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
✍ Scribed by Alynne I. MacLean; Leonidas G. Bachas
- Publisher
- Elsevier Science
- Year
- 1991
- Tongue
- English
- Weight
- 581 KB
- Volume
- 195
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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.