Production of monoclonal antibodies ABHILASHA TIWARI ANKITA DWIVEDI

Production of monoclonal antibodies………. • ABHILASHA TIWARI • ANKITA DWIVEDI • JASMINE KAUR • ARIJIT BISWAS

�Monoclonal anitbodies are homogeneous population of antibody molecules, derived from a single antibody producing cell, in which all antibodies are identical and of the same precise specificity for a given epitope.

Steps in production of monoclonal antibodies……

Immunisation…………. Mice are immunised with Ag … Antigen must be greater than 3000 -5000 Da…or linked to a hapten or a carrier protein…… Spleen cell, lymphocytes suspension is obtained….

Fusion……. Lymphocytes are fused with myeloma cells Myeloma cells are malignant lymphocytes……. Cells are fused using PEG(polyethyleneglycol) to promote membrane fusion…………. .

Culture in HAT…………. The medium in which the culture is done contains…. hypoxanthine, aminopterin, thymidine…. HAT kills myeloma cells…. . Spleen are short lived i. e, their life span is 7 -10 days, so they die gradually…. . Only hybrid cells survive……. Myeloma cells are engineered to be deficient in an enzyme…hypoxanthine guanine phosphoribosyl transferase(HGPRT)…. . Hence the myeloma cells would not survive in culture…unless this enzyme is added to the media………. .

The cells are cultured in HAT medium which is lacking the enzyme HGPRT… The powerful toxins of the media block the metabolic pathway “Denovo pathway”. . essential for purine metabolism…in DNA synthesis…… Fused cells survive ……… Hybridoma cells consisting of immortalized plasma cells fused with B cells will survive in the absence of supplemented HGPRT in culture medium…… Non fused, - HGPRT plasma cells and all non fused B cells die soon…….

Screening…………. �ELISA �RIA �SOLID PHASE IMMUNOASSAY………. .