Homogeneous enzyme immunoassay for lipoic acid based on the pyruvate dehydrogenase complex: a model for an assay using a conjugate with one ligand per subunit.
A homogeneous enzyme immunoassay for lipoic acid was developed by using an enzyme-ligand conjugate containing only one ligand per enzyme subunit. Theoretical studies have shown that the traditional use of multisubstituted enzyme-ligand conjugates has limited the detection limits and sensitivity obtainable with these assays. The use of conjugates with a smaller number of ligands should allow for improved assays. The pyruvate dehydrogenase complex was chosen for this study because each polypeptide chain of dihydrolipoyl transacetylase contains one lipoic acid as a covalently attached prosthetic group. Thus, the naturally occurring enzyme can be considered as an enzyme-lipoic acid conjugate. Anti-lipoic acid antibodies were developed in New Zealand White rabbits to be used as the analyte-specific binders. Association and binder dilution curves were prepared in order to optimize the reagent concentrations and the analytical conditions. Unexpected inhibition by free lipoic acid resulted in a biphasic dose-response curve with a detection limit of 5 x 10(-6) M lipoic acid. This technique has several advantages over previous electrochemical and chromatographic techniques for lipoic acid determination.
MacLean
A I
AI
Department of Chemistry, University of Kentucky, Lexington 40506-0055.
Bachas
L G
LG
D000906:Antibodies; D004305:Dose-Response Relationship, Drug; D007124:Immunoenzyme Techniques; D011768:Pyruvate Dehydrogenase Complex; D013053:Spectrophotometry; D008063:Thioctic Acid / Q000032:analysis*
D000906:Antibodies; D011768:Pyruvate Dehydrogenase Complex; D008063:Thioctic Acid