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 language | English (US) |
|---|---|
| Pages (from-to) | 303-307 |
| Number of pages | 5 |
| Journal | Analytical Biochemistry |
| Volume | 195 |
| Issue number | 2 |
| DOIs | |
| State | Published - 1991 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
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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. / MacLean, Alynne I.; Bachas, Leonidas G.
In: Analytical Biochemistry, Vol. 195, No. 2, 1991, p. 303-307.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Homogeneous enzyme immunoassay for lipoic acid based on the pyruvate dehydrogenase complex
T2 - A model for an assay using a conjugate with one ligand per subunit
AU - MacLean, Alynne I.
AU - Bachas, Leonidas G
PY - 1991
Y1 - 1991
N2 - 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.
AB - 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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UR - http://www.scopus.com/inward/citedby.url?scp=0025736626&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(91)90334-P
DO - 10.1016/0003-2697(91)90334-P
M3 - Article
C2 - 1750684
AN - SCOPUS:0025736626
VL - 195
SP - 303
EP - 307
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -