Analysis of HbA1c by Immuno Assay Procedures

A typical method uses a specific antibody (usually monoclonal) to the glucose and the first 5 to 10 amino acids of the β-chain. This antibody is latex coated. There is also an agglutinator which is a synthetic polymer with multiple copies of the HbA1c portion that the antibody targets. The agglutinator reacts with the antibody to give a scattering of light and an increase in absorbance.

The HbA1c from the blood sample then competes with the agglutinator for the antibody latex binding sites and thus reduces the scattering of light and the absorbance. So the greater the HbA1c in the blood, the less the absorbance will be from scattered light. From this the amount of HbA1c is calculated, and the total hemoglobin can be determined by measuring at or near the Soret absorption band of hemoglobin (410 – 420nm) or by Drabkins method (oxidation and conversion to cyanmethemoglobin) at about 540nm, or using the alkali hematin assay.

Assay platforms using an immuno assay procedure include; Siemens Healthcare Diagnostics DCA 2000 and Advia 1650, Dimension, Roche Cobas and Hitachi, and Beckman analyzers. Most of the reactions on hemoglobin from aging or other chemical or free radical type reactions do not occur at the HbA1c site where the antibody in this method group is targeted. For this reason this type of method is generally more tolerant of aging/degradative reactions and HbA1c controls would be expected to have longer useable reconstituted or open vial times than ion exchange HPLC procedures.