endowed with a zinc-
endopeptidase activity specific for SNARE proteins. SNARE proteins
are involved in the fusion of synaptic vesicles with the plasma
membrane and therefore the catalytic activity of the light chain is to
prevent exocytosis and neurotransmission.
Mendieta et al. (2009) Evaluated the effects of local
administration of Hc-TeTx on motor behavior and the dopamine (DA)
levels in the striatum of MPP+-treated rats. Recently it has been shown
that the C-terminus fragment of the tetanus toxin (Hc-TeTx) is
transported retrogradely and had shown neuroprotective effects,
preventing neuronal death by apoptosis. This could be a new alternative
preventing ongoing cell death and restoring the motor function in
Parkinson’s disease (PD), which is characterized by dopaminergic
neurodegeneration. The study shows that Hc-TeTx improves different
Review of literature
﴾ 28 ﴿
motor behavior strongly, which favors the hypothesis of the Hc-TeTx
fragment enhancing survival pathways that result in amelioration of the
dopaminergic system of rats with a dopaminergic lesion.
Turillazzi et al. (2009) Reported a case of a 65-year-old man
with a right pre-radial cutaneous laceration associated with a Colles'
fracture. His status for tetanus immunization was uncertain; so a course
of antitetanus treatment was immediately started. Two days after
admission the man suddenly developed severe nucal pain, rigidity and
dysphagia. A brain CT scan was negative. Cultures from the wound
were negative for Clostridium tetani; the CSF analysis was negative.
On the 9th day after admission, the man died. An immunohistochemical
study was conducted with an antibody directed against tetanus toxin
fragment C (TTC). By immunohistochemical evaluation, large motor
neurons in the ventral horn were immunopositive for TTC. High power
magnification of the ventral horn of spinal cord gray matter samples
showed TTC immunoreactivity in motor neuron axons and cell bodies,
using a confocal laser scanning microscope. The correct diagnosis
could be established on the basis of pathological examination with TTC
immunostaining.
PREPARATION OF TETANUS ANTITOXIN:
Harlow and Lane (1988) defined the antibodies as host
proteins produced in the presence of foreign molecules in the body.
This response is the combination of series of interaction between
macrophages; T-lymphocytes and B-lymphocytes, all reacting in the
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﴾ 29 ﴿
presence of foreign antigen. The end product of this response is the
production of large numbers of antibody molecules. They added that
the first exposure to an antigen induced a relatively weak reaction
known as primary response which prepares the animal for any
subsequent exposure to the same antigen when the antigen is
reintroduced; the secondary response is typically rapid and intense.
When the animal is given a second injection with the same antigen, a
much faster, more potent and more persistent response occurs. Because
the memory cells are long-live, then the secondary response can takes
place months or years after the primary response.
Blood et al. (1983) stated that the prophylactic dose of tetanus
antitoxin is 1500 – 3000 IU in horses. On farms where the incidence of
tetanus in lambs was high, antitoxin was usually given at docking and a
dose rate of 200 IU has been effectively used. The immunity was short
transient, persisting for only 10-14 days.
PURIFICATION OF TETANUS ANTITOXIN:
Favreau et al. ( 1983) Reported that heterologus antitoxic sera
have long been used for the treatment of toxic-infections: diphtheria
and tetanus, as well as snake and scorpion envenoming. With the
purpose of eliminating the risk of adverse reaction, whole sera have
been subjected to pepsin digestion which results in the destruction of
the Fc fragment responsible for the reactogenicity of the antibody
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﴾ 30 ﴿
molecule. Purified digested heterlogus sera proved to be more readily
tolerated than crude whole sera.
Michella and Parkinson (1987) Described a simple two step
procedure to purify the immunoglobulin G(IgG) fraction from
mammalian sera and ascites fluid. In the first step , albumin and other
non- IgG proteins are precipitated with caprylic acid (octanoic acid). In
the second step , the IgG fraction is precipitated with ammonium
sulphate. The procedure can be used to purify IgG fraction of serum
from rabbit, sheep, goat, horse, rate and mouse as well as monoclonal
antibodies from mouse ascites fluid. Greater than 80% of the IgG in
serum could be isolated with a purity equal to rabbit IgG purified by
anion-exchange chromatography. The method is simple and low cost.
Vaz et al. (1988) injected horse sera containing anti-tetanus
whole IgG molecules, bivalent F(ab')2 fragments and monovalent Fab'
fragments in 24 groups of 10–20 mice to compare their protective
activity. When tetanus was induced in the mice, either with toxin or
with spore suspension of Clostridium tetani 24 or 32 h prior to the
injection of the antitoxins, monovalent Fab' was significantly more
efficient in conferring protection against tetanus than F(ab')2 or IgG.
Mohanty and Elazhary (1989) Purified Immunoglobulin G
(IgG) from bovine serum raised against Aeromonas Salmonicida by
ammonium sulphate precipitation (ASP) or caprylic acid treatment
followed by ammonium sulphate precipitation (CAAS). Purity of IgG
samples prepared by both methods were examined by High
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﴾ 31 ﴿
Performance Gel Permeation Chromatography, electrophoresis and
antibody activity assay. Results suggest that IgG prepared by ASP is
better than that obtained by CAAS method in terms of the yield of the
IgG monomers and the recovery of the antibody activity.
Smith et al. (1992) Developed a novel antivenom for treating
patients with Vipera berus bite. The immunoglobulin fraction is
precipitated with sodium sulphate then cleaved with papain to produce
Fab' fragments. Those Fab' fragments are purified by affinity
chromatography on columns comprising V. berus venom coupled to
cyanogen bromide activated sepharose 4B . The resultant product is
some three times more effective than the non-purified Fab' in protecting
mice against the lethal venom effect.
Rea and Ultee (1993) Discovered that 0.5-0.8 M ammonium
sulphate has a beneficial effect on the peptic digestion of several
antibodies. Beside preventing precipitation, ammonium sulphate
usefully modulated the digestion rate, accelerating the digestion of
some antibodies and reducing it for others. Rabbit, goat and sheep
immunoglobulins G (IgG) were more temperature sensitive than the
murine antibodies digesting very slowly at room temperature but quite
rapidly at 37◦c. ammonium sulphate slightly reduced the digestion rate
of rabbit IgG, but had little effect on the digestion of the goat and sheep
IgGs. However it effectively suppressed the precipitation caused by the
addition of pepsin to the IgG.
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﴾ 32 ﴿
Rawat et al. (1994) Indicated the low toxicity of Fab' by their
ability to be used successfully at higher concentrations than the other
products tested (intact IgG F(ab)'2 and without deleterious effects. The
small size (55,000 Da) of ovine Fab' also results in faster kinetics, and a
greater volume of distribution around the body than IgG. Fab'-based
antivenom is likely to be effective sooner after administration and to
bind venom components in the body compartments unavailable to the
large IgG molecules.
Rojas et al. (1994) described a simple methodology for
hyperimmune horse plasma fractionation, based on caprylic acid
precipitation. Studied optimal conditions for fractionation; the method
gives best results when concentrated caprylic acid was added to plasma,
whose pH had been adjusted to 5.8, until a final caprylic acid
concentration of 5% was reached. The mixture was vigorously stirred
during caprylic acid addition and then for 60 min, afterwards the
mixture was filtered. Non-immunoglobulin proteins precipitated in
these conditions, whereas a highly enriched immunoglobulin
preparation was obtained in the filtrate, which was then dialysed to
remove caprylic acid before the addition of NaCl and Phenol.
Laing et al. (1995) produced a new antivenom, a polyclonal
ovine Fab preparation which provides superior protection, both in vivo
and in vitro for treatment of carpet viper (Echis ocellatus). Fab
fragments, which have the advantages of large volumes of distribution
and, theoretically, low immuno-reactivity, are produced by a reusable
solid-phase papain matrix which eliminates enzyme contamination of
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﴾ 33 ﴿
the product and reduces cost. The antivenom is lyophilized for
increased stability and extended shelf-life in tropical climates where it
is often impossible to keep such products cool.
Sheoran and Holmes (1996) described purification of the
subisotypes of equine IgG. Equine IgG possesses four well-defined
subisotypes, designated IgGa, IgGb, IgGc and IgG(T) on the basis of
their increasing anodal mobility in electrophoresis. Purification of IgGa
and IgGb was achieved by the separation of a ‘fall-through’ peak from
ion-exchange chromatography consisting of IgGa and IgGb into two
fractions (peaks C and D) by FPLC protein A and protein G affinity
chromatography. Peak C consisted of IgGb and peak D consisted of
IgGa exhibiting slightly faster cathodal migration than peak C in IEP
analysis. Affinity chromatography using protein A and G columns also
indicated that there may be two different components of IgG(T); one
with a low affinity for protein G and the other having a greater affinity
for protein G.
Guidolin et al. (1997) treated equine antisera raised against
rabies virus, Bothrops venoms and diphtherial toxin with β-
propiolactone treatment induce a reduction in complement activation,
tested ''in vivo'', without significant loss of biological activity.
Rodrigues-Silva et al. (1997) reported that when antivenoms are
exposed to high temperatures they develop turbidity with time which is
caused by the formation of high mol. Wt. protein aggregates. Some
hyperimmune horse antivenoms might also become turbid if frozen or
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﴾ 34 ﴿
lyophilized. Immediate adverse reactions to antivenom therapy have
been described. Immunoglobulin-containing aggregates could activate
serum complement and induces deleterious effects. Increasing the
thermal stability of protein preparations is a practical goal that may
improve the shelf-life of antivenoms. Sorbitol (1.0 M) was used as an
osmolyte (a natural substance or an organic compound capable of
stabilizing proteins) and decreased the formation of protein precipitates
in solutions of antibodies. Sorbitol was shown to be capable of
stabilizing antibodies at high temperatures, with no significant
perturbation in the secondary structure or affinity.
Lee (2000) Used High-performance sodium dodecyl sulfate-
capillary gel electrophoresis SDS-CGE to separate antibodies and their
fragments according to size. The magnitude of the apparent
fragmentation is temperature-dependent and is more pronounced with
rabbit, sheep and bovine immunoglobulin G’s than murine monoclonal
antibodies. In addition to temperature, pH and buffer also affect the
fragmentation. Without heat treatment during the preparation of the
SDS–antibody complexes, the observed fragments become nearly
absent; however, some murine monoclonal antibodies exhibit several
peaks that group near the expected migration time of an
immunoglobulin G, presumably due to their anomalous interaction with
sodium dodecyl sulfate. This high-performance electrophoretic
technique is suitable for quality control as well as the characterization
of the antibodies under experimental conditions.
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﴾ 35 ﴿
Kumpalume et al. (2002) Discribed a simple process for the
manufacture of polyclonal F (ab)2 fragments that might be adopted for
the facile preparation of antivenoms. The production of polyclonal F
(ab)2 fragments from serum commonly involves the initial purification
of IgG’s prior to their proteolytic cleavage and further purification. To
reduce the number of processing steps the authors have compared the
digestion of whole serum by free and immobilized pepsin with that of
pure IgG. It was observed that with equal units of pepsin activity,
caprylic acid pre-purified IgG was digested more rapidly than whole
serum but that the overall retention of antigen binding activity was
significantly greater in the latter case though the immobilized pepsin
lost about 40% of its proteolytic activity after first use.
Salwa et al. (2002) Stated that the main objective of antitoxic
plasma purification was to obtain a stable and highly purified antitoxin,
rich in specific antibodies, free of immunologically irrelevant plasma
proteins or gross proteins. Their results indicate that the most potent
yield and purified F (ab')2 antivenom preparation was obtained when
the first discarded precipitate was washed with 14% ammonium
sulphate saline; then after the second addition of ammonium sulphate,
the mixture was stirred overnight followed by precipitation of most
non-immunoglobulin proteins with the aid of caprylic acid to produce
antivenom rich in specific antibodies with higher yield and potency
compared to the method commonly used.
Boushaba et al. (2003) Assessed alternative route for the
production of polyclonal F (ab')2 fragments that might be adopted for
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﴾ 36 ﴿
the facile preparation of antivenoms. The method involves the digestion
of whole serum by free pepsin, which results in reduction of the
number of processing steps commonly in use, because it avoids the
initial purification of IgG's prior to their proteolytic cleavage by the
enzyme. It was observed that with equal units of pepsin activity,
caprylic acid prepurified IgG was digested more rapidly than whole
serum but that the overall retention of antigen binding activity was
significantly greater in the latter case. The results obtained from this
technique confirm and quantify previous observations that pepsin
digestion of whole serum is slower and easier to control than digestion
of pure IgG and results in higher recovery of antigenic binding activity.
Cheung et al. (2003) Used the Gradiflow (a preparative
electrophoresis instrument designed to separate molecules on the basis
of their size and charge) to purify antibody Fab and F(ab')2 fragments.
The method described is charge based, utilizing the difference in the pI
between the antibody Fab/ F(ab')2 fragments and antibody Fc
fragments that occur after enzyme digestion of whole antibody
molecules. This method of purification was successful across a range of
monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments
were purified from a number of mouse monoclonal antibodies (both
IgG1 and IgG2a isotypes) and Fab fragments were purified from egg
yolk IgY polyclonal antibodies. This is a rapid purification method
which has advantages over alternative methods that usually comprise
ion exchange and gel filtration chromatography. This method may be
applicable to most antibody digest preparations.
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﴾ 37 ﴿
Jones and Landon (2003) proposed a simple, high yield
protocol for processing serum to highly purified F(ab')2 and also, avoids
the need for an initial or subsequent salt precipitation and can be
utilized for either bench or large scale production. The protocol include
the digestion of ovine antiserum under acidic conditions (pH 3.5) by
pepsin which reduce all unwanted serum components to low molecular
weight (≤ 13 kDa)fragments while leaving the ~ 100 kDa F(ab')2 intact.
The pH is then raised to 6 to stop further digestion and the reaction
mixture centrifuged or filtered to remove any insoluble contaminants.
Next unwanted low molecular weight fragments are removed by
diafiltration with a 30 kDa nominal molecular weight cut-off membrane
leaving an F(ab')2 solution contaminated only with some pepsin and a
small amount of the aggregated low molecular weight fragments.
Raweerith and Ratanabanangkoon (2003). Studied a
combined process of caprylic acid (CA) precipitation and ion-exchange
chromatography on SP-Sepharose as a mean to fractionate pepsin-
digested horse antivenom F(ab')2 antibody. In the CA precipitation, the
optimal concentration for fractionation of F(ab')2 from pepsin-digested
horse plasma was 2%, in which 89.61% of F(ab') 2 antibody activity
was recovered in the supernatant with 1.5-fold purification. A
significant amount of pepsin remained active under these conditions.
An analytical cation exchanger HPLC column was tested to establish
optimal conditions for the effective separation of IgG, albumin, pepsin
and CA from the F(ab')2 product. The total recovery of antibody was
65.56% with 2.91-fold purification, which was higher than that
achieved by ammonium sulfate precipitation. This process removed
Review of literature
﴾ 38 ﴿
residual pepsin, high molecular weight aggregates and CA and should
be suitable for large-scale fractionation of therapeutic equine
antivenoms.
Cresswell et al. (2005) describe a rapid method for the
determination of optimum conditions for papain digestion of polyclonal
ovine IgG (purified by Na2SO4 precipitation) for the production of bio-
therapeutic Fabs (antigen-binding fragments). To determine the
optimum conditions for digestion, a factorial approach to the design of
experiments was undertaken. The results and methods suggest that,
provided the time and temperature are maintained at the high settings
evaluated (24 h, 40 degrees C), the modelled data predict IgG digestion
close to 100% for all the papain concentrations used. Provided papain is
used at >2.5% (w/w), either time and/or temperature may be reduced.
The results and methods described in the present paper may also be
applicable to the generation of therapeutic Fab fragments from other
immunoglobulins, including monoclonal antibodies purified from
mammalian cell culture.
Herrera et al. (2005) Adapted caprylic acid purification of IgG,
currently used in the manufacture of horse-derived antivenoms, for the
preparation of sheep and camel IgG. Sheep IgG had a molecular mass
of 150 kDa, whereas camel IgG presented two bands of molecular
masses of 160 and 100 kDa. Horse, sheep and camel IgGs were
compared to predict their potential for induction of early and late
adverse reactions. Horse and sheep IgGs showed a higher
Review of literature
﴾ 39 ﴿
anticomplementary activity than camel IgG, and also elicited a higher
anti-IgG response than camel IgG, when injected in mice. Horse IgG
agglutinated human type O erythrocytes, whereas no such reactivity
was observed in sheep and camel IgG preparations. Overall, camel IgG
showed the lowest potential for the induction of adverse reactions
among the three IgGs tested.
Morais and Massaldi (2005) studied the effect of the harsh
conditions prevailing during the digestion step on the activity of the
F(ab')2. To this purpose, the recovery of the activity of anti-Bothrops
hyperimmune equine plasma was determined after pepsin digestion
under different sets of processing conditions. The balance between pH
level and reaction time was found to be critical, reflecting a
compromise between complete cleavage of immunoglobulins and
strong denaturation of the F(ab')2 fragments. For pH in the range 2.8-
3.2, 30-65% of the initial activity was lost depending mainly on the
processing time. In conclusion, for equine F(ab') 2 antivenom
production, it seems convenient to carry out digestion at pH values
sufficiently low to ensure that total IgG breakdown is achieved in the
shortest time compatible with precise operation in the production scale.
Redwan (2006) Indicated that animal derived therapeutic
antibodies represent the best and only choice source of antitoxins,
especially in developing countries. Recently, several laboratories
changed their production protocol from ammonium sulfate (AS)
protocol to caprylic acid (CA) fractionation. This study showed that
using the CA protocol leads to improvement in the product quality, as
Review of literature
﴾ 40 ﴿
assessed by the albumin and protein content decrease (from 4.75 to
3.54 g/dL and 0.64 to 0.18 g/dL, respectively), which yielded a purer
antitoxin product. The F(ab)2 protein aggregate formation and turbidity
have been significantly reduced, 4.60 versus 2.55 and 0.046 versus
0.021 (p<0.01), respectively. However, the anti-complementary activity
was also reduced, from 42 to 33. The total IgG content was higher in
CA fractionated products than AS materials.
Burnouf et al. (2007) carried out a study to determine whether
pre-existing antivenom production steps may reduce viral risks
contamination. Two typical manufacturing steps were studied: (a) a pH
3.3 pepsin digestion of diluted plasma at 30◦C for 1 h, and (b) a
caprylic acid treatment of a purified F(ab)2 fragment fraction at 18◦C
for 1 h. Three lipid enveloped (LE) viruses [bovine viral diarrhoea virus
(BVDV), pseudorabies virus (PRV), and vesicular stomatitis virus
(VSV)] and one non-lipid-enveloped (NLE) virus
[encephalomyocarditis virus (EMC)] were used as models. The pH 3.3
pepsin digestion resulted in complete clearance of PRV and in almost
complete reduction of VSV, and in a limited inactivation of BVDV.
The caprylic acid treatment resulted in complete inactivation of the 3
LE viruses tested. For EMC no significant reduction was obtained.
Therefore the current manufacturing processes already include
production steps that can ensure robust viral inactivation of LE viruses
and moderate inactivation of a NLE virus.
Morais and Massaldi (2007) presented a comparative bench-
scale study of endotoxin contamination for two common processes of
Review of literature
﴾ 41 ﴿
immunoglobulin purification from equine plasma: ammonium sulphate
fractionation of F(ab')2 fragments and caprylic acid precipitation of
non-IgG proteins. To this end, both processes were carried out under
normal sterile conditions, using sanitized material and equipment and
optimal water quality in a clean but open environment. It was found
that exogenous contamination preferentially came from endotoxins
already present in reagents and/or raw materials, whereas
contamination from the environment was minimal. It is concluded that
sterility is not a sufficient condition to obtain an endotoxin-free
product. Only with proper sanitization of material, and by applying the
caprylic acid purification process with a starting plasma below 4-5
EU/mL, would it be possible to achieve a final product within the norm.
Mpandi et al. (2007) stated that caprylic acid is of great interest,
by providing the advantage of purifying mammalian immunoglobulins
and clearing viruses infectivity in a single step. To evaluate the
effectiveness of caprylic acid for the removal/inactivation of viruses,
spiking studies were carried out. The data show that the treatment with
caprylic acid 5% (v/v) can effectively be used as well to purify or to
ensure viral safety of immunoglobulins. Caprylic acid precipitation was
very efficient in removing and/or inactivating enveloped viruses (PRV,
BVDV) and moderately efficient against non-enveloped viruses
(MVM, ECMV). However the combination with the pasteurization
ensured an efficient protection against both enveloped and non-
enveloped viruses. Its a simple and non-expensive manufacturing
process of immunoglobulins and easily validated.
Review of literature
﴾ 42 ﴿
Fernandes et al. (2008) discussed a novel purification technique
for chromatographic purification of anti-rabies immunoglobulin G
(IgG) fragment F(ab)2 from horse serum. F(ab)2 was purified by two
successive chromatography steps using Cellufine A-200 and ProSep-
vA Ultra media. The purified F(ab)2 was characterized using
biochemical and biophysical methods and shown to be pure and
homogeneous. The purified F(ab)2 was reactive to rabies antigen in
immuno-electrophoresis and diffusion tests. The purified F(ab)2 was
biologically functional and was found to show a potency of 1500
IU/ml. Comparative analysis of the purity with commercially available
F(ab)2 by HPLC analysis and SDS-PAGE indicated that the present
product is better in purity.
Hervé et al. (2008) Used both natural polyamines with
increasing net charge valencies (putrescine, PUT; spermidine, SPD; and
spermine, SPM) and a synthetic polyamine (hexamethylenediamine,
HMD) to cationize antibodies. This study describes the covalent
modification of antitetanus F(ab)2 with these four polyamines using
different reaction conditions, and compares the effects of these
modifications on antibody interaction with cultured HL60 cells.
Cationization was shown to enhance cell interaction of the F(ab)2. It
was found that coupling the F(ab)2 to the SPD and SPM polyamines
had greater effect on cell interaction than coupling the F(ab)2 to the
PUT and HMD diamines. SPD and SPM were more effective than PUT
and HMD in terms of intracellular delivery of the F(ab)2. It follows
from all these results that electrostatic interaction involving charge
Review of literature
﴾ 43 ﴿
density plays a predominant role in the endocytic transport mechanism
of the F(ab)2 modified with these polyamines.
Wang et al. (2008) discussed a membrane based enhanced
hybrid bioseparation technique for efficient and scalable purification of
equine immunoglobulin G (IgG) from horse serum. In the presence of a
high antichaotropic salt concentration, equine IgG is selectively and
reversibly captured within a stirred cell membrane module from horse
serum, partly due to precipitation and microfiltration, and partly due to
hydrophobic interaction based membrane adsorption, while the
impurities are washed out from the device. The reversibly sequestered
IgG is then released by lowering the salt concentration which favor
both dissolution of the precipitated IgG and desorption of the
membrane bound IgG. The equine IgG purity obtained under optimized
conditions was 88% and its recovery was over 90%, both being
significantly higher than corresponding values obtained using currently
used purification techniques.
Grodzki and Berenstein (2010) Indicated that ion exchange
chromatography techniques are a powerful method for the purification
of proteins and monoclonal antibodies. The technique can separate
biomolecules that have minor differences in their net charge, e.g., two
protein molecules differing by a single charged amino acid. Given the
amphoteric character of proteins the pH of the solution is important in
the determination of the type of ion exchanger used. Immunoglobulins,
although they can be purified by either cation or anion exchange
Review of literature
﴾ 44 ﴿
chromatography, are most frequently purified by anion exchange with
DEAE resins.
Wang et al. (2010) Discussed a continuous two-stage cascade
ultrafiltration bioreactor-separator system for fragmentation of
immunoglobulin G (IgG) by pepsin and purification of F(ab)2 fragment.
The 10 kDa MWCO membrane of the first stage bioreactor retained
pepsin, IgG and F(ab)2 while allowing degraded Fc sub-fragments
through and the 70 kDa membrane of the second stage retained both
IgG and F(ab)2 while allowing pepsin through. The first bioreactor
therefore primarily carried out IgG digestion while the second stage
primarily served as a separator for pepsin and F(ab)2. The two-stage
system was first assessed using pure equine IgG as feed. Under
optimized feed and sweep flow rates, 97% IgG conversion with 93%
pure F(ab)2 product were obtained. When the bioreactor system was
operated with unpurified equine serum as feed, close to 95% IgG
conversion was observed. The results demonstrated the suitability of
the two-step cascade membrane bioreactor for production of F(ab)2
from both IgG and unpurified equine serum.
MATERIAL AND METHODS
﴿ ﴾
45
3-MATERIAL AND METHODS
3.1. MATERIALS
3.1.1. Strain:-
Clostridium tetani Harvard strain 49205 was originally obtained
from the New York State Department of Health. This strain was obtained
as a lyophilized ampoule from the Anaerobic Department, Veterinary
Serum and Vaccine Research Institute, Abbassia, Cairo.
3.1.2. Media:-
i-Blood agar medium
(Cruickshank et al.1975 and Oxoid manual,1998)
In 1 liter of distilled water 40g of blood agar base No.2, Oxoid
was suspended and brought to the boil to dissolve completely. Sterilized
by autoclaving at 121˚c for 15 minutes. Left to cool to 45-50˚c and 7%
sterile defibrinated sheep blood was added. Mixed with gentle rotation
and poured into Petri dishes.
ii-Thioglycolate medium U.S.P. (Brewer,1940)
Patent preparation obtained from Oxoid, LTD.
iii-Sabouraud dextrose agar.
Patent preparation obtained from Oxoid, LTD.
Used for sterility test for determination of fungal growth.
iv-Nutrient agar 1.5% agar, Oxoid.
6ml in tubes, slant agar.
MATERIAL AND METHODS
﴿ ﴾
46
Used for sterility test for determination of contamination by aerobic
organisms.
v-Modified Mueller and Miller (1954):(Wafaa, 2005)
Composition of one liter of medium.
Pancreatic digest of casein 22.5g
Beef heart infusion 50 ml
Glucose 11 g
NaCl 2.5g
Na2HPO4 2g
KH2 PO4 0.15g
KCl 0.1g
MgSO4 0.15 g
Cystine 0.25g
Ca-pantothenate 1.0mg
Uracil 2.5mg
FeCL3.6H2O 32mg
Distilled water to 1000 ml
The pH was adjusted to 7-7.2
Stock solutions for amino acids and vitamins were prepared and
stored separately at 4˚C for maximally 4 weeks.
Preparation of beef heart infusion:
One kg of minced defatted beef heart was suspended in one liter distilled
water. Brought rapidly to boiling and boiled for two minutes. Filtered
MATERIAL AND METHODS
﴿ ﴾
47
through filter paper and stored at 4˚C for maximally one week. Filtered
again just before adding to the medium.
Preparation of pancreatic digest of casein solution:
The amount of pancreatic digest of casein (commercially available as N-Z
case ,Sigma chemicals, U.S.A) needed was dissolved in distilled water, to
obtain a 10% w/v solution by heating. Cooled down to 40˚C
approximately; 1.25g charcoal was added per liter. The solution was
stirred for 20 minutes and filtered twice through filter paper.
Stock solutions:-
1) Cystine
2.5g was suspended in 12 ml distilled water, and then 6.2 ml
concentrated HCl was added and the solution was diluted with distilled
water to a final volume of 25 ml. Then 2.5 ml was added per liter of
medium.
2) Uracil
0.1 g of Uracil was dissolved in 10 ml HCl, and then the solution
was diluted with distilled water to a final volume of 40 ml. Then one ml
was added per liter of medium.
3) Ca-pantothenate
0.1 g ca-pantothenate was dissolved in 100 ml ethanol 25%.
From the above solution one ml was added per liter of medium.
MATERIAL AND METHODS
﴿ ﴾
48
3.1.3. Antisera and toxoids:
-Tetanus toxoid for flocculation test
Was purchased as a standard solution containing 1300 Lf/ml from
Holding Company for Biological Products and Vaccines-AGOUZA,
Cairo.
- Commercial antitetanic serum obtained from Holding Company
for Biological Products and Vaccines-AGOUZA, Cairo.
Each ampoule contains 1500 I.U/ml. It was used for flocculation test.
3.1.4. Experimental animals:-
i-Albino Swiss mice
A total of (200) albino Swiss mice weighing 15-22 gm were used
for the determination of the minimum lethal dose (MLD) of toxins and
protective activity of antitetanic serum fractions. These mice were
obtained from the farm of the Serum and Vaccine Research Institute,
Abbassia, Cairo.
iii-Horses
A total of 10 apparently healthy adult horses between 3-5 years,
used for routine antitetanic serum production in Serum and Vaccine
Research Institute, Abbassia, Cairo. They were kept under hygienic
measures receiving balanced ration and adequate water.
3.1.5. Apparatuses and equipments:
a- Seitz filter. Single sheet stainless-steel mechanical filter with reservoir
of two liters.
MATERIAL AND METHODS
﴿ ﴾
49
b- Beckman J2-Mc high speed cooling centrifuge(16000 rpm), Beckman
Instruments Inc. Irvine, California, USA.
c- Dynatech Immunoassay System MR 7000-Dynatech medical products
USA. Used for reading microplates in ELISA test and in measuring
protein.
d- LPLC system (pumb model Mini Plus3, detector uv/VIS-151 FC
203B,Gilson) were used for chromatography of tetanus toxin.
e - Milton Roy Spectronic 601 Spectrophotometer. Used for measurement
of A 280 nm of tetanus toxin.
3.1.6. Buffers and solutions
3.1.6.1. Sterile 0.85% physiological saline
It was used for dilution of toxins and antisera in flocculation test.
3.1.6.2. Sterile peptone saline.
1% w/v peptone and 0.5% w/v sodium chloride. It was used for
dilution of toxin for MLD determination.
3.1.6.3. Bovine serum albumine (2mg /ml )
Used for protein measurement .
3.1.6.4. Tris-HCl, buffer 0.1M containing 1M NaCl pH 8.0.
5.30 g tris-base and 8.88 g Tris-HCl were added to one liter of
distilled water. Used for elution of tetanus toxin in chromatography.
MATERIAL AND METHODS
﴿ ﴾
50
3.1.6.5. 10 mM sodium phosphate buffer pH 7.4.
Used for dialysis of tetanus toxin purified from culture filtrate.
3.1.6.6. 0.1 M phosphate buffer pH 6.5 containing 1 mM Na EDTA and 1
mM of cystein-HCl. Used in digestion of tetanus toxin.
3.1.6.7. Buffers and solutions used for purification of antitetanic
serum:
-0.36 M Hydrochloric acid.
-Pepsin solution: 50 mg/ml pepsin in distilled water sub-aliquoted
and stored frozen at -20˚c until required.
-Piperazine base solution (50m M).
- Buffer A (20 m M Piperazine, 150 m M NaCl, adjust to pH 6.0
with conc. HCl).
3.1.6.8. Reagents used for ELISA:
The ELISA buffers and reagents were prepared according to Leslie
and Frank (1989) as follows:
Antigen coating buffer:
Carbonate-bicarbonate buffer pH 9.5.
Stock solution "A":
0.2 M solution of anhydrous sodium carbonate 21.2 g in 100ml.
Stock solution "B":
0.2 M solution of sodium hydrogen carbonate 16.7 gm in 100 ml.
Stock solution "A" 16 ml
Stock solution "B" 34 ml
Distilled water up to 200 ml
pH was adjusted to 9.6
MATERIAL AND METHODS
﴿ ﴾
51
Washing solution:
(Phosphate buffered saline-tween) pH 7.2-7.4
Sodium chloride 8.0 g
Potassium chloride 0.2 g
0.008 M disodium hydrogen phosphate 1.15 g
Potassium dihydrogen phosphate 0.2 g
Tween 20 0.5 ml
Distilled water 1000 ml
Blocking solution:
Bovine serum albumin 1.0 g
Washing solution 100 ml
Conjugates:
Rabbit poly anti-horse IgG (H&L), HRP conjugate, KOMA Biotech Inc.
0.1 M citrate buffer, pH 5:
0.1 M citric acid, C6H8O7, 1 H2O 21.01 g/l
0.1 M disodium hydrogen phosphate 17.8 g/l
Equal mixture of citric acid and phosphate is added to obtain
phosphate-citrate buffer pH 5.
Substrate solution:
O-Phenylene diamine 34 mg
0.1 M citrate phosphate buffer 100 ml
Hydrogen peroxide 50 µl
MATERIAL AND METHODS
﴿ ﴾
52
Stopping solution: Sulphuric acid 12.5 ml
Distilled water up to 100 ml
3.1.6.9. Reagents used in SDS-PAGE:- (According to
Oconner, 2006) *Stock acrylamide solution: 30% acrylamide, 0.8% bis-acrylamide.
Filter through filter paper and store at 4˚c.
*Separating gel (10% , in 0.37 M Tris, pH 8.8)
-1.5M Tris-HCl, pH 8.8 2.5 ml
-20% (w/v) SDS 0.05 ml
-Acrylamide, Bis-acrylamide 3.3 ml
(30% , 0.8 % w/v) -10% Ammonium persulphate 0.05 ml
-TEMED 0.005 ml
-Distilled water 4.1 ml
*Stacking gel (4 % gel, 0.125 M Tris, pH 6.8)
-0.5 M Tris, pH 6.8 1.25 ml
-20% (w/v) SDS 0.025 ml
-Acrylamide, Bis-acrylamide 0.67 ml
(30% , 0.8 % w/v) -10% Ammonium persulphate 0.025 ml
-TEMED 0.005 ml
-Distilled water 3.075 ml
MATERIAL AND METHODS
﴿ ﴾
53
*Running buffer:-
5 × diluted to 1× before use
5 × running buffer, pH 8.3(1 liter)
Tris Base 15 g
Glycine 72 g
SDS 5 g
Distilled water to 1 liter
*Protein stain:-
0.1% Coomasie brilliant blue R250 in 50% methanol, 10% glacial
acetic acid. The dye was dissolved in methanol and water first then
glacial acetic acid was added then filtered.
*Destain solution:-
10% methanol, 7% glacial acetic acid.
The gel was preserved in 2% glycerol.
*protein markers:
- SpectraTM Multicolor Broad Range protein ladder of ten bands (10, 15,
25, 35, 40, 50, 70, 100, 140 and 260 kDa)
-InvitrogenTM See blue prestained standard (210, 78, 55, 45, 34, 23, 16,
7, 4 kDa)
MATERIAL AND METHODS
﴿ ﴾
54
3.2. METHODS. 3.2.1. Preparation of tetanus toxin.(According to Mueller and
Miller1954, Maria et al. 1997)
The content of freeze dried ampoule of Clostridium tetani strain
was reconstituted in few amounts of thioglycolate broth (Oxoid) and
incubated at 36˚C for 24-48 hrs. Sterility test was performed using
nutrient agar tubes and blood agar plates. 2 ml of the thioglycolate broth
culture was used to inoculate 500 ml of production media present in 1
liter cylindrical pyrix jars which have been covered with a thin layer of
cotton between 2 layers of cheese cloth, held firmly in place by a
tightened strip of flexible metal. In this cover was inserted a long syring,
sterilized with the medium and serve to introduce the inoculum. The
media was incubated for 7-10 days at 36˚C, at the fourth day sample was
taken, centrifuged and checked for toxin production by flocculation test.
This was repeated daily until two similar successive Lf (Limits of
flocculation)values were obtained. Incubation was stopped by exposing
the cultures to a 4˚C temperature to promote bacterial lysis. Two days
later, the completeness of cell disruption was verified by Gram stain.
Glycine 5 g / liter media, dissolved in hot water was added to the culture
as stabilizer. The culture fluid was filtered through Seitz filter and
sterility test was performed to the filtrate.
3.2.2. Evaluation of the prepared tetanus toxin:-
3.2.2.1. Determination of minimal lethal dose of tetanus toxin
(M.L.D) (According to Nadia, 1992).
Ten fold serial dilutions of tetanus toxin using a solution of 1%
peptone and 0.5% NaCl were prepared .From each dilution 2 mice were
MATERIAL AND METHODS
﴿ ﴾
55
injected s/c at the root of the tail towards the right side with 0.5 ml and
observed for 4 days. The dilution that killed the two mice at the fourth
day was determined. One M.L.D is the smallest amount of toxin that
when injected s/c would kill the mice within 96 hours.
3.2.2.2. Determination of Lf (Limits of flocculation) value of toxin or
toxoid. (According to WHO, 1997 and Demain et al., 2006)
The flocculation test is an in vitro method based on the observation
that tetanus toxin or toxoid and tetanus antitoxin aggregate and form
visible floccules when mixed in certain proportions in test-tubes. The
precipitate is developed more rapidly when equivalent amounts of toxin
and antitoxin are present than when an excess of either toxin or antitoxin
is available.
Test procedure:
-Water bath was adjusted to 45°C.
- The flocculation tubes were labeled 1-10.
-The reference antitoxin was diluted to contain 100 Lf/ml by using
physiological saline 0.85%. Increasing amounts of the 100 Lf/ml
reference antitoxin were pipetted into each set of 10 tubes in the
following amounts: 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70,
0.75, and 0.80 mls.
-Normal saline was added to each tube to bring the volume of each
flocculation tube to one ml.
-One ml of test toxin was added quickly to have the same start time for all
tubes.
- Mixed thoroughly by gentle shaking of each tube.
- All tubes were incubated in racks in the water bath with 1/3 of the
MATERIAL AND METHODS
﴿ ﴾
56
reaction mixture immersed in the water of the water bath, so that stirring
by virtue of the convection currents will occur.
- Time was recorded and each vial was observed closely for flocculation
every 3 minutes. The time of the first 3 tubes showing flocculation was
recorded. Usually 20-40 minutes.
Tube No. 1 2 3 4 5
Reference antitoxin 100 Lf (ml) 0.40 0.50 0.60 0.70 0.80
Normal saline (ml) 0.60 0.50 0.40 0.30 0.20
Unknown toxin (ml) 1 1 1 1 1
F3 F1 F2
F1 and F2 and F3 show the order of flocculation, F1 being the first
tube to flocculate and F3 the last .
The concentration of toxin in the tube in which flocculation was first
initiated was designated LfFF. The concentration of toxin in the tube in
which flocculation was initiated second was designated LfF.
Calculation was done as follows:
Lf toxin / ml = LfFF - (LfFF – LfF ) when LfFF > LfF
LfFF +(LfF – LfFF ) when LfFF < LfF
3.2.3. Purification of tetanus toxin:- (According to Shone and Tranter,
1995)
Culture supernatant fluid obtained by filtration was the starting
material.
-To a stirred culture supernatant fluid at 4˚C, solid ammonium
sulphate was added slowly to 43% saturation (250 g / liter). After stirring
for a further 30 minutes the resulting precipitate was recovered by
centrifugation 16 000 x g for 10 minutes and dissolved in 10 m M sodium
MATERIAL AND METHODS
﴿ ﴾
57
phosphate buffer pH 7.4. The ammonium sulphate precipitation was then
repeated on this solution, raising final concentration to 46 %. The
resulting pellets dissolved in 10 mM sodium phosphate buffer pH 7.4 and
dialysed thoroughly against the same buffer.
3.2.4. Measuring protein by Bradford method: (According to
Bradford,1976 and Walker, 2002)
The Bradford assay relies on the binding of the dye Coomassie
Blue G 250 to protein. The binding of the dye to protein causes a shift in
the absorption maximum of the dye from 465 to 595 nm, and it is the
increase in absorption at 595 nm which is monitored.
Reagents: The assay reagent was made by dissolving 100 mg of
Coomassie Blue G 250 in 50 ml of 95% ethanol. The solution was then
mixed with 100 ml of 85% phosphoric acid and made up to one liter with
distilled water. The reagent should be filtered through filter paper and
then stored in an amber bottle at room temperature. It is stable for several
weeks. Protein standard is BSA (bovine serum albumin) solution contain
2mg / ml of protein. Samples and standard were diluted with distilled
water.
-Ten microliters of standard or sample were pippeted into each
well in the microplate using fixed-volume pipette.
-Two hundred microliter of dye reagent were added into each well
using the multichannel pipet. The sample/standard was thoroughly mixed
with dye by depressing the plunger repeatedly to mix the reagent and
MATERIAL AND METHODS
﴿ ﴾
58
sample in the well. The microplate was incubated at room temperature for
5-60 minutes and the absorbance at 595nm was measured using a
microplate reader.
The absorbance values of the standard were plotted versus their
corresponding protein concentration to prepare a calibration curve
(Fig.1). The protein concentration of the samples was determined from
the calibration curve.
Fig. ( 1 ) Bovine serum albumin standard curve.
* Chromatography of purified tetanus toxin: (according to Page and
Thorpe,1998)
- The sephadex G-100 was soaked with Tris-HCl, pH 8 containing 1 M
NaCl buffer overnight.
- The Pharmacia column (40×2 cm) was packed by sephadex G-100 after
its soaking.
- Purified tetanus toxin was mixed with blue dextran dye and loaded on
MATERIAL AND METHODS
﴿ ﴾
59
the top of the column and 34 fractions were collected (about 4-6 ml of
each fraction) fractionation was performed using LPLC system (pump
model Mini Plus3,detector uv/VIS-151 FC 203B,Gilson)
- The absorbance of the fractions was measured at 280 nm (A 280 ).
- Fractions with high protein content were collected and concentrated
using ethylene glycol.
3.2.5. Digestion of tetanus toxin: (According to Robinson, 1988 and
Goretzki and Habermann,1985)
-To purified chromatographed toxin in 0.1 M phosphate buffer pH
6.5 containing 1 m Na EDTA, 10 mM of cysteine- HCl, about one u of
papain /one mg protein of toxin was added in about one-tenth of the
volume of the toxin solution.
- The mixture was incubated at 45 ˚C for 1hr, after which the temperature
was raised to 55˚C for 2 hrs .
-the mixture was then cooled and chromatographed in a column of
sephadex G-100 and eluted with Tris-HCl, pH 8 containing 1 M NaCl.
* Chromatography of digested tetanus toxin: (according to Page and
Thorpe, 1998)
- The sephadex G-100 was soaked with Tris-HCl, pH 8 containing 1 M
NaCl buffer overnight.
- The Pharmacia column (40×2 cm) was packed by sephadex G-100 after
its soaking.
- The digested tetanus toxin was mixed with blue dextran dye and loaded
on the top of the column and fractions were collected (about 4-6 ml of
MATERIAL AND METHODS
﴿ ﴾
60
each fraction) fractionation was performed using LPLC system (pump
model Mini Plus3,detector uv/VIS-151 FC 203B,Gilson)
- The absorbance of the fractions was measured at 280 nm (A 280 ).
3.2.6. Evaluation of fragment C:-
3.2.6.1. Polyacrylamide gel electrophoresis in SDS buffer (According to
Oconnor, 2006).
* Preparation and pouring of the separating gel (10%):
a- The vertical slab gel unit was assembled in the casting mode by
using the 1.5 mm spacers.
b- The prepared solution of the separating gel 10% was pipetted in to
the previously prepared spacer to the level about 4-5 ml from the
top.
c- Degassing was done using a vacuum pump for 3 minutes.
d- Ten µl TEMED was added to initiate polymerization.
e- The running gel was poured using a Pasteur pipette up to the level
of the pen mark
f- A thin layer of butyl alcohol was added on the top of the gel to
avoid contact with oxygen immediately after one hour. It is better
to be left overnight for ripening of the gel.
g- The gel was washed with deionized water and the assembly was
inverted to drain.
* Preparation of the stacking gel (4%):
1- Degassing was done using a vacuum pump for 3 minutes.
2- Five µl TEMED were added to initiate polymerization.
MATERIAL AND METHODS
﴿ ﴾
61
3- After polymerization, about 1-2 ml of the stacking gel solution
was poured onto the surface of the polymerized gel.
4- A clean Teflon comb was inserted immediately into the staking
gel.
5- The gel was placed in a vertical position at room temperature
and left for polymerization for one hour.
6- Before removing the comb a mark at the bottom of each well
was made on the glass plate that help to locate the wells during
sample loading , wells were washed thoroughly with deionized
water.
7- The wells and the upper electrode chamber were filled with tank
buffer.
* Preparation of the samples and markers:
1) Dilution of each sample of tetanus toxin and its fragments with
equal volume of 1x sample loading buffer.
2) Dilution of the SpectraTM Multicolor Broad Range protein ladder
of ten bands (10, 15, 25, 35, 40, 50, 70, 100, 140 and 260 kDa)
with equal volume of 1x sample loading buffer.
3) The previous mixture was boiled for 5 minutes in a water bath
then the samples and markers were kept to be cold.
* Loading and running the gel:
1)Ten µl of the marker were applied in the first well while 20 µl of
each sample was applied in each well by using Hamilton syringe.
2) The gel was removed from the assembly pouring stand and
placed in the running apparatus.
3) The remainder of the tank buffer was poured into the lower
MATERIAL AND METHODS
﴿ ﴾
62
electrode chamber.
4) A lid was placed on the top of the lower buffer chamber to
enclose the cell.
5) The gel was electrophoresed at a constant current of 80 mA per
gel for 4 hours ± 30 minutes in vertical slab gel electrophoresis
apparatus until the tracking dye had migrated 1 cm down the length
of the separation gel.
* Protein visualization:
1) After electrophoresis, the current was switched off, the cell lid
was removed carefully, the tank was poured off and the gel
assembles were removed from the tank and the plates were
separated.
2) The gel was transferred carefully to an appropriate tray and
stained overnight in a staining solution with gentile agitation.
3) De staining was done in the de staining solution (methanol\
acetic acid solution) with gentle shacking for 30-45 minutes.
Destaining was repeated several times (3-4 times) till the
background of the gel become clear and the protein bands were
obvious.
4) The gel was exposed to UV lamp to detect protein bands and
the results were analyzed using a program (Alpha Ease Fc Stand
Alone v.3.1.2).
The gel was then preserved in 20% glycerol.
3.2.6.2 Toxicity test:
Several dilutions (0.1, 0.2, 0.3, 0.4, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1
mg in 0.5 ml) of fragment C in 1% peptone saline were injected into 3
MATERIAL AND METHODS
﴿ ﴾
63
mice weighing 18-22 g. Deaths were recorded on the 4th day.
3.2.7. Production of antitetanic serum by using crude tetanus toxin:
-Horses used for routine antitetanic serum production were used.
They received increasing amounts of alum precipitated tetanus toxin
every week. They receive 1200 Lf in the 1st week, 2000 Lf in the 2nd
week, 3000 Lf in the 3rd week, 4000 Lf in the 4th week , 5000 Lf in the 5th
week, 5600 Lf in the 6th week then horses were bled after 9 days.
3.2.8. Purification of antitetanic serum:
3.2.8.1.Preparation of IgG:- (According to Guidolin et al., 1997)
Ammonium sulfate: A volume of 24% (w/v) ammonium sulfate
solution (ASS) was slowly added to a volume of plasma under constant
agitation. After addition of 0.3% (v/v) of toluol, the pH of the mixture
was adjusted to 6.92 (with HCl 2.5 M). The mixture was then submitted
to stirring during one hour at room temperature and maintain at rest
overnight at 5 ± 1º C.
After eliminating the precipitate, 50% (w/v) ASS was added to the
mixture until it reached a final concentration of 33%. The pH was
adjusted to 6.95 (with NaOH 5N) and the mixture was submitted to
stirring during two hours at room temperature and maintained at rest
overnight at 5 ± 1º C. The precipitate was then collected, washed twice
with 30% (w/v) ASS, and dissolved in a 0.85% NaCl solution (SS)
corresponding to, approximately, 50% of the plasma initial volume. Then,
the material was dialysed until negative to the presence of ammonium
sulfate using barium chloride solution. After the pH was adjusted to 5.4
with a 10% (v/v) solution of acetic acid, the serum was maintained at rest
overnight at 5 ± 1º C .
MATERIAL AND METHODS
﴿ ﴾
64
3.2.8.2. Preparation of IgG by caprylic acid (According to Rojas et al.,
1994):-
Two parameters will be studied in order to find optimal conditions:
caprylic acid concentration and pH.
Caprylic acid concentration:
caprylic acid was added drop wise directly to 50 ml of undiluted
crude plasma whose pH has been adjusted to 4.5 by the addition of 1.76
N acetic acid. Caprylic acid was added up to a final concentration 1%,
2%, 3%, 4%, 5%, 6%,7%, 8% ( v/v) followed by vigorous stirring for one
hour at room temperature before filtration. The mixture was filtered
through Whattman filter paper. The filtrate was dialyzed for 48 hours
against distilled water. Afterwards, NaCl and tricresole were added to a
final concentration of 0.15M and 0.35%, respectively. pH was adjusted to
7.2 by adding of 1N NaOH. The preparation was sterilized by filtration
through 0.22-µm membranes.
pH: 50 ml of plasma were prepared and their PH adjusted to 4.0, 4.5,
5.0, 5.5, 5.8, 6.0, 6.5, 7.0 and 7.5 with 1.76 N acetic acid. Then caprylic
acid was added to give a final concentration of 5% and plasma was
fractionated.
3.2.8.3. Preparation of F(ab')2 : (According to Jones and Landon,
2003)
Crude tetanus antisera was filtered and allowed to equilibrate to
room temperature.
-Fourty ml of antisera was adjusted slowly to pH 3.0, 3.25, 3.5, 4.0, 4.5
MATERIAL AND METHODS
﴿ ﴾
65
and 5.0 with 0.36 M HCl while mixing with a magnetic stirrer.
*Pepsin digestion:-
-Acidified antisera was warmed in a water bath at 37˚C for 30 min.
Pepsin was added to give a ratio of 4294 enzyme units per ml of original
antiserum while mixing on a magnetic stirrer. Then allowed on stirrer for
5 min and the digestion was allowed to proceed for 18-24 hr at 37 ˚C.
-The digestion was stopped by adjusting the pH to 6.0 with the Piperazine
base solution while mixing .
-The mixture was centrifuged at 2750 x g (4-12˚C).
- Then the precipitate was dialysed against buffer A.
3.2.8.4. Preparation of F(ab')2 : (According to Salwa et al.2003)
pH of crude plasma was adjusted to 3.3 by 1.76 N acetic acid
followed by the addition of 3.5g pepsin/liter plasma. Digestion was
performed at 22-25ºC for 1 hour, and the pH was elevated to 3.6 using 1N
NaOH for 30 min. Afterwards plasma pH was readjusted to 5.8 and the
mixture was incubated for 15 min. at 56ºC followed by centrifugation for
10 min. at 900 x g to remove fibrinogen. Caprylic acid was added drop
wise to the undiluted plasma to obtain a final concentration of 5% (v/v).
The mixture was stirred vigorously for 24 h, The mixture was filtered .
The filtrate was dialyzed for 48 hours against phosphate buffered saline
(PBS) at pH 7.2 to remove caprylic acid. Afterwards, NaCl and tricresole
were added to a final concentration of 0.15M and 0.35%, respectively.
The preparation was sterilized by filtration through 0.22-µm membranes.
MATERIAL AND METHODS
﴿ ﴾
66
3.2.8.5.Preparation of Fab fragments:(According to Smith et al.1992
and Walker, 2002)
Fab antitoxin was obtained by cleaving samples of IgG with
minimal albumin contamination and retained the binding capacity of the
whole serum with papain in a concentration of 1:20. L-cysteine and
EDTA were added in a concentration of 2 µm to activate papain.
Digestion was performed at 37ºC for4 hours, 6 hours and 24 hours. The
reaction was terminated by addition of iodoacetamide in a final
concentration of 0.03M.
3.2.9. Evaluation of the prepared fragments(IgG, Fab2 and
Fab):- I-Protein concentration was determined ( according to Bradford,1976).
II-Albumin concentration determination: (Diamond Diagnostics)
*Principle:
Albumin in the presence of bromocresol green at a slightly acid
pH, produces a color change of the indicator from yellow green to green-
blue. The intensity of the color formed is proportional to the albumin
concentration in the sample.
*Procedures:
- Spectrophotometer was adjusted to 630 nm.
-The following volumes were pipetted in the cuvette.
Blank Standard Sample
R2(ml) 2.5 2.5 2.5
Standard(µl) _ 10 _
sample(µl) _ _ 10
MATERIAL AND METHODS
﴿ ﴾
67
- Mixed and incubated for 10 minutes at room temperature.
- The absorbance (A) of the samples and standard was read
against the blank.
*Calculations:
(A) sample
× 4 (standard concentration) = g/dl
(A) standard
III-Turbidity: Was assessed by recording absorbance at 600 nm.
Iv - Determination of Lf (Limits of flocculation) value.
V -SDS-PAGE for the purified serum fragments:-(According to
Oconnor, 2006)
The purified serum solution was separated by SDS-PAGE, by
pouring 10% separating gel in the electrophoresis unit, then 4% stacking
gel poured on the separating gel and the serum solution was applied
across the length of the lane and electrophoresed, the gel was then stained
by coomasie blue stain overnight and the stain then was substituted by the
destain. The gel was then preserved in 20% glycerol.
VI- ELISA for assaying the yield of each fragment(IgG, Fab2 and Fab):
( According to Maloy, 1990).
-The microtitre plates were coated with 100 µl of tetanus toxoid
containing 60 µg/ml as protein in carbonate-bicarbonate buffer (9.6 pH),
incubated at 4oC overnight.
-The plates were washed three times with washing solution.
-The plates were blocked by adding 100µl of blocking buffer per well,
MATERIAL AND METHODS
﴿ ﴾
68
then to be incubated at 37oC for one hour.
-Then the plates were washed as mentioned above.
-Tested serum samples (IgG, Fab2 and Fab) were diluted to 1/1000, then
were added in 100µl to microtitre plates.
-Also, negative serum of dilution 1/1000 was added as same as serum
samples which were used as control.
-Standard positive serum containing 1500 IU/ml were diluted in washing
buffer to give concentration of 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 and 1
IU/ml, respectively, then 100µl from each concentration were added to
microtitre plates.
-Each of serum samples, negative control and standard positive serum
were added in duplicated wells.
-After that, the plates were incubated for one hour at 37oC, then washed
as described above.
-Anti-horse IgG conjugated horseradish peroxidase was diluted to
1:60,000 then 100 µl was added to the microtitre plates.
-Then, the microtitre plates were incubated at 37oC for one hour, then
washed as mentioned above.
- substrate buffer 100 µl (O-Phenylene diamine) were added and the
colour was allowed to develop for 15-20 minutes after being kept in room
temperature in dark place.
-The reaction was stopped by addition of 50 µl of stopping buffer per
well.
-The absorbance was measured at 490 nm.
MATERIAL AND METHODS
﴿ ﴾
69
VIII- The protective activity :( According to Vaz et al. 1988)
Protective activity of the antitetanus preparations was determined
from the percentage of survival of mice. Tetanus was induced in groups
of 8 mice (18-22 gm) by injection into the right hind leg of 4 MLD of
tetanus toxin. After 24 hours mice were injected into the left hind leg with
different doses (1, 5, 10, 15, 20 and 25 I.U) of antitoxin IgG, F(ab')2 or
monovalent Fab'.
RESULTS
﴿ ﴾ 70
4.RESULTS I -Purification of tetanus toxin from culture filtrate using
ammonium sulphate. The obtained results in table (1) show that toxin produced from
culture filtrate (70 Lf/ml) by precipitation with ammonium sulphate; in a
two step procedure first subjected to ammonium sulfate in a concentration
of 43% then 46%, it gave toxin 2000 Lf /ml , 16.4 mg /ml protein content
and 2.624 mg protein Nitrogen per ml.
RESULTS
﴿ ﴾ 71
Table No. (1) Purification of tetanus toxin from culture
filtrate using ammonium sulphate.
mg protein
per ml
mg protein
Nitrogen
per ml
Lf/ml
Crude
Toxin
46.96
13.1
70
Purified
toxin
16.4
2.624
2000
RESULTS
﴿ ﴾ 72
II - Chromatography of tetanus toxin The purified toxin sample (about 7 ml) was filtered by 0.22 µm
filter and chromatographed on a column of Sephadex G-100 and yield 34
fraction (4-6 ml each)and their absorbance was measured at wave length
280 nm.
Result in table (2) and figure (2) show a peak of tetanus toxin at
fractions number 12-17.
RESULTS
﴿ ﴾ 73
Table No. (2) Chromatography of tetanus toxin
Fraction number OD at 280 nm
1 0.119 2 0.049 3 0.08 4 0.205 5 0.237 6 0.245 7 0.242 8 0.261 9 0.33
10 0.488 11 0.72 12 1.051 13 1.638 14 2.923 15 5.74 16 5.48 17 2.17 18 0.526 19 0.191 20 0.117 21 0.111 22 0.083 23 0.045 24 0.059 25 0.012 26 0.01 27 0.02 28 0.015 29 0.016 30 0.007 31 0.016 32 0.032 33 0.059 34 0.024
RESULTS
﴿ ﴾ 74
Fig.(2) Chromatography of tetanus toxin
01234567
1 4 7 10 13 16 19 22 25 28 31 34
fraction number
(OD
) abs
orba
nce
280
RESULTS
﴿ ﴾ 75
III -Chromatography of papain digested tetanus toxin
Fractions number 12-17 were collected, concentrated then
digested by papain enzyme and chromatographed on a column
of sephadex G- 100. Results in table (3) show the presence of 4 fractions (9-12) which
had high protein content and gave the following readings on 280 nm
absorbance: 3.59, 6.27, 8.15, 2.94.
RESULTS
﴿ ﴾ 76
Table No. (3) Chromatography of papain digested tetanus
toxin
Fraction number OD at 280 nm
1 0.026 2 0.006 3 0.024 4 0.149 5 0.568 6 0.974 7 1.15 8 1.7 9 3.59
10 6.27 11 8.15 12 2.94 13 0.36 14 0.05 15 0.032 16 0.032 17 0.028 18 0.017 19 0.006 20 0.009 21 0.011 22 0.006 23 0.006 24 0.01 25 0.1 26 0.001 27 0.002
RESULTS
﴿ ﴾ 77
IV-SDS polyacrylamide gel electrophoresis of tetanus toxin
digestion fragments. Fractions number 9, 10, 11 and 12 resulted from chromatography
of digested tetanus toxin were subjected to SDS polyacrylamide gel
electrophoresis 10% to determine its molecular eight.
Results in fig. ( 3 ) revealed that lane one and two is the native
tetanus toxin with a molecular weight of approximately 150 kDa and lane
number 3 have a molecular weight of 100 kDa and lane number 4 have a
molecular weight of 48 kDa.
RESULTS
﴿ ﴾ 78
Fig.( 3 ) SDS polyacrylamide gel electrophoresis of tetanus
toxin digestion fragments
M Standard Spectra Multicolor protein marker
(260, 140, 100,70, 50,40,35,25,15,10)
Lane 1 Fraction No. 9
Lane 2 Fraction No. 10
Lane 3 Fraction No. 11
Lane 4 Fraction No. 12
RESULTS
﴿ ﴾ 79
V - Toxicity test of tetanus toxin and fragment C The toxicity of fragment C is measured by injecting several
dilutions (0.1, 0.2, 0.3, 0.4, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mg protein in
0.5 ml) made in 0.15 M NaCl containing 0.5% peptone into hind leg of 3
mice. Animals were observed for 4 days.
Tetanus toxin in 0.1 mg (0.5 ml) was injected for comparison.
Result in table (4) indicated that fragment C is atoxic even at 1 mg
protein while tetanus toxin contain 1.2 ×106 MLD/ml.
RESULTS
﴿ ﴾ 80
Table No. (4): Toxicity test of tetanus toxin and fragment C
Mg protein
administered
Toxicity
MLD/mg protein
Tetanus toxin 0.1 mg (0.5 ml) 1. 2 × 106
Fragment C 0.1 mg
0.2 mg
0.3 mg
0.4 mg
0.5 mg
0.6 mg
0.7 mg
0.8 mg
0.9 mg
1.0 mg
No toxicity
,, ,,
,, ,,
,, ,,
,, ,,
,, ,,
,, ,,
,, ,,
,, ,,
,, ,,
RESULTS
﴿ ﴾ 81
VI -Preparartion of IgG using different concentrations of
caprylic acid.
Caprylic acid in different concentrations 1-8% is used at pH 4.5.
Results in table (5) reveal that digestion of antitetanic serum with caprylic
acid at 3% gave the highest protein content; 75.5 mg/ml and that 8% gave
the lowest protein content; 12.4 mg/ml. Caprylic acid at 2% gave the
highest albumin content; 2.91, while 5% and 1% gave the lowest albumin
content; 1.31 mg / ml.
Caprylic acid at 4% gave the highest titer 520 Lf/ml and 1%
gave the lowest titer; 300 Lf/ml while 8% gave the highest turbidity; 0.98
and 2% gave the lowest turbidity 0.47.
RESULTS
﴿ ﴾ 82
Table No. (5): Preparation of IgG using different
concentrations of caprylic acid.
Protein mg/ml
Albuminmg/ml
Lf/ml Turbidity ELISA titer
1 % 50.67 1.31 300 0.48 210.2
2 % 52.3 2.91 440 0.47 410.56
3 % 75.5 1.83 430 0.58 390.25
4 % 44.5 1.78 520 0.70 450.12
5 % 35.5 1.31 400 0.090 410
6 % 36.4 1.40 500 0.84 510.10
7 % 25.5 1.42 420 0.888 500.23
8 % 12.4 1.44 310 0.98 470.33
Ammonium sulphate
29.6 1.97 400 0.176 430.1
Crude serum 81.1 27.5 660 0.280 700
RESULTS
﴿ ﴾ 83
VII - Preparation of IgG using caprylic acid at different pH
values. Results in table No. (6) show that caprylic acid digestion at pH
4.25 gave the highest protein content ; 50 mg/ml and pH 5.8 gave the
lowest protein content ; 27.4 mg/ml
It also point that pH 4.0 gave the highest albumin content; 3.96
and pH 5.8 gave the lowest albumin content; 0.52 mg /ml.
Caprylic acid digestion at pH 5.0 gave the highest titer; 540
Lf/ml and that pH 4.0 gave the lowest titer; 300 Lf/ml by flocculation
test and by ELISA test pH 5.0 gave the highest ELISA titer; 410.32 and
that pH 7.0 and 7.5 gave the lowest ELISA titer; 70. pH 4.0 gave the
highest turbidity; 0.137 and pH 7.0 gave the lowest turbidity; 0.052.
Ammonium sulphate precipitation of antitetanic serum produce
1.97 mg/ml albumin and the resultant supernatant is very turbid; 0.176
when read at 600 nm
RESULTS
﴿ ﴾ 84
Table No.(6): Preparation of IgG using caprylic acid at
different pH values.
Protein
mg/ml Albumin mg/ml
Lf /ml Turbidity ELISA titer
pH 4 36.4 3.96 300 0.137 270.23
pH 4.5 35.5 1.31 400 0.090 410
pH 5 30.96 1.04 540 0.087 410.32
pH 5.5 32.2 0.93 500 0.057 300.56
pH 5.8 27.4 0.52 520 0.056 230.72
pH 6 30.3 0.96 500 0.056 200.63
pH 6.5 30.5 1.00 480 0.057 90.31
pH 7 28.3 1.12 400 0.052 70.41
pH 7.5 36.8 1.25 400 0.080 70.5
Ammonium sulphate
29.6 1.97 400 0.176 430.1
Crude serum 81.1 27.5 660 0.280 700
RESULTS
﴿ ﴾ 85
VIII - Preparation of F(ab)2 using pepsin enzyme at different
pH values The pepsin enzyme is used in a fixed concentration and 6 different
pH values (3, 3.25, 3.5, 4, 4.5, 5 ) to digest antitetanic horse serum.
The results obtained in table (7) indicate that pH 3.25 has the lowest
protein content ; 0.82 and the lowest albumin content 0.13while digestion
at pH 4.0 gave the lowest turbidity; 0.54 and digestion at pH 4.5 gave the
highest titer 590 Lf /ml.
RESULTS
﴿ ﴾ 86
Table No. (7): Preparation of F(ab)2 using pepsin enzyme
at different pH values.
Protein mg/ml Albuminmg/ml
Lf /ml Turbidity ELISA Titer
pH 3 1.914 0.40 200 0.057 150
Ph 3.25 0.82 0.13 270 0.056 430
pH 3.5 3.35 1.25 320 0.056 450
pH 4 2.01 0.26 560 0.054 510
pH 4.5 3.22 1.04 680 0.081 590
pH 5 0.948 0.34 200 0.055 170
RESULTS
﴿ ﴾ 87
IX - SDS-PAGE of F(ab)2 and IgG products of antitetanic
serum The purification of serum samples using caprylic acid or pepsin
is monitored by SDS-PAGE. SDS is performed out at 10 % acrylamide.
SDS results in Fig.(4) and table No.(8) show that pepsin digestion
produce F(ab)2 (molecular weight of about 98 ± 3 kDa) at pH 3.25 with
less contaminants.
The result in Fig. (4 and 5) and table No.(8 and 9) also show
that caprylic acid digestion at pH 5.8; lane 23 and
pH 5.5; lane 10 give IgG (molecular weight of about 169-178 kDa) with
little albumin contamination.
RESULTS
﴿ ﴾ 88
Fig. (4) SDS of F(ab)2 and IgG products of antitetanic
serum. (lanes 1-13)
M Standard protein marker Lane 1 F(ab)2 by pepsin digestion at pH 3.0
Lane 2 F(ab)2 by pepsin digestion at pH 3.25
Lane 3 F(ab)2 by pepsin digestion at pH 3.5
Lane 4 F(ab)2 by pepsin digestion at pH 4.0
Lane 5 F(ab)2 by pepsin digestion at pH 4.5
Lane 6 F(ab)2 by pepsin digestion at pH 5.0
Lane 7 IgG by caprylic acid digestion at pH 4.0
Lane 8 IgG by caprylic acid digestion at pH 4.5
Lane 9 IgG by caprylic acid digestion at pH 5.0
Lane 10 IgG by caprylic acid digestion at pH 5.5
Lane 11 IgG by caprylic acid digestion at pH 6.0
Lane 12 IgG by caprylic acid digestion at pH 6.5
Lane 13 IgG by caprylic acid digestion at pH 7.0
RESULTS
﴿ ﴾ 89
Table No. (8): Analysis of SDS-PAGE results of IgG and F(ab)2
Lanes marker lane1 lane 2 lane 3 lane 4 lane 5 lane 6 Lane7 lane 8 lane 9 lane 10 lane 11 lane 12 lane 13
Rows Mol.w. r1 238.84 r2 210 205.35 205.35 175.55 176.55 178.55 175.36 179.36 173.76 178.36
r3 98.11 99.77 98.66 101.46 93.818 96.48 95.406 95.94 98.64 102.61 89.211 91.23 95.406
r4 78 72.73 71.29 81.116 r5 71.29 r6 55 51.44 52.38 52.38
r7 48.7 48.406 48.406 49.29 50.2 r8 45 45.55 r9 40.08 38.68 36.671 42.09 r10 34 34.92 34.45 35.23 35.54
32.811 33.4 34.15
r11 27.21 32.08 31.94 28.58 29.88
26.62 26.73
RESULTS
﴿ ﴾ 90
Fig. (5): SDS of F(ab)2 and IgG products of antitetanic
serum. (lanes 14-25).
M Standard protein marker
Lane 14 IgG by caprylic acid digestion at pH 7.5
Lane 15 IgG by caprylic acid digestion at 2% concentration
Lane 16 IgG by caprylic acid digestion at 3% concentration
Lane 17 IgG by caprylic acid digestion at 4% concentration
Lane 18 IgG by caprylic acid digestion at 5% concentration
Lane 19 IgG by caprylic acid digestion at 6% concentration
Lane 20 IgG by caprylic acid digestion at 7% concentration
Lane 21 IgG by caprylic acid digestion at 1% concentration
Lane 22 IgG by caprylic acid digestion at 8% concentration
Lane 23 IgG by caprylic acid digestion at pH 5.8
Lane 24 IgG by caprylic acid digestion at pH 4.5
Lane 25 Ammonium sulphate purified antitetanic serum
RESULTS
﴿ ﴾ 91
Table No. (9): Analysis of SDS-PAGE results (lanes 14-25)
25 24 23 22 21 20 19 18 17 16 15 14 M Rows (mol.w.) (mol.w.) (mol.w.) (mol.w.) (mol.w.) (mol.w.) (mol.w.) (mol.w.) (mol.w.) (mol.w.) (mol.w.) (mol.w.) (mol.w.) r1 215.39 217.41 212.02 214.71 215.39 213.37 216.06 216.73 r2 212.02 208.65 211.35 207.31 210 208.65 209.33 210 208.65 210 r3 199.9 201.24 199.9 198.55 199.22 199.9 199.22 199.9 r4 173.63 173.63 173.63 177 175.65 177 177 173.63 179.69 r5 169.59 172.96 172.29 172.96 170.27 r6 131.88 135.92 r7 125.14 127.16 125.14 124.47 123.12 122.45 121.1 121.1 120.43 121.1 122.45 123.8 r8 110.33 115.04 114.37 113.69 115.04 115.04 r9 78 r10 55 r11 50.714 50.429 50.429 50.429 51.571 50.714 51.286 51 50.714 51.286 50.143 50.429 r12 44.833 44.167 44.5 45.571 46.143 45.571 44.5 45 r13 40.5 r14 34
RESULTS
﴿ 92 ﴾
X- Preparation of F(ab)2 using pepsin enzyme and caprylic
acid. F(ab)2 was prepared by pepsin digestion at pH 3.5 then
precipitated by caprylic acid 5%.
The results in table (10) show a comparison between F(ab)2 prepared by
pepsin digestion only at pH 3.25 and F(ab)2 prepared by pepsin+ caprylic
acid which give higher protein content (2.61mg/ml) and higher albumin
(0.72 mg/ml) with lower yield 45 Lf/ml and more turbid solution.
RESULTS
﴿ 93 ﴾
Table No. (10):- Preparation of F(ab)2 using pepsin enzyme
and caprylic acid.
F(ab)2 with
Protein mg/ml
Albumin mg/ml
Lf/ml Turbidity ELISA Titer
Pepsin +caprylic
acid
2.61 0.72 45 0.075 30
Pepsin only
at pH 3.25
0.82 0.13 270 0.056 430
RESULTS
﴿ 94 ﴾
XI - Preparation of F(ab) fragment by papain digestion at
different digestion time. The obtained result in table (11) indicate that digestion of
antitetanic IgG (produced by caprylic acid digestion at pH 5.8 and 5%
concentration) with papain in 4 hours gave higher protein content; (15.92
mg/ml) and at 24 hours gave the least protein content; (9.3 mg/ml). At 6
hours it gave the least albumin contamination;(0.19 mg/ml) and at 24
hours gave the highest albumin contamination; (0.37mg/ml). Digestion
for 4 hours gave the highest titer both by flocculation and by ELISA (300
Lf/ml and 200.1 ELISA titer), while at 24 hours gave the least titer(180
Lf/ml and 50.5 ELISA titer)
RESULTS
﴿ 95 ﴾
Table No. (11): Preparation of F(ab) fragment by papain
digestion at different digestion time.
Protein
mg/ml Albumin mg/ml
Titer Lf/ml ELISA titer
F(ab) at 4 hr 15.92 0.32 300 200.1
F(ab) at 6 hr 13.30 0.19 250 110.3
F(ab) at 24 hr 9.3 0.37 180 50.5
IgG (starting material)
27.4 0.52 520 230.7
RESULTS
﴿ 96 ﴾
Fig. (6) SDS-PAGE for papain digested serum
M Standard protein marker
Lane 1 Digested serum with pepsin and caprylic acid.
Lane 2 F(ab) by papain digestion for 4 hour
Lane 3 F(ab) by papain digestion for 6 hour
Lane 4 F(ab) by papain digestion for 24 hour
Lane 5 Crude antitoxin serum
Lane 6 Ammonium sulphate purified antitoxin serum
RESULTS
﴿ 97 ﴾
Table No. (12) Analysis of Fig. (6)
M Lane1 Lane2 Lane3 Lane4 Lane5 Lane6 r1 260 r2 r3 213 213 r4 187 r5 166 r6 162 155 r7 140 144 r8 130 r9 124 127 r10 112 r11 100 96 88 100 r12 70 64 r13 r14 60 r15 r16 50 52 r17 40 48 48 49 35 22
RESULTS
﴿ 98 ﴾
XII -Evaluation of the Protective activity of prepared IgG,
F(ab)2 and F(ab). Groups of mice were injected with 4 MLD tetanus toxin and after
24 hours injected with 1, 5, 10, 15, 20 and 25 IU per mice from each
fragment.
The results in table (13) indicate that in case of IgG injecion all
mice died in the dose of 5 IU and only 25% survived at 10 IU. While
75% of mice survived when injected with either 20 or 25 IU.
In case of F(ab)2 injection 75% of mice survived with 5 IU, 20 IU and 25
IU, while F(ab) gives 50% protection with 1 IU and 100% protection in
the dose 15-25 IU.
RESULTS
﴿ 99 ﴾
Table No.(13): Percentage of survival of mice treated with IgG, F(ab)2 or F(ab) 24 hr after the inoculation of tetanus toxin.
IgG F(ab)2 F(ab) Antitoxin(LF)
S/D Survival
%
S/D Survival
%
S/D Survival
%
1 0/8 0 % 0/8 0 % 4/8 50%
5 0/8 0 % 2/8 25 % 6/8 75%
10 2/8 25 % 4/8 50 % 6/8 75%
15 4/8 50 % 6/8 75% 8/8 100%
20 6/8 75 % 6/8 75 % 8/8 100%
25 6/8 75 % 6/8 75 % 8/8 100%
DISCUSSION
﴿ ﴾
101
5. Discussion
Although preventive medicine has progressed in recent decades,
tetanus infection remains a life-threatening condition and is still an
important health issue worldwide. Tetanus is caused by Clostridium
tetani which is an anaerobic, motile, gram positive rod found worldwide
in the soil, as well as in animal and, occasionally, human feces. Although
tetanus is ubiquitous, infections in certain developing regions of the
world are associated with high mortality and morbidity largely because of
a lack of rigorous immunization programs and available treatment
options. Consequently, tetanus has become one of the target diseases of
the World Health Organization (WHO) Expanded Program on
Immunization. ( Hatamabadi et al., 2010).
Tetanospasmin or neurotoxin is one of the most known powerful
exotoxins known. This heat-labile protein (molecular weight 150,000 kD)
is produced by growing cells and released during autolysis. The
neurotoxin is responsible for the characteristic spastic paralysis of
tetanus.(Carter and Darla ,2004).
The common tetanus toxin vaccines for active immunization
contain almost exclusively an antigen which has been prepared by
inactivation of tetanus toxin with formaldehyde. This substance (toxoid)
is provided with a large number of antigenic determinants, only a few of
which is important, however for the production of antibodies that protect
against tetanus. The elimination of those determinants which are not
DISCUSSION
﴿ ﴾
102
required for protection is desirable in order to obtain antigenic and/or
immunogenic substances of which- due to a narrower spectrum of
determinant groups- an increased specificity and an improved
compatibility may be expected. (Duffy, 1980)
Tetanus toxin may be degraded by papain. This enzyme splits a
polypeptide bond approximately in the middle of the heavy chain subunit,
yielding fragment C, corresponding to the carboxy terminal portion of the
heavy chain, with a molecular weight of about 47,000 dalton and
fragment B, comprising the N-terminal part of the heavy chain and the
entire light chain polypeptide, with a molecular weight of 95,000. Peptide
C can bind to ganglioside, show retrograde transport in axons, is atoxic,
and a good immunogen producing antibodies which neutralize tetanus
toxin. ( Burns, 2002).
The present study was directed toward two main goals including
the preparation of tetanus toxin with higher titer to prepare highly
immunogenic peptide C and preparation of purified antitetanic serum
using different methods with a comparative evaluation of the efficacy of
antitetanic whole IgG, F (ab)2 and F(ab).
Within the present study tetanus toxin fragment C was prepared
and assessed. Tetanus toxin as the starting material for preparation of
fragment C was obtained by cultivation of Clostridium tetani in a
modified Muller and Miller (1954) medium followed by isolation as
culture filtrate. The culture filtrate was purified by ammonium sulphate in
a two step procedure, 43% then 46% precipitation.
DISCUSSION
﴿ ﴾
103
The tabulated results in table (1) showed that the purified tetanus
toxin contains about 2000 Lf/ml , 16.4 mg protein/ml and 2.624 mg
protein Nitrogen / ml, such toxin was further purified by gel filtration
chromatography on a column of sephadex G-100.
Gel filtration chromatography (also called size exclusion
chromatography) was used to separate protein molecules according to
size. In gel filtration, a protein mixture (the mobile phase is applied to a
column of small beads (Sephadex G-100) with pores of carefully
controlled size (the stationary phase). The movement of the solute is
dependent on the flow of the mobile phase, and the Brownian motion of
the solute molecules causes their diffusion into and out of the
chromatographic bed. Large proteins, above the exclusion limit of the gel,
will not enter the beads and so move with the advancing solute front,
while small molecules enter the beads and must traverse this space as
well as the volume around the beads. Proteins are therefore eluted in
order of decreasing molecular size. A very large dye that cannot enter the
gel, called dextran blue is used to determine the point at which fractional
measurements should be taken of the eluant. (Page and Thorpe,1998)
Fractions eluted from chromatography were monitored by
measuring absorbance value at wave length 280 nm, The obtained result
in table (2) and fig. (2) showed that the toxin was eluted in fractions
number 12-17 with reading at 280 of 1.051, 1.638, 2.92, 5.74, 5.48 and
2.17 respectively.
DISCUSSION
﴿ ﴾
104
The fractions mentioned above were collected and concentrated by
ethylene glycol 6000 to 5ml and then digested by papain enzyme in the
presence of L-cysteine and EDTA as inactivating agents for the enzyme.
The demonstrated results in table (3), revealed that purified tetanus
toxin digested by papain yield 27 fraction on chromatography by
sephadex G-100 column. Fractions number 9-12 when read at 280 nm
showed the readings: 3.59, 6.27, 8.15 and 2.94 which were subjected to
SDS-PAGE.
On SDS-PAGE 10% tetanus toxin has a molecular weight of about
150 kDa and fragment C has a molecular weight of about 48 kDa as
shown in fig.(3). These results agree with the findings of Helting and
Zwisler, (1977). who produced fragment C with a molecular weight of
47±5% , Goretzki and Habermann, (1985), Caleo and Schiavo, (2009)
who pointed out that fragment C has a molecular weight of 50 kDa and
Weller et al. ,(1989) who showed that fragment C has a molecular
weight of 52.1 kDa.
To study the toxicity of fragment C; several dilutions were
prepared in 0.15 M NaCl containing 0.5% peptone starting from 0.1 mg
to 1 mg protein and injected into hind leg of mice .The obtained results in
table (4) showed that fragment C is atoxic even in concentrations as high
as 1 mg ,while the native tetanus toxin is lethal in 0.1 mg and lower
concentrations. These results come in agreement and supported by those
of Fishman et al. (1992), He et al. (2000) who pointed out that
immunization of mice with fragment C resulted in the production of
DISCUSSION
﴿ ﴾
105
antibodies that were able to protect mice against a challenge with tetanus
toxin, furthermore Francis et al. (2004) stated that fragment C is non-
toxic and could be considered as a vector to enhance delivery of
potentially therapeutic proteins to motor neurons from the periphery
following an intramuscular injection.
Antibodies, also know as immunoglobulin are proteins that are
used by the immune system to identify and neutralize foreign structures,
such as bacteria and viruses. Because of versatility of antibodies,
antibody based therapies may be developed against any pathogen. The
serum therapy was firstly described in 1890. In the next years, antibodies
were largely produced and used to control a wide range of infectious
disease (Wang et al., 2010)
IgG, the main serum antibody and the intact format almost
exclusively used in therapeutic antibodies, is a Y-shaped, multidomain
protein with antigen-binding sites located on the two Fab tips and
recruitment of effector functions mediated by the stem Fc domain.
To date, animal derived therapeutic antibodies represent the best
and only choice source of antitoxins, especially in developing countries.
Furthermore, this industry needs to develop a production protocol to
achieve safer products.(Redwan, 2006)
During the present study, antibody purification was carried out
using different techniques in order to optimize the purification protocol to
yield the highest specific antibody concentration and purity.
DISCUSSION
﴿ ﴾
106
Caprylic acid has been used for the purification of IgG from serum
and ascitis fluid. Although the molecular basis of this fractionation
remains unclear, it has been shown that caprylic acid precipitates non-
IgG serum proteins under particular physicochemical conditions, leaving
in solution a highly enriched IgG preparation (Rojas et al., 1994).
Several factors influence the precipitation of IgG by caprylic acid
of which pH and caprylic acid concentration seem to be the most striking
parameters, So different concentrations of caprylic acid at fixed pH; 4.5
(recommended by Michella and Parkinson, 1987)and different pH of
the plasma at a fixed caprylic acid percent of 5% were studied.
For comparison, IgG purified by precipitation of antitetanic serum
with ammonium sulphate was prepared.
In defining optimal conditions for antitoxin production, variables
such as yield, turbidity, protein and albumin concentrations and
electrophoretic profile in samples of antitoxin fractionated with caprylic
acid were taken into consideration.
Results in table (5) revealed that digestion of antitetanic serum
with caprylic acid at 3% gave the highest protein content (75.5 mg/ml)
and that 8% gave the lowest protein content (12.4 mg/ml). Caprylic acid
at 2% gave the highest albumin content (2.91 mg/ml) and 6% gave the
lowest albumin content (1.31 mg/ml).
DISCUSSION
﴿ ﴾
107
Caprylic acid at 4% gave the highest titer (520 Lf /ml) and 1%
gave the lowest titer ( 300 Lf /ml). Caprylic acid at 8% gave the highest
turbidity 0.98 and 2% gave the lowest turbidity 0.47.From the above
mentioned result it is concluded that caprylic acid at a concentration of
5% and pH 4.5 is considered the best concentration. These results agree
with the result of Rojas et al., (1994) and disagree with the findings of
Redwan et al., 2005 who used caprylic acid at a concentration of 15%.
Such agreement and disagreement could be attributed to the followed
technique used for preparation of antitetanic serum, the used animal
species, the animal health condition and the time of serum collection post
animal inoculation.
In case of using different pH in caprylic acid precipitation of non
IgG compounds, results in table (6) show that caprylic acid digestion at
pH 7.5 gave the highest protein content (36.8 mg/ml) and pH 5.8 gave the
lowest protein content ( 27.4 mg/ml). It also point that pH 4.0 gave the
highest albumin content; 3.96 and pH 5.8 gave the lowest albumin
content; 0.52. Caprylic acid digestion at pH 5.0 gave the highest titer; 540
Lf/ml and that pH 4.0 cave the lowest titer; 300 Lf/ml by flocculation test
and by ELISA test pH 5.0 gave the highest titer; 410.32 , while pH 7.0
and 7.5 gave the lowest titer; 70.41 and 70.5 respectively, pH 4.0 gave
the highest turbidity; 0.137 and pH 7.0 gave the least turbid fluid; 0.052.
On balancing between albumin content, protein content and
yield, pH 5.8 was suggested to be the optimum pH for caprylic acid
fractionation of hyperimmune horse plasma.
DISCUSSION
﴿ ﴾
108
Ammonium sulphate purification has been used for purification
of antitetanic serum in most laboratories during the last decades, but the
result in table (6) show that caprylic acid is superior to yield, albumin
contamination and turbidity. This finding is in accordance with Butler,
(1972), who speculated that short chain fatty acid may stabilize the
antibodies against loss of activity and also agree with the findings of
Redwan et al.,( 2005), but disagree with the results of Mohanty and
Elazhary,(1989) who suggest that IgG prepared by ammonium sulphate
precipitation is better than that obtained by caprylic acid method in terms
of yield and recovery of antibody activity.
These results are supported by the data obtained from SDS –
PAGE analysis in table No. (9) and fig.(5). The data indicate that pH 5.8
gave the best purity with IgG at a molecular weight of 173.63 - 169.59
kDa and no protein aggregates with higher molecular weights are present
, less albumin contamination and less low molecular weight digestion
products. These results agree with the findings of Salwa et al., (2003)
who adjusted plasma at pH 5.8 before using caprylic acid to precipitate
non IgG fractions after pepsin digestion and disagree with the results of
Rojas et al.,( 1994), Michella and Parkinson,( 1987) who choose pH
5.5 and 4.5 respectively as optimum for the digestion of hyperimmune
horse plasma with caprylic acid.
The Fc domain recruits cytotoxic effector functions through
complement and/or through interactions with γFc receptors (Fc receptors
for gammaglobulins) and can provide long serum half-lives (>10 days)
through interaction with the neonatal Fc receptor (FcRn), which acts as a
DISCUSSION
﴿ ﴾
109
salvage receptor (binding and transporting IgGs in intact form both within
and across cells and rescuing them from a default degradative pathway).
There is a range of applications, however, in which the Fc-mediated
effects are not required and are even undesirable. For example, a long
serum half-life results in poor contrast in imaging applications, and
inappropriate activation of Fc receptor–expressing cells can lead to
massive cytokine release and associated toxic effects. To remove the Fc
domain (and associated effects), IgGs have been dissected into
constituent domains, initially through proteolysis (with such enzymes as
papain and pepsin) and later genetically engineered into either
monovalent (Fab, scFv, single variable VH and VL domains) or bivalent
fragments (Fab′2, diabodies, minibodies, etc.). (Holliger and Hudson,
2005)
The pepsin enzyme is used in a fixed concentration and 6 different
pH values (3, 3.25, 3.5, 4, 4.5, 5 ) to digest antitetanic horse serum.
The results obtained in table (7) indicate that pH 3.25 has the lowest
protein content ; 0.82 and the lowest albumin content 0.13while digestion
at pH 4.0 gave the lowest turbidity; 0.54 and digestion at pH 4.5 gave the
highest titer 590 Lf/ml.
pH 3.25 was chosen as the best pH for pepsin digestion due to its
lower albumin and protein content so it means less allergens and less
anaphylactic reactions. It also has the highest titer by flocculation and
elisa tests. These results are also supported by data obtained from SDS-
PAGE as shown in fig.(4) and table (8).These results come in close
agreement with Salwa et al (2003) who used a pH of 3.3 to produce
DISCUSSION
﴿ ﴾
110
F(ab)2 and disagree with the result of Jones and Landon (2003) who
proposed a pH of 3.5 to be optimum for the production of F(ab)2 .
Combination between both pepsin digestion and caprylic acid
precipitation resulted in a very poor yield as shown in table (10) and data
of SDS-PAGE in fig. (6) and its analysis in table (12) as lane 1 which
also show that the procedure result in excess digestion and it result in the
production of F(ab) fragment (molecular weight approximately 48-52
kDa) along with the F(ab)2 (molecular weight approximately 96 kDa).
In order to compare the protective activity of intact IgG, F(ab)2, and
F(ab) Against the lethal action of tetanus toxin, F(ab) was produced by
limited papain digestion at a ratio of 1:20 but the time of digestion needs
to be optimized so the digestion is performed for 4 , 6 and 24 hours.
As shown in table (11) and fig.(6) it is seen that digestion for 4
hours gave the best yield of 300 Lf /ml and 200 ELISA titer but it has
more protein and albumin than the digestion at 6 hr and 24 hr.
Digestion for 24 hour gives the lowest yield; 180 IU/ml and the
highest albumin contamination. So digestion for 6 hours is considered to
be the best time for production of F(ab) as it balance between titer and
purity. These results were also supported by the results of SDS-PAGE in
fig. (5) and table (12) which indicated that F(ab) molecule is represented
by one band about 48-49 kDa.
DISCUSSION
﴿ ﴾
111
F(ab) doesn't induce anaphylaxis because it has only one binding
site and doesn't crosslink to form immune complex. This gives an
advantage to F(ab) preparations.
The results in table (13) demonstrate the percentage of survival of
mice treated with different doses of IgG, F(ab)2 or F(ab) 24 hr after the
inoculation of 4 MLD of tetanus toxin. This dose was chosen because it
reproduce in mice the course of the disease induced by 10 MLD of spores
(Smith and MacIver, 1975).
It was found that IgG at a dose of 5 IU did not protect mice and at
10 IU protect only 10% of the mice, even at a dose of 25 IU didn’t
achieve complete protection against tetanus toxin.
On the other hand F(ab)2 at a dose of 5 IU protect only 25% of
mice ,but at 10 IU protect 50% of mice and didn’t give full protection
even at 25 IU
The result also indicate that F(ab) induced 50% protection with 1
IU and 100% protection in the dose 15-25 IU.
So it is clear that F(ab) fragment is more protective than F(ab)2 and
IgG. These results agree with the findings of Smith et al.,(1992) and Vaz
et al. (1988) and disagree with the findings of Redwan, (2005) who
demonstrated that F(ab) fragment is less immunogenic than F(ab)2 and
IgG .
DISCUSSION
﴿ ﴾
112
Conclusion:
From the present results it could be concluded that:
-Purified tetanus toxin from culture filtrate can be fragmented with papain
enzyme after gel chromatography to yield 4 fractions containing fragment
C.
- Fragment C which has a molecular weight of about 48 kDa is atoxic
when inoculated into mice at a dose of 1 mg.
- IgG antitetanic serum is the best achieved using caprylic acid at pH 5.8.
- F(ab)2 can be produced in a good yield;( 270 Lf at pH 3.25) using
pepsin enzyme.
-Digestion of IgG for 6 hours with papain enzyme yield F(ab) fragment in
a good yield.
- F(ab) fragment is more protective against tetanus toxin than F(ab)2 and
IgG.
SUMMARY
﴿ ﴾
113
Recent studies on toxins and serum of Clostridium tetani
Tetanus disease is one of the most dramatic and globally prevalent
disease of humans and vertebrate animals. The global fatality rate of
tetanus has been estimated as 30-50%. Fortunately, it is successfully
controlled through immunization with tetanus toxoid. Hyperimmune
serum obtained from sheep or horse confers effective protection in
unimmunized animals and humans.
The results of the experiments included in the present study showed
that:
1- Tetanus toxin was produced in a modified Mueller and Miller (1954),
media and gave a titer of 70 Lf/ml.
2- Toxin was purified from culture filtrate by ammonium sulphate and
produce an Lf content of 2000 Lf/ml, protein content of 16.4 mg/ml and
2.624 mg protein Nitrogen per ml.
3- Purified tetanus toxin was further purified by chromatography on
sephadex G-100 and yield one peak.
4- purified toxin was digested by papain enzyme and then applied to
chromatography on sephadex G-100 to separate the yield of digestion.
6. Summary
SUMMARY
﴿ ﴾
114
5- Fragment C was inoculated in mice to assess its toxicity and was
atoxic even in large amounts.
6- Antitetanic serum was fractionated to yield IgG by two method;
ammonium sulphate and caprylic acid.
7- Caprylic acid was used in different concentrations and the best
concentration was 5% which produce good yield and good purity.
8- Caprylic acid was used in different pH values and the best pH was 5.8
9- F(ab)2 was produced by pepsin alone and by pepsin and caprylic acid
and resulted in that the best pH was 3.25 and that pepsin alone give better
yield and better purity.
10- F(ab) was produced by limited papain digestion for different time and
was found that 6 hours give a good balance between yield and purity.
11- IgG, F(ab)2 or F(ab) were compared for their protective capacity
against 4 MLD of tetanus toxin and it was found that F(ab) give the best
protection.
From the above mentioned study we conclude the following:-
- Tetanus toxin can be digested with papain enzyme and
chromatographed to yield fragment C.
- Fragment C with a molecular weight of approximately 48 kDa is atoxic
when injected in mice .
SUMMARY
﴿ ﴾
115
-IgG is best produced from antitetanic horse serum by caprylic acid at 5%
concentration and at pH 5.8.
- Pepsin enzyme produce F(ab)2 in good yield ;270 IU at pH 3.25.
- Twenty five units of F(ab) give 100% protection to mice injected with 4
MLD of tetanus toxin, while IgG and F(ab)2 produce 75% protection at
the same dose.
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١
********************
دول الم وبخاصة في ال ع انحاء الع شار في جمي يعد مرض التيتانوس من األمراض الواسعة األنت
ه ضها النامي ي بع ا ف د متوطن ي يع انوس . والت اه بالتيت سبة الوف غ ن ال % ٥٠-٣٠وتبل ل األطف يمث
د التيتانوس للحاالت يمكن السيطره على مرض التيتانوس باستخدام التحصين بتوآسي . منها% ٥٠
األآثر تعرضا لالصابه وباستخدام مصل التيتانوس المحضر في الخيول واألغنام آعالج للحاالت
.المصابه وآوقايه قبل العمليات الجراحيه
-:تم في هذه الدراسهة وحدة ٧٠(ة سمي ىو قد اعط ١٩٥٤ر ــــلر و ميل وميديا م إنتاج توآسين التيتانوس على - /تندفي
. ) مللي
ة - انوس تنقي سين التيت ة الت توآ د سلفات األب سيب ر باستخدام طريق وم و ق سي أعطىموني و آ نالت
ة٢٠٠٠ دة تندفي ي / وح ي ملل وى بروتين م١٦٫٤و محت م/ ملج ي ث صل ت تنقيملل طة الف ة بواس
.الكروماتوجرافي الزالة الشوائب
ة- انوس تجزئ سين التيت ابين ث توآ زيم الب تخدام ان ى باس ة م المنق زاء الناتج صل األج طة ف بواس
.الفصل الكروماتوجرافي
ام في فئران لحقنه ) ج(و بعد ذلك تم اخذ الجزء - ر س التجارب لتقييم مدى سميته وقد وجد انه غي
).ملجم١( حتى عند الجرعات العاليه
ه من سيرم التيتانوس الخيلي بواسطة بعد ذلك تجزئة تم - زات مختلف ك ترآي د حمض الكابريل وق
و ز ه ضل ترآي د ان اف تخدامو، %٥وج دروجيني باس ات اس هي د درج ك عن حمض الكابريل
.٥٫٨مختلفه وجد ان افضل درجه هي
٢
اج ٣٫٢٥ آما اتضح ان درجة اس هيدروجيني - سين وانت زيم البيب هي الدرجه الفضلى لعمل ان
F(ab)2 من سيرم التيتانوس الخيلي.
اج الجزء ساعات هي الفترة ال٦ فترة - ابين F(ab) مناسبة النت زيم الب حيث اعطت باستخدام ان
اقل نسبة البومين
تعطي F(ab) وجد ان على الحمايه من توآسين التيتانوس F(ab)2,IgG , F(ab)قدرة قارنة مب -
.حمايه فقط% ٧٥ التي تعطي F(ab)2,IgG وحده دوليه بعكس٢٥تخدام س عند احمايه%١٠٠
-:نتج ما يليومن هذه الدراسة نستذه األجزاء س يمكن تجزئة توآ - بواسطة الفصل ين التيتانوس بعد تنقيته الى عدة اجزاء وفصل ه
.الكروماتوجرافي
. آيلو دالتون غير سام عند حقنه في فئران التجارب٤٨ذو الوزن الجزيئي ) ج( الجزء -
ي IgG أفضل طريقه ألنتاج- انوس الخيل ز هي باستخدام ح من سيرم التيت ك بترآي مض الكابريل
.٥٫٨وعند درجة اس هيدروجيني % ٥
.٣٫٢٥عند درجة اس هيدروجيني F(ab)2 انزيم البيبسين يعمل بطريقة افضل ألنتاج -
. وحده دوليه٢٥عند استخدام من توآسين التيتانوس% ١٠٠يحمي بنسبة F(ab) الجزء-