Detection of HbA(1c) by boronate affinity immunoassay using bacterial magnetic particles.

We have developed a boronate affinity immunoassay system using m-aminophenylboronic acid (mAPB) coupling to bacterial magnetic particles (BMPs). Homobifunctional crosslinker, Bis-(succcimidyl)suberate (BS3), was employed for preparation of mAPB-BMPs conjugates (mAPB-BMPs). Quantities of HbA(1c) on mAPB-BMPs were evaluated based on luminescence from alkaline phosphatase-conjugated anti-Hb antibody (ALP-antibody) binding to HbA(1c) on the BMP surface. The binding of HbA(1c) to mAPB-BMPs occurred gradually and was almost completed within 10 mm. The coupling reaction is enhanced due to static electric interaction between the positive charges on HbA(1c) and negative charges on BMPs. The amount of HbA(1c) binding to mAPB-BMPs increased with increasing sodium chloride concentrations in the range of 0-100 mM. However, the amount of Hb binding to mAPB-BMPs also increased in high concentration of sodium chloride. The Hb binding to mAPB-BMPs was detached from mAPB-BMPs when Hb-mAPB-BMPs were washed with low salt buffer. This indicates that Hb is nonspecifically adsorbed onto the surface of mAPB-BMPs in high concentration of sodium chloride. These results suggest that selective separation of HbA(1c) using mAPB-BMPs can be achieved with these conditions. A dose-response curve was obtained between luminescence intensity and HbA(1c) concentration using a fully automated boronate affinity immunoassay. A linear relationship between luminescence intensity and HbA(1c) concentration was obtained in the range of 10-10(4) ng/ml.