Immunochemical Determination of Hemoglobin-A1c Utilizing a Glycated Peptide as Hemoglobin-A1c Analogon

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URI: http://nbn-resolving.de/urn:nbn:de:bsz:21-opus-3738
http://hdl.handle.net/10900/48263
Dokumentart: Teil einer Konferenzveröffentlichung
Date: 2001
Source: http://barolo.ipc.uni-tuebingen.de/biosensor2001/
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Sonstige - Chemie und Pharmazie
DDC Classifikation: 540 - Chemistry and allied sciences
Keywords: Biosensor , Immunoassay , Diabetes mellitus
Other Keywords: Hämoglobin A1c
Other Contributors: Gauglitz, Günter
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Abstract:

We describe the development of a heterogeneous affinity-matrix based immunoassay for the determination of HbA1c which could in future be applicable to analytical devices. We developed an immunoenzymometric assay (IEMA) where the glycated pentapeptide Val-His-Leu-Thr-Pro (VHLTP) as HbA1c analogon is immobilized either to the surface of a microtiter plate by adsorption or to an amino-modified cellulose membrane by covalent linkage. The immobilized analogon competes together with the HbA1c in the sample for the antigen binding sites of the anti-HbA1c antibodies. Glucose oxidase-labeled antibodies have been used to indicate the antigen-antibody reaction indirectly and enzyme activity was detected optically. Calibration curves for HbA1c were obtained with a linear range of 1,5-10 µg ml-1 (23-155 nM). In a mixture of non-glycated and glycated hemoglobin with a total hemoglobin concentration of 30 µg ml-1 (465 nM) a linear range was obtained between 5-50 % HbA1c. Since the glycated peptide shows a high affinity for the anti-HbA1c antibody (Kd = 0,3 nM) only a low contact time (< 1 min) between the modified solid support and the preincubated mixture of HbA1c and anti-HbA1c antibody was required. Regeneration of the affinity-matrix was carried out with 10 mM HCl for 3 min without loss of antibody binding activity.

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