Microdetermination of rabbit immunoglobulin allotypes by ELISA using specific antibodies conjugated with peroxidase or with biotin

Abstract

Enzyme-linked immunoadsorbent assays (ELISA) specific for the allotypes of rabbit heavy chain variable region (group a) and light chain constant region (group b) have been developed. These assays utilize affinity-purified allogeneic anti-allotype antibodies to coat polyvinyl microtiter plates. After test samples are added to the plates, the same affinity-purified antibody, coupled to the enzyme peroxidase, is used to detect binding of allotype-positive IgG samples to the coated plate. These assays can detect as little as 40 ng/ml of IgG of the appropriate allotype; the assays are highly specific and allow quantitative determination of allotype-defined IgG samples. In addition, the procedure can be modified by coupling of the antibody with biotin followed by development with avidin-peroxidase; this adaptation avoids the difficulties encountered in antibody-peroxidase conjugations. The ELISA assay is not influenced by the presence of anti-allotype antibody in the test sample, giving a distinct advantage over solid-phase radioimmunoassay procedures conventionally used for the quantitative determination of rabbit allotypes.