Prof. Dr. ALI MOHAMED SOLIMAN EL-GED Prof. Dr. AHMED AHMED EL-BASIOUNY Prof. Dr ...

146 Pages·2011·1.23 MB·English

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)
٣٢ ﻪﻳﻷا :ةﺮﻘﺒﻟا ةرﻮﺳ
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Acknowledgement

First of all, many thanks to the All Merciful God, who gave me every
thing I have.
Dr. Ali
I wish to express my sincere appreciation and gratitude to
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.

Dr. Ahmed Ahmed El-
I wish to express my great gratitude to
Basiouny,
Chairman of Hygiene and Preventive Medicine, Kafr El-
Sheikh, Tanta University for his stimulating supervision, encouragement and
interest in this work.

Dr. Fayka Kamal Ahmed,
I am deeply indebted to Chief
Researchers of Microbiology, Veterinary Serum and Vaccine Research
Institute, Abbassia, Cairo for scientific advice and facilitation of requirements
for achieving this work.

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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
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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) …….... 64
2
3.2.8.4. Preparation of F(ab) (pepsin+caprylic)…….. 65
2
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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LIST OF TABLES

Table Title Page
1 Purification of tetanus toxin from culture filtrate using 71
ammonium sulphate.
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 82
Caprylic acid
6 Preparation of IgG using caprylic acid at different pH values. 84
7 Preparation of F(ab) using pepsin enzyme at different pH 86
2
values
8 analysis of SDS-PAGE results of IgG and F(ab) (lanes1-13) 89
2
9 Analysis of SDS-PAGE results of IgG and F(ab) (lanes 14- 91
2
10 Preparation of F(ab) using pepsin enzyme and caprylic acid. 93
2
11 Preparation of F(ab) fragment by papain digestion at different 95
digestion time.
12 Analysis of Fig.(5) 97
13 Percentage of survival of mice treated with IgG, F(ab) or 99
2
F(ab) 24 hr after the inoculation of tetanus toxin.

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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) and IgG products of antitetanic 88
2
serum(lanes 1-13)
5 SDS of F(ab) and IgG products of antitetanic serum. 90
2
(lanes 14-25)
6 SDS-PAGE for papain digested serum 96

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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
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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 (H ) binds
C
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

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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:
1-Production of highly potent tetanus toxin.
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.

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