MICROFLUOROMETRIC IMMUNOASSAYS WITH ANTIGENS BOUND TO SEPHAROSE BEADS

Abstract

Sepharose beads, to which protein had been coupled, were used in the development of two types of microfluorometric immunoassays. The first is an inhibition assay: a fixed amount of antiserum was absorbed with different amounts of antigen. This was accomplished in both direct and indirect incubation schemes. The system has been studied with human IgG and mouse IgM as an example. The second is a competition assay: fluorescein-labeled ovalbumin competea with unlabeled unlabeled ovalbumin for the binding sites of a rabbit antiovalbumin. This antiovalbumin was not bound directly to Sepharose but to Sepharose-coupled ovalbumin. In the indirect fluorescence inhibition and the competition assay, the sensitivity levels were determined. They proved to be the same, namely, 25 ng. This means that these fluorescence immunoassays are 10 times less sensitive than conventional radioimmunoassays and 10 times more sensitive than conventional single radial immunodiffusion.