An immunochemical assay model system for the sensitive detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylatingn subunit pyruvate dehydrogenase (E1)

Abstract

An immunochemical enzyme immunoassay model system was developed and compared for maximun sensitivity with a radioimmunoassay method and the classic enzyme activity method for the detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit, pyruvate dehydrogenase (E1), isolated from Eschericha coli. Cross-linked large molecular weight antibody-enzyme conjugate systems are compared with heterobifunctional singular antibody conjugates substituted with high levels of horseradish peroxide. Both polyclonal and monoclonal antibodies generated to the Escherichia coli PDHc and E1 antigens were used to develop a double-antibody sandwich microtiter plate enzyme-linked immunosorbent assay. It is demonstrated that a double sandwich immunochemical assay system can be quantitative for PDHc, can detect PDHc in crude cell lysates and has levels of sensitivity of 2.0 · 10 −16 mol for the detection of PDHc. This assay model systems provides specific antibody selection criteria and coupling methods needed to select specific antisera that cross-react with human PDHc. This rapid and sensitive immunochemical assay method clearly demonstrates that sensitive mass assay systems can be developed for the detection of PDHc. Different from Western blot, this methodology could be used to generate mass assays which could be applied to the rapid detection of mammalian antigens (employing the corresponding antibodies) implicated in a number of pyruvate dehydrogenase deficiencies associated with human disorders.