Hybridoma Screening by Antibody Capture: Enzyme-Linked Detection (Indirect ELISA) in Polyvinyl Chloride Wells

In this antibody capture assay for hybridoma screening, the antigen is immobilized on a solid substrate (the surface of the wells in a polyvinyl chloride [PVC] microtiter plate), and antibodies in the hybridoma tissue culture supernatant are incubated with the antigen. Unbound antibodies are removed by washing, and antibody-antigen complexes are detected by secondary antibody conjugated to alkaline phosphatase (AP), which catalyzes the conversion of a chromogenic substrate to a blue/green product. Alternative secondary antibodies are necessary for experiments in which immunoglobulin-fusion proteins have been used as immunogens or screening proteins.