2.8 HbA1c enzymatic assay
For HbA1c enzymatic assay three reagents were used. The reagent 1 was
consisted of 0.1% Triton X-100, reagent 2 was composed of Alkaline
protease, 3,3-5,5-Tetramethylbenzidine (TMB 0.5 mM), and suitable
buffer and, reagent 3 was included purified FPOX and Peroxidase (5
U/ml).The blood cells were hemolyzed by mixing 500 µL of reagent 1, and
25 µL of blood sample. Torelease Fru-ValHis, 12 µL of hemolyzed sample
was added to the 180 µL of reagent 2 and incubated at 37 °C, and after 5
min its absorbance was read at 652 and 800 nm (sub wavelength), next, 60
µL of reagent 3 was added to the sample then incubated at 37 °C for 20
minand the absorbance was read at the same wavelengths.