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

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

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

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