Hybridoma technology monoclonal antibodies

Hybidoma technology for production of monoclonal antibodies

1. PRODUCTION OF MONOCLONAL ANTIBODIES BY HYBRIDOMA TECHNIQUE VASANT KUMAR M.Sc. BIOTECHNOLOGY (3911)

2. HISTORY 1964 : A way is developed to isolate hybrid cell from two parent cell lines using the Hypoxanthine- aminoprotein - thymidine (HAT) selection media 1975 : Georges J.F. Köhler and and César Milstein used hybridoma to generate monoclonal antibodies. Awarded with Nobel prize ( 1984 ) 1975 : Leonard Herzenberg coined the term hybridoma. 1986 : First monoclonal antibody was licenced by FDA, Orthoclone OKT 3 ( muromonab - CD3 ) Which was approved for the use in Preventing kidney transplant rejection. 1988 : Greg Winter gave technique to Humanize monoclonal antibodies .

3. WHAT IS HYBRIDOMA TECHNOLOGY? Hybridoma are formed via fusion between a splenocyte cells with tumorous myeloma cells. It can divide continuously by the quality derived from myeloma cells. Expresses a large amount of one specefic Monoclonal antibodies . Can be Cryopreserved to meet never ending supply of important Mab .

4. MONOCLONAL ANTIBODIES Identical immunoglobulins , from single B-Cell clone of a single parent or a single hybridoma cell line. Targets a Single Epitope . They bind to Specefic diseased or damaged cells. Treat a wide range of medical conditions.

5. DIFFERENCE BETWEEN MONOCLONAL AND POLYCLONAL ANTIBODIES MONOCLONAL ANTIBODIES POLYCLONAL ANTIBODIES Homogenous population of antibodies. Mixture of immunoglobulin molecules. Produced by the same clones of plasma B cells. Produced by different clones of plasm a B cells. Interact with a particular epitope on the antigen. Interact with different epitopes on the same antigen.. Posses less cross reactivity. Posses comparatively high cross reactivity.

6. OUTLINE OF PRODUCTION OF MONOCLONAL ANTIBODIES

7. IMMUNIZATION SCHEDULE Desired protein should be in an adequate quantity (a few milligrams) and pure . Mice must be immunized with antigen 6–10 weeks before fusion. Collection of Pre-immune serum is required prior to immunization to use as a Baseline control for antibody screening. Immunization schedule includes Intra- peritonial injections of 2–4 adult mice ( eg , BALB/c mice) with 20–100 µg of purified antigen in a total volume of 200 µL ( ie , 200 µL of a 1:1 emulsion of antigen in saline: adjuvant ).

8. MYELOMA CELL LINE CULTURE Myeloma cells are cultured in presence of 8-azaguanine so they are unable to synthesize the HGPRT enzyme . By using Aminopterin , de novo synthesis pathways are blocked in myeloma cells (where salvage pathway was previously blocked) die. Hybridomas have a functional salvage pathway (derived from the spleen cells of mouse) and can grow Elective culture medium is HAT medium hypoxanthine, aminopterin , and thymidine

9. FUSION The Parental myeloma cells should be Mycoplasma -free and fuse well eg : SP2/0 and X63Ag8. 653 B cells are mixed with HGPRT negative myeloma cells and a fusing agent, such as Polyethylene glycol. Sendai virus is commercially available and still used in experiments with hybridoma production.

10. GROWTH AND SELECTION OF MONOCLONAL ANTIBODY 7–14 days after fusion, Interleukin 6(IL-6) is added ( Hybridoma growth factor .) Unfused myeloma cells die in presence of Aminopterin. Qualitative and Quantitative levels screened by flow cytometry or ELISA. Selected cultures are cloned and re-cloned to achieve a pure clone population.

11. LONG-TERM MAINTENANCE AND CRYO PRESERVATION OF MAB S Selected hybridomas are Cryo -preserved in Liquid Nitrogen in ampules . Evaluate the quality of the produced antibody regularly. ANTIBODY PRODUCTION AFTER FUSION Using Surface expanded tissue culture flasks or hollow fibre systems , such as Technomouse . Growing the hybridomas in mice as an Ascitic tumor. Yields ascitic fluid with antibody concentrations of nearly 1–5 mg/ mL .

12. APPLICATIONS OF MONOCLONAL ANTIBODIES DIAGNOSTIC TOOLS IN RESEARCH AND LABORATORY: a) Western blot, immunodot blot, ELISA, radioimmuno assay (RIA) etc. GENE CLONING: a) Probe for detecting those cells that make the product and therapy to detect the gene. TO IDENTIFY CELL TYPES: a) Various types of T-cells carry cell surface antigens on their surfaces. b) Helpful in defining changes in T and B-cells during development. PROTEIN PURIFICATION: a) MAb -antigen complex has a Single binding affinity it is possible to elute the required protein in a single.

13. DIFFERENTIAL THERAPEUTIC MAbs

14. CANCER DIAGNOSIS AND THERAPY Diagnosis of Solid tumors , particularly the carcinomas of the lung, breast, colon, and rectum. MAbs binds complement proteins, which leads to direct cell toxicity that is Complement Dependent Cytotoxicity (CDC). Block growth factors released by tumor cells by blocking Growth Factor Receptor.

15. AUTOIMMUNE DISEASES Used for autoimmune diseases include infliximab and adalimumab , which are effective in rheumatoid arthritis, Crohn’s disease, and Ulcerative Colitis. Due to their ability to bind to and inhibit Tumor Necrosis Factor (TNF), TNF-α. The first therapeutic MAb (murine IgG 2a CD3- specific) (FDA-approved) was a transplant rejection drug, OKT3 (or muromonab , orthoclone ) in 1986.

16. ANTIBODY ENGINEERING HUMANIZATION OF MURINE ANTIBODIES Chimeric antibodies with human constant region and mouse variable region were constructed. Transgenic mice containing human immunoglobulin germ line locus may be used as an alternative strategy for producing Human Mabs . Humira is the first fully human MAb drug used for treatment of rheumatoid arthritis and was launched in 2003.

17. SIDE-EFFECTS AND LIMITATIONS OF MAbs Side effects include fever, headache, weakness, chills, nausea with vomiting and diarrhea, and low blood pressure. Bevacizumab (used against tumor blood vessel growth) can have side effects such as Kidney damage. MAb therapies are a financial burden on patients.

18. REFERENCES Monoclonal Antibodies : a tool in clinical research Waliza Ansar1 and Shyamasree Ghosh - Indian Journal of Clinical Medicine Corresponding author email: [email protected] Monoclonal antibodies: Paul Nelson University of Wolverhampton , Gary Reynolds QE Hospital & University of Birmingham Article  in  Molecular Pathology · June 2000 https://www.researchgate.net/publication/264309886

19. THANK YOU  A Presentation by VASANT KUMAR STAY SAFE AND WEAR MASK