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

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 (K