Monoclonal antibody (MAb)

Topics included :- Introduction to monoclonal antibody; Principle for creation of hybridoma cells and steps involved in it; Second generation monoclonal antibodies; Advantages, disadvantages and applications (Diagnostic and therapeutic) of MAbs.

1. MONOCLONAL ANTIBODY ( MAb ) SUBJECT : IMMUNOLOGY BY- S. DASH

2. OVERVIEW :

3. INTRODUCTION : Monoclonal antibody is a single type of antibody that is directed against a specific antigenic determinant(epitope). Natural source : Found in patients suffering from multiple myeloma. In 1975, George Kohler and Cesar Milstein achieved a large scale production of MAbs . The pair won the 1984 Nobel Prize in Medicine and Physiology for this discovery. The production of monoclonal antibodies by the hybrid cells is referred to as Hybridoma Technology .

4. PRINCIPLE FOR CREATION OF HYBRIDOMA CELLS The mutated cells deficient in the enzyme HGPRT are grown in a medium containing hypoxanthine, aminopterin and thymidine (HAT medium), they cannot survive due to inhibition of de-novo synthesis of purine nucleotides. Thus cells lacking the HGPRT enzyme, grown in HAT medium die. The hybridoma cells possess the ability of myeloma cells to grow in vitro with a functional HGPRT gene obtained from lymphocytes (with which myeloma cells are fused). Thus, only the hybridoma cells can proliferate in HAT medium and this procedure is used for selection.

5. STEPS OF PRODUCTION OF MONOCLONAL ANTIBODIES Immunization Cell fusion Selection of hybridomas Screening of products Cloning and production Characterization and storage.

6. IMMUNIZATION The animal is immunized using an appropriate antigen. The antigen along with an adjuvant (mainly Freund’s complete) is injected subcutaneously. The injections are administered at multiple sites to increase the production of B-lymphocytes, which are responding to the antigen. Three days prior to the sacrifice of the animal, a final dose of antigen is given intravenously. This allows the synthesis of antibodies to it’s maximum level. The concentration of the desired antibodies is assayed in the serum of the animal at frequent intervals during the course of immunization. When the serum concentration of the antibodies is optimal, the animal is sacrificed. The spleen is aseptically removed and disrupted by mechanical or enzymatic methods to release the cells. The lymphocytes of the spleen are separated from the rest of the cells by density gradient centrifugation.

7. CELL FUSION The thoroughly washed lymphocytes are mixed with HGPRT defective myeloma cells. The mixture of cells is exposed to polyethylene glycol (PEG) for a short period of time. PEG is removed by washing and the cells are kept in a fresh medium. These cells are composed of a mixture of hybridomas (fused cells), free myeloma cells and free lymphocytes.

8. SELECTION OF HYBRIDOMAS Within the 7-10 days of culture, only the hybridoma cells are able to grow whereas the rest starts disappearing. Selection of a single antibody producing hybrid cells is very important. This is possible if the hybridomas are isolated and grown individually. The suspension of hybridoma cells is so diluted that the individual aliquots contain on an average one cell each. These cells, when grown in a regular culture medium, produce the desired antibody.

9. SCREENING OF PRODUCTS The hybridomas must be screened for the secretion of the antibody of the desired specificity. The two techniques namely ELISA and RIA are commonly used for this purpose. In both the assays, the antibody binds to the specific antigen and the unbound antibody and other components can be washed off. Thus, the hybridoma cells producing the desired antibody can be identified by screening.

10. CLONING AND PROPAGATION The single hybrid cells producing the desired antibody are isolated and cloned. Two techniques are commonly employed for cloning hybrid cells- Limiting dilution method Soft agar method LIMITING DILUTION METHOD The suspension of hybridoma cells are serially diluted and the aliquots of each dilution are put into microculture wells. The dilutions are so made that each aliquot in a well contains only a single hybrid cell. This ensures that the antibody produced is monoclonal. SOFT AGAR METHOD The hybridoma cells are cultured in soft agar. It is possible to simultaneously grow many cells in semisolid medium to form colonies. These colonies will be monoclonal in nature.

11. CHARACTERIZATION AND STORAGE The monoclonal antibody has to be subjected to biochemical and biophysical characterization for the desired specificity. It is also important to elucidate the Mab for the immunoglobulin class or sub-class, the epitope for which it is specific and the number of binding sites it possesses.

12. SECOND GENERATION MONOCLONAL ANTIBODIES By employing site-directed mutagenesis, cysteine residues are introduced at the predetermined positions on the Mab. These cysteine residues which facilitate the isotope labelling. This would be more useful in diagnostic imaging and radioimmunotherapy.

13. ADVANTAGES Monoclonal antibodies truly represent a homogenous state of a single molecular species. Each Mab is specific to a given antigenic determinant. DISADVANTAGES Laborious and time-consuming. Since they are produced against a single antigenic determinant, therefore they cannot differentiate the molecules well. Mab for human use should be totally free from all pathogenic organisms, including viruses, but since there is no guarantee it poses a great danger.

14. APPLICATIONS DIAGNOSTIC APPLICATIONS Biochemical analysis for the diagnosis of pregnancy, cancers, hormonal disorders, infectious diseases. Diagnostic imaging for the detection of myocardial infarction, deep vein thrombosis, cancers, atherosclerosis, bacterial infections. THERAPEUTIC APPLICATIONS Direct use as therapeutic agents to destroy disease causing organisms in the treatment of cancers, AIDS, autoimmune diseases and organ transplantation. As targeting agents in therapy as immunotoxins (for treatment of cancers) in drug delivery, for dissolving blood clots in radioimmunotherapy. PROTEIN PURIFICATION BY IMMUNOAFFINITY TECHNIQUES MISCELLANEOUS APPLICATIONS INCLUDES CATALYTIC AGENTS LIKE ABZYMES, USED IN AUTOANTIBODY FINGERPRINTING.

15. REFERENCES : Textbook of Biotechnology by U. Satyanarayana. Edition-2013 ; Publisher-Books & Allied Ltd. Textbook of Immunology ( Kuby ) by Judith.A . Owen, Jenni Punt, Sharon.A . Stanford and Patricia.P.Jones . Edition-7 th ; Publisher – W.H. Freeman and Company. www.Wikipedia.com www.genescript.com

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