Double-antibody Sandwich ELISA Method for Erythropoietin in Serum and Development of the Kit and Its Study on the Technology

Objective To develop a double antibody sandwich ELISA method for detecting erythropoietin(EPO),establish ELISA kit of the serum EPO,then initially study the research methodology of its clinical detection. Methods Coat the 96-microwell plate with EPO antibody was diluted with buffer,the each well of the 96-microwell plate should be dropped with 150 μL antibody per well. The test sensitivity,recovery,linearity,and stability using ELISA method,with serum from healthy indiv-iduals and patients as clinical samples. Results The optimal antibody concentration was 1∶600,the best working concentration of antigen was 1∶100,and the best working concentration of enzyme labeled antibody was 1∶5 000. The standard protein curve regression equation was y=0.017x+0.300 2,r=0.971 0,P0.05. The sensitivity was 0.46 U/L,and the mean recoveries of samples with high and low concentrations were 106.74%,104.78% respectively. The coefficient of variation of intra-assay ranged were 3.044% ,7.964% and the coefficient of variation of interassay ranged were 1.379% ,12.915% ,the serum sample was 20,80 U/L. Conclusion Double-antibody sandwich ELISA assay of serum EPO has good sensitivity,specificity,accuracy,and provides the experimental evidence for quick,accurate,and convenient clinical EPO detection.