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

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