MicroELISA assays of anti‐HLA activity and isotype of human monoclonal antibodies

Both monoclonal human antibodies to HLA‐DR antigens and supernatants from oligoclonal B‐cell lines can be conveniently screened for activity by microELISA assays which use 1/10 of the volume of reagents used in conventional ELISA assays. Target cells are fixed to the bases of wells in Terasaki plates, 5 μl volumes of supernatants incubated in these wells, and target bound antibody detected by peroxidase‐conjugated anti‐immunoglobulin followed by the substrate ABTS(2,2′‐azino‐di‐(3‐ethylbenzthiazoline sulphonate). The plates are read on a micro EIA reader. Supernatants can also be assayed for immunoglobulin content and isotype in Terasaki plates by coating the wells with isotype‐specific anti‐immunoglobulin, adding test supernatant and developing with appropriate peroxidase‐conjugated anti‐immunoglobulin sera and ABTS. When assaying for immunoglobulin content, the plates can be read either with a reader or by eye. Advantages and modifications of these procedures are discussed. There are no apparent practically important disadvantages to these procedures as compared with more conventional ELISA assays.