Microplate chemiluminescence enzyme immunoassay for the quantitative evaluation of carbohydrate antigen 72-4 in human serum

A highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) was developed for the quantitative evaluation of carbohydrate antigen 72-4 (CA72-4) in human serum, using luminol-H₂O₂ catalyzed by horseradish peroxidase (HRP) as the chemiluminescence system. The simple and quick determination was accomplished through a sandwich reaction mode. Several physicochemical parameters of the immunoreaction, including incubation conditions, antibody coating conditions, dilution ratio of anti-CA72-4-HRP conjugate, and chemiluminescence reaction time, were studied and optimized. The proposed method exhibited a linear range of 0–200 U/mL with correlation coefficient and detection limit of 0.9995 and 0.18 U/mL, respectively. The inter-assay and intra-assay coefficients of variation (CV) were both less than 10%. The average recovery of two clinical sera with low and high concentration CA72-4 was 99.3% and 98.7%, respectively. Normal tumor markers, including AFP, CEA, CA24-2, CA19-9 and CA15-3, did not cross-react with each other. The method’s stability was evaluated by assessing its analytical performance after storing the immunoreagents at 4°C and 37°C for 7 days. Little difference was found, indicating satisfactory stability of the method. The present method has been successfully applied to the detection of CA72-4 human serum, and showed a good correlation with the commercially available ELISA kit (r ²=0.9383). This method showed great potential in the fabrication of diagnostic kit for CA72-4, and could be well used in diagnosis of cancer in clinical practice. Supported by the National Basic Research Program of China (Grant No. 2007CB714507) and the National High Technology Research and Development Program of China (Grant No. 2006AA02Z4A8)