The study of a chemiluminescence immunoassay using the peroxyoxalate chemiluminescent reaction and its application

The paper presented a novel chemiluminescence (CL) immunoassay method, which combines the advantages of traditional enzyme-linked immunosorbent assays (ELISA) and bis (2,4,6-trichlorophenyl) oxalate (TCPO)–hydrogen peroxide CL detection system. A fluorescent product 2,3-diaminophenazine (DAPN) was produced by reaction between o-phenylenediamine (OPDA, 1,2-diaminobenzene) and H2O2 catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO–H2O2 chemiluminescent reaction, and led to CL. The dependence of the CL intensity on the concentrations of antigen was studied. As analytical application, the proposed method was used for determination of recombinant human interleukin 6 (rHu IL-6) and β-human chorionic gonadotropin (β-HCG). Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of rHu IL-6 in the range of 4.0–625.0 pg/ml, and β-HCG in the range of 12.5–400.0 mIU/ml. The detection limits were 0.5 pg/ml for rHu IL-6 and 3 mIU/ml for β-HCG with relative standard deviation of 2.3 for 78.0 pg/ml rHu IL-6, and 3.9 for 50.0 mIU/ml β-HCG. This method has been applied to the determination of rHu IL-6 in human serum and β-HCG in urine with satisfactory results.