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Hybridoma Technology HYBRIDOMA TECHNOLOGY Subject : Dr. KRISHNA KUMAR V M.sc,Ph.D,PGDCS Govt. Degree College ,NAIDUPET Email. Id : [email protected] HYBRIDOMA TECHNOLOGY LEARNING OBJECTIVES • TO UNDERSTAND THE NATURE OF ANTIBODY • DISTINGUISH BETWEEN POLYCLONAL AND MONOCLONAL ANTIBODIES • TO LEARN THE TECHNIQUE INVOLVED IN HYBRIDOMA TECHNOLOGY • STEPS INVOLVED IN I) CELL- FUSION III) ANTIBODY PRODUCTION INTRODUCTION ANTIBODY : • Antibodies are specialised proteins that are recruited by the immune system in response to an antigenic stimulus. • Antibodies identify and recognise the foreign antigens and remove them from the body. • Antibodies are produced B lymphocytes (or) B-cells • The production of antibodies is the main function of the humoral response (or) adaptive Immune system TYPES OF ANTIBODIES POLYCLONAL ANTIBODIES: • Antibodies derived from different B lymphocyte cell lines. • Heterogeneous in nature collected from multiple B cell clones. • Recognise and bind to many different epitopes of single antigen. • Primary role is to detect unknown antigens. Monoclonal antibody MONOCLONAL ANTIBODIES (Mabs) : • Antibodies are identical as they are produced by one type of immune cell (or) Clones of single parent cell. • Homogeneous in nature with affinity towards a specific antigen MONOCLONAL ANTIBODIES (Mabs) • Monoclonal antibodies possess two important characteristics, namely high specificity and high reproducibility. • widely used in diagnostic, clinical and therapeutic applications. • Plays a key role in immunotherapy HYBRIDOMA TECHNOLOGY MONOCLONAL ANTIBODIES : • Production of monoclonal antibodies using hybridoma technology was discovered in 1975 by GEORGE KOHLER & CESAR MILSTEIN. • They shared the NOBEL PRIZE in the year,1984. nobelprize.org • In 1990 MILSTEIN produced the first monoclonal antibodies HYBRIDOMA TECHNOLOGY HYBRIDOMA TECHNIQUE : • Well established method to produce monoclonal antibodies specific to desired antigens. • Hybridomas are generated by fusion of B-cell(short lived) with myeloma cell(immortal cell) • Hybridomas exhibit two important characteristics, ability to grow continuously and to produce huge amounts of antibodies. • Hybridomas can be cloned to produce large no. of daughter clones. • The daughter clones secrete the desired antibody. GENERATION OF MAbs IMMUNIZATION OF MICE Preparation of Screening myeloma cells of sera ISOLATION OF SPLEN CELLS CELL -FUSION TISSUE CULTURETissue Screening of hybridomas Expansion and selection of cultures to be cloned Isolation of clones Purification and testing of MAbs HYBRIDOMA TECHNOLOGY The technique involves two major events a) Cell-fusion b) production of antibodies. a) CELL –FUSION : • Isolation, purification and characterization of the desired antigen in adequate quantities. • Inoculation of mice with the purified antigen. • Removal of B-cells from immunised mice • Isolation of myeloma cells. • Fusion of B-cells with myeloma cells( cell-fusion) with the help of poly ethylene glycol(PEG) HYBRIDOMA TECHNOLOGY HYBRIDOMA CELL : • Formed by the fusion of B lymphocytes with myeloma cells www.adaltis.net • Hybridomas are immortalized cells. • Multiply rapidly in cultures. • Cloned to produce many identical daughter clones • Daughter clones produce large no. of antibodies. HYBRIDOMA TECHNOLOGY HYBRIDOMA TECHNOLOGY b) ANTIBODY PRODUCTION : Selection of hybrid cells in HAT(hypoxanthine-aminopterin-thymidine) medium: • Three types of cells appear during fusion: (1) unfused myeloma cells that are deficient in HGPRT enzyme (2) unfused spleen cells (3) hybridoma cells. • Only hybridomas survive in HAT medium • Growth of hybridomas occur with the addition of Inter-leukin-6 as media supplement HYBRIDOMA TECHNOLOGY HYBRIDOMA TECHNOLOGY b)ANTIBODY PRODUCTION • Screening of hybridomas by ELISA (enzyme-linked immunosorbent assay) • Screening process selects hybridomas that produce specific antobodies • Hybridomas are cultured in culture medium, supplemented with insulin, ethanol, amine and other hormones. • Hybridoma cell lines are cryopreserved for future use • Selected clone cells are grown by in vitro or in vivo conditions. HYBRIDOMA TECHNOLOGY b) PRODUCTION OF ANTIBODIES : i) in vivo method : • The hybridoma cell is injected in to the peritoneal cavity of mice through injection. • They grow in a constant manner and continuously secrete antibodies in the ascitic fluid. • Ascites are collected and purified . • It contains high concentration of antibodies. • This method is quicker,easier and economical. HYBRIDOMA TECHNOLOGY ii) in vitro method: Bioreactors(hollow fiber bio-reactor) • Supports biologically active environment • Fiber cell systems-ideally suited for in vitro production • Cells are cultured in hollow fibers • Fresh culture media and wastes are recirculated • Antibodies are produced in high concentrations in cell compartment • Can be easily purified • Collected at different time points • Production upto 1grm quantity of Mabs HYBRIDOMA TECHNOLOGY KEY POINTS: • KOHLER & MILSTEIN(1975) discovered the principle for production of monoclonal antibodies.“ • “ Hybridoma technology” is a technique for the production of a large number of identical antibodies • Hybridoma technique: a) Cell fusion b) Production of antibodies. • Cell fusion results in hybridoma cell which is immortal. • Hybridomas are cloned to produce large no. of antibodies. • Hybridomas are cryopreserved for future use..
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