Monoclonal Antibodies and their applications

A specific and single type of antibody that is produced by hybridoma cells.
This technique was achieved by George Kohler and Cesar Milstein in 1975. (Nobel Prize, 1984)
Antibodies (or) immunoglobulins is a protein - produced by B- lymphocytes (Plasma cell) in mammals.
Antibodies used by immune system to identify and neutralize foreign objects like bacteria and viruses.
Each antibody recognizes a specific antigen unique to its target.

1. Production of Monoclonal Antibodies ( MAbs ) and their Applications (Hybridoma Technology) By Dr. S.SELVARAJ Assistant professor Dept. of Zoology Vivekananda College Tiruvedakam West 1

2. Synopsis Introduction Principle Production of MAbs Immunization Cell fusion Selection of hybridomas Screening the products Cloning and propagation Characterization and storage Applications of MAbs Diagnostic Applications Therapeutic Applications Protein purification Miscellaneous Applications References 2

3. A specific and single type of antibody that is produced by hybridoma cells. This technique was achieved by George Kohler and Cesar Milstein in 1975. (Nobel Prize, 1984) A ntibodies (or) immunoglobulins is a protein - produced by B- lymphocytes (Plasma cell) in mammals. A ntibodies used by immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target. 3 Introduction

4. Fusion between myeloma cell ( malignant plasma cell) and spleen cell from suitable immunised animal. Myeloma cells - Growth and multiplying B- lymphocytes- Secrete their own antibodies Myeloma cells cannot synthesize their own antibodies because, lack of HGPRT enzyme. Mutant myeloma cell can not grow in HAT medium. Only hybrid cell can grow in HAT medium and produce monoclonal antibody. Principle 4 Hypoxanthine Guanine phosphoribosyl Transferase

5. 5 Production of Monoclonal Antibodies ( MAbs )

6. Production of MAbs 1. Immunization Immunize the animal with appropriate antigen Inject multiple site and repeated several times B-lymphocytes stimulation increased against antigen Antibodies synthesized against specific antigen Spleen is aseptically removed Spleen cells are removed using mechanical (or) enzymatic B-lymphocytes of spleen cells are separated 6

7. 2. Cell fusion Fusion of B-lymphocytes to myeloma cells is induced using polyethylene glycol (PEG) After few minutes 1. Mixture of Hybridoma (Fused cells) 2. Free myeloma cells 3. Free lymphocytes (The production of hybrid cells is r eferred to as H ybridoma technology ) 7

8. 3. Selection of Hybridoma Cells are cultured in HAT medium (Specific) Hybridoma cells only grow , other cells slowly disappear (7-10 days of culture) Select the single antibody producing hybrid cells Hybridomas are isolated and grown individually 8 Hyphoxanthine Aminopterin Thymidine that blocks one pathway for nucleotide synthesis Cells lacking HGPRT enzyme, grown in HAT medium die

9. Screened the desired antibody producing cells Two techniques namely ELISA and RIA In both the assays, the antibody binds to the specific antigen and other components can be washed off Hybridoma cells producing the desired antibody can be identified by screening. Antibody secreted by the hybrid cells is referred to as monoclonal antibody. 4. Screening the products 9

10. Desired antibody are isolated and cloned Two techniques are commonly used for cloning hybrid cells 1. Limiting dilution method – Serial dilution method 2. Soft agar method – Semisolid medium- to form colonies 5.Cloning and propagation 10

11. Biochemical and biophysical characterization for desired specificity MAbs of the immunoglobulin class or sub-class , the epitope for which it is specific and the number of binding sites it possesses. Desired cell lines are frozen in liquid nitrogen for future uses. 6.Characterization and storage 11

12. Applications of MAbs 1. Diagnostic applications Biochemical analysis for pregnancy, cancer, hormonal disorder. Diagnostic imaging for myocardial infarction, bacterial infections. 2. Theraputic applications Treatment of cancer, Asthma, Viral infections. 3. Protein purification by immunoaffinity techniques. 4. Miscellaneous applications as catalytic agents, in autoantibody fingerprinting . 12

13. https://www.slideshare.net/YashikaSaini7/monoclonal-antibody-146466667 . https://www.slideshare.net/ArchanaSoni3/hybridoma-technology-93942798. Dr . U. Satyanarayana (2011) Biotechnology, Books and Allied (P) Ltd. Kolkata, India. References 13