SPECIFIC FLOW-INJECTION SANDWICH BINDING ASSAY FOR IGG USING PROTEIN A AND A FUSION PROTEIN

A sandwich-type flow-injection binding assay for quantitation of vario us IgG's was developed. The assay is based on the pseudoimmunological reaction between protein A from Staphylococcus aureus and immunoglobul in G from different species. Protein A immobilized on a solid support and a fusion protein of protein a and beta-galactosidase from Escheric hia coli are used for detection. The fusion protein is produced with a temperature-inducible recombinant E. coli strain. A sandwich is forme d by subsequent injection of IgG and fusion protein into the buffer st ream flowing through the immobilized protein A column. The amount of e nzyme activity bound is proportional to the amount of IgG bound and is measured by pumping a lactose solution as substrate for beta-galactos idase through the protein A column. Lactose is converted to glucose an d galactose. The detector is an enzyme thermistor that measures the he at evolved in the enzymatic conversion of glucose by coimmobilized glu cose oxidase and catalase. The assay takes 16 min at a flew sate of 0. 6 mL min(-1) with a lower detection limit of 33 pmol per injection of rabbit IgG. The precision of replicate measurements has a standard dev iation of 4-5%, and the column can be used for more than 50 cycles.