Hybridoma technology

HYBRIDOMA TECHNOLOGY OVERVIEW

1. HYBRIDOMA TECHNOLOGY BY: K.Kavya Priya. M.Sc. Biochemistry. Semester III. Department of Biochemistry and Bioinformatics. GITAM Institute of science. GITAM University.

2. CONTENTS: HISTORY. INTRODUCTION. METHODOLOGY : CELL FUSION SELECTION SCREENING CULTURING APPLICATIONS.

3. HISTORY: The production of monoclonal antibodies was invented by César Milstein and Georges J. F. Köhler in 1975. They were awarded noble prize for their work on monoclonal antibodies in 1984.  The term hybridoma was coined by Leonard Herzenberg. Leonard Herzenberg.

4. WHAT IS HYBRIDOMA TECHNOLOGY??? Hybridoma technology is a method for producing large numbers of identical antibodies also called as monoclonal antibodies. In this technique monoclonal antibodies are generated by fusing B-cells with myeloma tumor cells which grow indefinitely in culture.  Such hybrid cell obtained by fusion of B-lymphocyte and myeloma cell is called a Hybridoma. HYBRIDOMA

5. The hybrid cell has the capacity of antibody production derived from B cells. It can divide continuously by the quality derived from Myeloma cells. By combining the desired qualities of both the cells, the technology ensures large scale antibody production of single specificity. Monoclonal antibodies

6. METHODOLOGY: Laboratory animals (mammals, e.g. mice) are first exposed to the antigen that an antibody is to be generated against. Usually this is done by a series of injections of the antigen, over the course of several weeks. These injections are typically followed by the use of in vivo  electroporation , which significantly enhances the immune response.  The spleenocytes from spleen and myeloma cells from bone marrow of the immunized mouse are isolated.

7. The fusion of the B cells with myeloma cells can be done using: Physical method: Electrofusion. Chemical method :  Polyethylene glycol. PEG mediated Cell fusion Electrofusion

8. Cell fusion leading to formation of Hybridoma cell.

9. Nucleotide synthesis: Nucleotide synthesis is essential for cell survival. The two p athways for nucleotide synthesis are : Denovo pathway . (synthesis of nucleotides from simple sugars, amino acids etc.) Salvage pathway . (synthesis of nucleotides from intermediate of nucleotide degradation pathways) An important enzyme of salvage pathway is HGPRT.

10. SELECTION: Process through which only hybridoma cells are selected by eliminating unfused myeloma cells and unfused B cells HAT medium is used for the selection of hybrid cells. Fused cells are incubated in HAT medium (hypoxanthine- aminopterin - thymidine  medium) for roughly 10 to 14 days. Elimination of unfused myeloma cells: Removal of the unfused myeloma cells is necessary because they have the potential to outgrow other cells, especially weakly established hybridomas The myeloma cells first selected for fusion are hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutant cells(raised by using 8-azaguanine which causes mutations ). Thus the unfused myeloma cells in the medium cannot carryout nucleotide synthesis through salvage pathway due to the deficiency of HGPRT.

11. Where as, Aminopterin blocks the denovo pathway. Hence, unfused myeloma cells die, as they cannot synthesize nucleotides. Elimination of unfused B cells: Unlike the unfused myeloma cells, the B cells can carryout nucleotide synthesis through Denovo pathway as they are not HGPRT mutants. But due to the lack of infinite cell division capability the B cells which survive in the HAT medium die within 7-10 days. Thus, only the Hybrid cells survive on HAT as they have HGPRT enzyme from B cells and the repeated cell division property of myeloma cells.  

12. SELECTION OF HYBRIDOMA

13. The incubated medium is then diluted into multi-well plates to such an extent that each well contains only one cell. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope , and are thus monoclonal antibodies. 96 WELL PLATE

14. SCREENING PROCESS: The next stage is a rapid screening process, which identifies and selects only those hybridomas that produce antibodies of appropriate specificity.  The screening technique used is called  ELISA . The hybridoma culture supernatant, secondary enzyme labeled antibodies, are then incubated, and the formation of a colored product indicates a positive hybridoma. ELISA SCREENING

15. CULTURING : Culturing of hybridomas: In vitro In vivo IN VITRO CULTURING: The hybridoma cell that produces the desired antibodies can be cloned to produce many identical daughter clones. Once a hybridoma colony is established, it will continually grow in culture medium produce antibodies. Multiwell plates are used initially to grow the hybridomas, and after selection, are changed to larger tissue culture flasks. This maintains the well-being of the hybridomas and provides enough cells for cryopreservation and supernatant for subsequent investigations. The culture supernatant can yield 1 to 60 µg/ml of monoclonal antibody, which is maintained at -20 °C or lower until required.

16. IN VIVO CULTURING: This procedure involves introduction of hybridoma cells into the peritoneal cavity if the animal. The ascetic fluid is isolated from the animal which is used for the isolation and purification of the antibodies. IN-VITRO AND IN-VIVO CULTURING

17. APPLICATIONS: BIOCHEMICAL ANALYSIS: Monoclonal antibodies are used in immunoassays like: RIA( radioimmuno assay ) ELISA (Enzyme linked immuno sorbent assay) These assay measure the concentrations of hormones like Insulin Thyroxine TSH etc. Blood grouping test Pregnancy test ( Human chorionic gonadotropin) CANCER: Estimation of plasma carcinoembryonic antigen in c olorectal cancer, Prostate specific antigen for prostate cancer.

18. DIAGNOSIS OF INFECTIOUS DISEASES: These antibodies could be used for detection of infections such as typhoid , syphilis etc. DIAGNOSTIC IMAGING: Radiolabelled monoclonal antibodies are used in the diagnostic imaging of diseases, this technique is called immunoscintigraphy . Applications in detection of Myocardial infraction, Deep vein thrombosis etc. Detection of Tumor markers for various cancers ex: carcinoembryonic antigen (CEA) - cancer of stomach, colon, pancreas. Alpha fetoprotein – cancer of Liver . CANCER TREATMENT: Monoclonal antibodies against the surface antigens of cancer cells mediate cancer cell death through ADCC ( antibody mediated cell dependent cytotoxicity ) Phagocytosis etc.

19. IMMUNOSUPRESSION: The monoclonal antibodies could be used as immunosupressive agent after organ transplantation . Ex: OKT3 against CD3 of T cells has been successfully used in renal and bone marrow transplantation.