Evaluation of carbohydrate antigen 50 in human serum using magnetic particle-based chemiluminescence enzyme immunoassay.

A magnetic particles (MPs)-based chemiluminescence enzyme immunoassay (CLEIA) with high sensitivity, specificity, rapidity, and reproducibility was proposed for the evaluation of tumor marker, carbohydrate antigen 50 (CA50) in human serum. The immunomagnetic particles coated with anti-fluorescein isothiocyanate (FITC) antibody was used as dispersed solid phase for the immunoassay, which was based on a sandwich immunoreaction of FITC-labeled anti-CA50 antibody, CA50 antigen, and alkaline phosphatase (ALP)-labeled anti-CA50 antibody, and was based on a subsequent chemiluminescence reaction of ALP with 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2'-adamantane) (AMPPD) solution. The CL emission intensity was directly proportional to the amount of analyte present in a sample solution. The effects of several physicochemical parameters, including the concentration of FITC-labeled anti-CA50 antibody, the dilution ratio of ALP-labeled anti-CA50 antibody, the volume of MPs and substrate, the immunoreaction time and other relevant variables upon the immunoassay were studied and optimized. The proposed method exhibited advantages in a lower minimum detectable concentration of 1.0 U mL(-1) with comparison to the commercially available immunoradiometric assay (IRMA), and showed a larger linear range of 0 to 300 U mL(-1), as well as less total assay time of only 50 min with comparison to both IRMA and microplate CLEIA. The coefficient of variation was less than 7 and 11% for intra- and inter-assay precision, respectively. This method has been successfully applied to the evaluation of CA50 in human serum with recoveries from 82 to 112%, and showed a good correlation with the commercially available CA50 IRMA.