Purification of Glycated Hemoglobin from Hemolysateof Blood Samples and Its Application in Immunoassay . 1

This study was embarked on, to produce working standards for HbA1c ELISA assay, using locally accessible materials.A pool of blood samples with high levels of HbA1c were used to make hemolysate. Estimation of Hemoglobin level and % HbA1c was done, by Bradford assay and Biorad D-10 respectively, prior to loading on Sephadex G-100 column. Peaks eluted for different hemoglobin variants were purified and confirmed by SDS PAGE. The range of working standards for in-house ELISA assay for HbA1c was made from purified fractions of HbA1c. The concentration of Hemoglobin and HbA1c were 3.3g/ml and 20% prior to loading on Sephadex G-100 column and was abridged to 3.0g/ml and 18% respectively as assessed by Bradford assay. The molecular weights of HbA1a, HbA1b, HbA1c and HbA0 peaks, eluted from the Sephadex G-100 column, were confirmed by SDS PAGE as 66KD-68KD. The in house working standards prepared from HbA1c purified fraction were in the range of 2% 18% and confirmed with the commercial HbA1c ELISA kit. The standard points, prepared for HbA1c ELISA assay, can be used at local clinical setups for routine screening and management of Diabetes Mellitus to make the test economical.