Establishment Of Homogeneous Chemiluminescent Immunoassay For Serum Human Chorionic Gonadotropin

Human Chorionic Gonadotrophin is an glycoprotein hormone composed of237amino acids produced by the syncytiotrophoblast with a molecular mass of39KDa. It is heterodimeric, with an alpha subunit and an beta subunit. The alpha subunit of this protein was similar with FSH, LH and TSH, but the beta subunit was specific. Recently, HCG was widely used in diagnosis of pregnancy,miscarriage,eetopic pregnancy and gestational trophoblastic disease.Luminescent oxygen channeling assay(LOCI) is a homogeneous immunoassay method capable of rapid, highly sensitivity and time resolved, robust, quantitative determination of a wide range of analytes, low luminescent background, without washing and easy to automatic compared with previously reported immunoassay. It is a new technology for quantity the beta-HCG in serum.AimHCG homogenous test was developed via LOCI on the plat form of AlphaScreen. Method evaluation and clinical performance were performed in our study.MethodsBeta-HCG polyclonal antibodies, beta-HCG monoclonal antibody and alpha HCG monoclonal antibody were bound to the latex microparticles. Biotinylated antibodies were prepared by coupling the beta-HCG polyclonal antibodies and beta-HCG monoclonal antibody with biotin-NHS ester. Checkboard Titration testing of antibodies determines the best combination of antibodies coating on microparticles and biotinylated antibodies and the optimum working concentration. Reaction buffer, adding sequence, proportion of different reagents and the reaction time were optimized. Standard does-response curve was established using four parameter logistic, and the concentration of unkonw HCG could be calculated through the standard curve. Method evaluation and clinical performance were performed through above method. ELISA Cal were used for statistical analysis.ResultsAlpha-HCG monoclonal antibody coating microparticles, biotinylated beta-HCG polyclonal antibodies and Streptavidin labeled donor micropaticle were used in the serum HCG test. The within-run precision and between-run precision were3.93%-4.40%and5.60%-8.39%respectively. The sensitivity was1.88IU/ml and recovery was99.5%-105.9%. Common interference components were tested, and the interference rate of hemolysis, bilirubin and RH were lower than10%. The cross reactivity among LH, FSH and TSH were all lower than3%.50clinical samples were used in method comparison, using CBOAS6000as reference instrument. The two methods have a good correlation(r2=0.974).Chi-square test and consistency analysis indicated there is no significant different between the established method and imported electrochemiluminescence measurement.Conclusions:The quantity measurement of human serum HCG established in this study was a promising approach for clinical diagnosis, with the advantage of high sensitivity, accurate and reliability, easy-to-handle and without washing.