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

Abstract

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 (r2=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.