Study On The Direct Elisa Of Detection For Hbalc

BackgroundDetection of blood HbA1c levels is the gold standard for evaluation of the blood sugar control of diabetes mellitus.Although there are have a variety of methods to detecting HbA1c, such as HPLC , cation exchange resin column chromatography, micro-column affinity chromatography method, and immune agglutination inhibition,these methods still have much space to improve in accuration, economicsane, and speed.ObjectiveThis method is based on the principle of Enzyme-linked immunosorbent assay and the chemical structure of glycosylated hemoglobin, and using of monoclonal antibodies for specificityβ-chain epitope of glycosylated hemoglobin. The study was signed to build competitive ELISA method to detection glycosylated hemoglobin, and evaluation the method.MethodsAccording to competitive rule,a test method of direct competition ELISA was designed following:antigen HbA1c labing by HRP based on sodium heptaiodic acid method,and then each reaction condition of ELISA was optimized.The standard product was diluted into 8 concentration gradients followed by ELISA test on the basis of optimized reaction conditions.We used the logarithm of standard product concentration as abscissa,inhibition ratio as ordinate to establish standard curve that induced equiation of linear regression and lowest detectable limit.This ELISA method was finally evaluated for it's stability, precision and accuracy. ResultsThe specifically enzyme-labelled antigen HRP-HbA1c binding with HbA1c monoclonal antibody were obtained, the conjugated ratios for enzyme-labelled antigen were 0.565.The optimum condition for the ELISA was as follows: Costar ELISA plate was radiation by UV. The HbA1c-McAb was diluted by 0.05 M carbonate buffer (pH9.6) to microtiter plates at a contration of 1:400(150μl/well)and incubation 24h at 4℃. Plate was washed three times with PBST for 3 minutes per times(the mode of washings is identical next), and 200μl 2% BSA was added to each well to elimate nonspecific binding by blocking the plate surface where protein was not bound, after 2h of incubation at 37℃, washing, and 1:40 enzyme-labelled antigen and varying concenteations of standard HbA1c (75μl/well) were added, after 2h of incubation at 37℃, washing, and 150μl/well TMB substrata solution was added , followed by the addition of stopping solution (2mol/l H₂SO₄, 50μl/well) after 30 minutes in the dark at room temperature. Absorbance at 450nm was determined by an enzyme immunoassay reader.Percent inhibiting was calculated from the absorbance obtained in the presence and absence of enzyme-labelled HbA1c in standard. A linear dose-response standard curve was prepared by plotting log[HbA1c] versus percent inhibiting. The regression equation of standard curve was y=37.823x-26.833, The correlation coefficient R²= 0.9802, the lower limit detection was 16μg/ml and a linear range was 16～1024μg/ml. The valid period of the ELISA kit in 4℃was longer than 6 month, the C.V. within-assay and between-assay was 2.78% and 6.22%, recoveries of added standard ranged from 96.5% to 100.3%.ConclusionsIn this study, the direct competition ELISA method of testing HbA1c establishment. This method is accurate and reproducible. For the basis to development of independent intellectual property rights in China .