Raising Polyclonal Antibodies Against Mycobacterium Toxoid, in Laboratory Animals

RAISING POLYCLONAL ANTIBODIES AGAINST MYCOBACTERIUM TOXOID IN LABORATORY ANIMALS

PROTOCOL FOR M.PHARM

DISSERTATION

SUBMITTED TO

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA.

BY

AVAD P. R

M PHARM, PART –I,

DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY.

BHARATHICOLLEGE OF PHARMACY

UNDER THE GUIDENCE OF

Dr.GURUKAR MATHEW.S,Ph.D

PROFESSOR,

DEPARTMENT OF PHARMACEUTICAL BIOTECHNOLOGY,

BHARATHICOLLEGE OF PHARMACY,

BHARATHI NAGARA, MANDYA-571422.

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA.

Annexure-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

NAME OF THE INSTITUTION

COURSE OF STUDY AND SUBJECT

TITLE OF TOPIC

MATERIALS AND METHODS

ENCLOSURE- I

6. BRIEF RESUME OF THE INTENDED WORK

6.1 Need For Study

Polyclonal antibodies are typically produced by immunization of a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces the B-lymphocytes to produce IgGimmunoglobulin specific for the antigen. This polyclonalIgG is polyclonal purified from the mammal’s serum1.

Many methodologies exist for polyclonal antibody production in laboratory animals. Institutional guidelines governing animal use and procedures relating to these methodologies are generally oriented around humane considerations and appropriate conduct for adjuvant that is agents which modify the effect of other agents while having few if any direct effects when given by themselves use2. This includes adjuvant selection, routes and sites of administration, injection volumes per site and number of sites per animal. Institutional policies generally include allowable volumes of blood per collection and safety precautions including appropriate restraint and sedation or anaesthesia of animals for injury prevention to animals or personnel1.

The primary goal of antibody production in laboratory animals is to obtain high titer, high affinity antisera for use in experimentation or diagnostic tests. Adjuvants are used to improve or enhance an immune response to antigens. Most adjuvants provide for an injection site, antigen depot which allows for a slow release of antigen into draining lymph nodes.

Such antigens by themselves are generally poor immunogens. Most complex protein antigens induce multiple B-cell clones during the immune response, thus, the response is polyclonal. Immune responses to non-protein antigens are generally poorly or enhanced by adjuvants and there is no system memory8.

Recent investigation showed that polyclonal antibodies, which is raised against mycobacterium toxoid, was used in used to detect mycobacterium in tissue with the use of immunohistochemical techniques, as vitamins are must for the production of antibodies, the production of antibody can be enhanced by studying, immunizing animal, with vitamins, and without vitamins, and injecting along with adjuvants, and can be detected by the Method of ELISA.

Use of polyclonal antibodies against carcinogen-DNA adducts in analysis of carcinogenesis.

6.2 Review Of Literature

1. LI QY. etal., ExtractedGliadins from three wheat varieties, viz., Henan and Nongda, were used as antigens to immunize in BALB/C mice, and the corresponding polyclonal antibodies were prepared, designated as Anti-A, Anti-B, Anti-C, Anti-Aa, Anti-Ab, Anti-Ag and Anti-Av, respectively. Of seven polyclonal antibodies tested, three (Anti-Ag, Anti-Av and Anti-A) displayed significant positive or negative associations between antibody binding and dough development time, strength, valorimenter value and stability time, but no significant correlations were observed with water absorption. These results suggest that polyclonal antibodies could be used for rapid prediction and screening of wheat quality paraK4.

2. Folwarczna J. etal., Reported the gene encoding the coat protein (CP) of a potato virus Y (PVY) was cloned into expression vector pMPM-A4Ω. PVY CP was expressed in Escherichia coli and the purified recombinant protein was used for raising rabbit polyclonal antibodies. The sera and antibodies were tested for the detection of PVY in the laboratory host Nicotiana tabacum CV. Petit Havana SR1 and in various cultivars of the natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies can be used for the detection of the whole strain spectrum of PVY by indirect plate trapped antigen ELISA and Western blot, but not by double antigen sandwich ELISA5.

3. Michael K. Schunk et al., Found the Immunization introduces an external substance to induce the host immune system to respond specifically. Typically an antigen is used, but DNA, or a primed, pre-existing leukocyte or antigen presenting cell, can also be used. Immunization is currently being used or investigated for the prevention and treatment of infectious diseases, cancer, addictions, allergies, pregnancy, and autoimmune diseases. The choice of immunization protocols is complex, and results may be affected by many factors such as dose and concentration of antigen, choice of adjuvants, time between inoculation and response measurement, and method of detection. The immune system responses to an antigen are also complex and continue to develop with advancing age. Anatomical, physiological, and immune system differences between species influence responses to immunization, as do the purity and presentation of the antigens and adjuvants. When directly comparing results, animals should be sourced from the same supplier. This review highlights the many uses of immunization techniques and introduces important considerations for the choice of protocols and animal models6.

4. Padmavathy L, et al., A study was undertaken to evaluate the utility of immuno-histochemical staining to demonstrate Mycobacterium tuberculosis antigen in tissue sections. This is based on the finding that the mycobacterium antigen is the last to disappear from the tissues and thus can be used as a marker of mycobacterial infections. Mycobacterial antigen was demonstrable in majority of cases of cutaneous tuberculosis using polyclonal antiserum. This test by itself cannot be considered as diagnostic. The results should be considered along with other findings7.

5. Simona,Montietal M, etal., Fusaproliferin (FP), a toxic metabolite of the world-wide maize pathogens Fusarium proliferatum and Fusarium subglutinans, was recently found to be a natural contaminant of maize. FP-hemiglutarate conjugated to modified bovine serum albumin was synthesized, characterized, and used as an antigen for raising polyclonal antibodies by immunizing rabbits. Indirect and competitive ELISA and immunoblotting analyses were performed to determine antibody specificity towards the mycotoxin. The determination of 10 µg of free FP/mL was achieved using antibodies purified by means of affinity chromatography on a FP-lysine-Sepharose column. This unsatisfactory detection limit is due to high background values; thus, this method is not competitive with traditional UV-HPLC methods.

6. Deepa Arora, et al., Reported reduced antibody response to tetanus toxoid (‘VT) was previously demonstrated during vitamin A deficiency but the response to bacterial lipopolysaccharide (LPS) was normal. We addressed whether anti-TT IgG responses are enhanced in vitamin A-sufficient and deficient rats by immunization with LPS plus TI’. Antibody responses in vitamin A-sufficient and deficient rats increased significantly after co immunization8.

7. Pasatiempo, Immunized the rats with polysaccharide antigens from Streptococcus pneumoniaor Neisseria men ingitidis, antibody production did not exceed 0-19% of the response of control rats. Vitamin A depletion also severely compromised the response to two T cell-dependent antigens, tetanus toxoid and sheep red blood cells. In striking contrast, retinol-depleted rats immunized with lipopolysaccharides from Pseudomonas aeruginosa and Serratia marcesens produced an antibody response indistinguishable from retinol-sufficient animals. These lipopolysaccharides could elicit antibodies in rat pups, whereas the capsular polysaccharide antigens could not9.to homogeneity from bovine

8. Hardy D et al.,Found,mice (Sobey) bred to select lack of antibody production against bovine serum albumin have been examined for their responsiveness over a wide range of antigen doses. With low doses of antigen both Sobey mice and an out bred line of Swiss White mice produce similar amounts of antibody. Antigen in amounts greater than 500 yg produce tolerance more effectively in Sobey mice and this is the reason for their unresponsiveness at high antigen dose levels11.

6.3 Objective Of The Study

The Overall aim of the proposed study is to production of purified polyclonal antibodies, against, antigen of mycobacterium tuberculosis,

1)Isolation and identification of Mycobacterium tuberculosis from clinical isolates

2)Preparation of Antigen from identified microorganisms.

3)Purification of polyclonal antibodies.

4)Characterization of purified antibodies.

ENCLOSURE II

7. MATERIAL AND METHODS

Isolation of Mycobacterium tuberculosis from clinical isolates

Identification of isolated bacteria,

Cultural characteristics studies.

Staining techniques (Acid fast staining technique).

Biochemical tests Aneja et al., 2008.

Preparation of Antigen from identified microorganisms.

Immunization of antigen into animals (Standard methods)

Intravenously

Intramuscularly

Subcutaneously

Purification of polyclonal antibodies

Collection or drawing of blood from animals

Serum separation

Salt precipitation technique

Dialysis

Ion exchange chromatography

Affinity chromatography

Characterization of purified antibodies

Ouchterlony double diffusion tests

Single radial immuno diffusion assay

Immunoelectrophorosis

Rocket Immunoelectrophorosis

SDS PAGE

Detection of antigen

ELISA

7.1 Source Of Data

1. Bharathi College of Pharmacy library, Bharathi Nagara.
2. E-Library from BharathiCollege of Pharmacy.
3. IISC Library, Bangalore.

7.2 Method Of Collection Of Data

The preliminary data required for the experimental study were obtained from

1. Internet
2. Scientific Abstract
3. Scientific journal
4. Relevant books

7.3 Does the study require any investigation or interventions to be conducted on patients or other humans or animals? If so please mention briefly.

-Yes-

7.4 Has ethical clearance been obtained from your institution in case of 7.3?

Yes, attached intuitional ethical clearance certificate.

ENCLOSURE III

8. LIST OF REFERENCES

1. Jennings VM review of selected adjuvants used in antibody production, ILAR Journal1995,37;119-125.

1. Jackson LR and Fox JG institutional policies and guidelines on adjuvants and antibody production, ILAR Journal 1995;37:141-153.

1. Ana Maria G. Pasatiempo, Makiko Kinoshita, Christopher E. Taylor,T and A. Catharine Ross,Antibody production in vitamin A-depleted rats is impaired after immunization with bacterial polysaccharide or protein antigens, The FASEB Journal1990; 4:2518-2527.

1. LI Q Y , Ji KM, Song XY , Zhang EY , Pei YH et al., Polyclonal Antibodies to Grain Gliadins and Their Application in Wheat Quality Prediction Cereal Research Communications200836(1),117–124.

1. J. Folwarcznaa,c, H. Plchovaa, T. Moraveca, H. Hoffmeisterovaa, P. Dedicb, N. Cerovskaa Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato Virus Y Folia Microbiol 2008;53(5):438–442.

1. Michael K. Schunk, Eileen Macallum GApplications and Optimization of Immunization Procedures, ILAR Journal2005;46(3):247-257.

1. Padmavathy, Lakshmana Rao L, Ramanadhan and Shakilamycobacterial antigen in tissues in diagnosis of cutaneoustuberculosis,Indian Journal of Tuberculosis 2005;52:31-35.

1. Deepa Arora , A CatharineRos,Diano M, Antibody response against tetanus toxoid is enhanced by lipopolysaccharide or tumor necrosis factor-alpha in vitamin A-sufficient and deficient rats13 Am J Clin Nutr1994;59:922-8.

1. Arturo Casadevall Passive Antibody Administration Immediate Immunity as a Specific Defense against Biological Weapons, Emerging Infectious Diseases 2002;8(8).

1. Czeslaw Lugowski, Malgorzata Kulakowska, Elzbieta Enterobacterial Common Antigen-Tetanus Toxoid Conjugate as Immunogen romanowska infection and immunity1983; 42(3):1086-1091.

1. Ronald Bach, John Oberdick, Yale Nemerson Immunoaffinity Purification of Bovine Factor VII From on April 10, 2008.

1. Adler HE and Damassa AJ, Vitamin A Adjuvant with Arizona Hinshawii Bacterin, Applied microbiology1972;24(5)849-850.

1. Mysore Pandurangaraj Urs Babitha, Devaiah Madhu Harishchandra Sripathi Prakash Hunthrike Shekar Shanty Production of polyclonal antisera to manganese superoxide dismutase expressed in downy mildew resistant pearl millet and its application for immunodiagnosis,Electronic Journal of Biotechnology2006;(9)5.

1. Aneja .KR, experiments in microbiology and plant pathology and biotechnology new age international publishers2008.

1. Keith Wilson, John Walker practical biochemistry, principles and techniques, 2003; fifth Cambridge edition.

1. Guangjie Liu, Jianping Wang, Jianchun Xiao, Howe Zhao and Yongtang Zheng, Preparations and Characterization of Three Monoclonal Antibodies Against HIV-1 p24 Capsid Protein Cellular And Molecular Immunology2007;4(3):203-208.

1. Mar lies Leenaars PPA, Coenraad F.M. Hendriksen, Wim A. de Leeuw,

Florina Carat, Philippe Delahaut, René Fischer, Marlies Halder, W.The Production

Of Polyclonal Antibodies in Laboratory Animals, ATLA1999; 27: 79-102.

1. Monique Diano, Andrele Bivic The protein protocols Hand book2002;2:945-952.

1. Jennings VM, Fox JG, UC Berkeley Animal Care and Use Committee 2006;1-3.