prof. dr. ali mohamed soliman el-ged prof. dr. … · veterinary serum and vaccine research...

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

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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

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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


﴾ 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


﴾ 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


﴾ 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


﴾ 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.


﴾ 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


﴾ 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


﴾ 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


﴾ 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


﴾ 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.


﴾ 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


﴾ 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


﴾ 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.


﴾ 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


﴾ 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.


﴾ 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


﴾ 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.


﴾ 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


﴾ 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


﴾ 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-


﴾ 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


﴾ 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


﴾ 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


﴾ 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


﴾ 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


﴾ 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


﴾ 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


﴾ 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


﴾ 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.


﴾ 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


﴾ 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


﴾ 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.


﴾ 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


﴾ 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.


﴾ 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


﴾ 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


﴾ 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


﴾ 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


﴾ 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.


﴾ 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


﴾ 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


﴾ 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.


﴿ ﴾

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


﴿ ﴾

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.


﴿ ﴾

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.


﴿ ﴾

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.


﴿ ﴾

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


﴿ ﴾

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


﴿ ﴾

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


﴿ ﴾

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)


﴿ ﴾

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


﴿ ﴾

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


﴿ ﴾

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


﴿ ﴾

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


﴿ ﴾

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


﴿ ﴾

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


﴿ ﴾

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.


﴿ ﴾

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


﴿ ﴾

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


﴿ ﴾

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 .


﴿ ﴾

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


﴿ ﴾

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.


﴿ ﴾

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


﴿ ﴾

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,


﴿ ﴾

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.


﴿ ﴾

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




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 7 IgG by caprylic acid digestion at pH 4.0







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 15 IgG by caprylic acid digestion at 2% concentration










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 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

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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

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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

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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

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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

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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

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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

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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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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-

prof. dr. ali mohamed soliman el-ged prof. dr. … · veterinary serum and vaccine research...

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