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

Alynne I. MacLean, Leonidas G Bachas

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17 Citations (Scopus)

Abstract

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 × 10-6 m lipoic acid. This technique has several advantages over previous electrochemical and chromatographic techniques for lipoic acid determination.

Original languageEnglish (US)
Pages (from-to)303-307
Number of pages5
JournalAnalytical Biochemistry
Volume195
Issue number2
DOIs
StatePublished - 1991
Externally publishedYes

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Pyruvate Dehydrogenase Complex
Thioctic Acid
Immunoenzyme Techniques
Assays
Ligands
Enzymes
Binders
Limit of Detection
Dihydrolipoyllysine-Residue Acetyltransferase
Electrochemical Techniques
Prosthetics
Dilution
Theoretical Models
Rabbits
Peptides
Antibodies

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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title = "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",
abstract = "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 × 10-6 m lipoic acid. This technique has several advantages over previous electrochemical and chromatographic techniques for lipoic acid determination.",
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