Immunization of the appropriate specie with antigen emulsified with adjuvant occurs on days 1, 14, and 28. Sera are tested for antibody titer in an ELISA assay (or with the investigator’s screening method) and if titers are high, hyperimmunization is performed and spleen cells are isolated and fused to myeloma cells with polyethylene glycol (PEG). Hybridoma cells are selected with HAT medium that kills non-fused myeloma and spleen cells. Hybridoma cells are subcultured in microwells and supernatants are screened for IgG secretion in the investigator’s antigen specific system. Cells from positive wells are cloned. A solid phase ELISA assay or FACS screening can be used to identify positive wells. The positive cultures are cloned and the culture supernatants rescreened. Frozen stocks are produced as well as the conditioned media that contains the monoclonal antibody.

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