Determination of the specific antibody titer

The amount of specific antibody present in polyclonal antiserum, ascites fluid, or hybridoma supernatant can be quantitated by either solid-phase radioimmunoassay (RIA) or by direct ELISA. In the solid-phase assay described here, serially diluted antiserum is incubated in microtiter wells previously coated with the relevant antigen. Bound antibody is detected by employing 125I-labeled anti-immunoglobulin antibodies. The amount of specific antibody in the antiserum is then determined from a standard curve generated with a specific antibody of known concentration. The unknown antiserum and the standard antibody are assayed in parallel. The support protocols describe the chloramine T and IODO-GEN procedures for radioiodination of the anti-immunoglobulin reagent. The use of the solid-phase RIA procedure to determine the light-chain and heavy-chain isotypes present in polyclonal antisera and fluids containing monoclonal antibodies is also described.