Development Of Chemiluminescent Immunoassay For Determination Sexual Steroid Hormones

Immunoassay has become popular in the field of life and environmental science because of the advantages of high sensitivity, wide linear range and easy automation. Immunoassay method includes radioimmunoassay (RIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA) and chemiluminescence immunoassay (CLIA). Especially, CLIA captures simultaneously the high sensitivity of chemiluminescent analysis and the high specificity of immunoassay, the study and application of CLIA on the analysis of the environment and human serum will have momentous academic and practice significance. Based on these points, the dissertation would lay stress on the development and application of CLIA methods for determination of some sexual steroid hormones.This thesis consists of five main chapters as follows:Chapter 1 Based on the introduction of categories, sources and functions of sexual steroid hormones, the analytical methods and technologies for these steroid hormones were reviewed. Also, the basic principles and the recent study progress of chemiluminescence immunoassay were described and its developmental prospect was expected.Chapter 2 A simple, fast, and highly sensitive CLEIA for 17β-estradiol (E2) in environmental water samples was developed, using magnetic particles (MPs) labeled with secondary antibody as both the immobilization matrix and the separation tools. The water samples were pretreated with solid-phase extraction using C18 cartridges for the removal of matrix effects. Several physicochemical parameters including the dilution ratios of E2-6-horseradish peroxidase (HRP) conjugate and anti-E2 PcAb, immunoreaction time, volume of chemiluminescent substrate and MPs, chemiluminescence reaction time, and pH of assay solution were studied and optimized. At optimal experimental conditions, it was found that the proposed method exhibited high performance with detection limit of 2.0 pg/mL, linear range of 20～1200 pg/mL, and total assay time of 45 min. Both inter-and intra-assay coefficient of variation (CV) were less than 10%. The average recoveries of three different spiked concentration samples ranged from 86.3 to 108%. The method was successfully applied to the determination of E2 in river, waste, and tap water, and showed a good correlation with the commercially available RIA kit.Chapter 3 A magnetic particles (MPs)-based CLIA with high sensitivity, specificity, and reproducibility was proposed for the evaluation of E2 in human sera. The MPs coated with secondary antibody were used as dispersed solid phase for the immunoassay, and HRP-luminol-H2O2 chemiluminescent system with high sensitivity was chosen as the detection system. The method showed specific recognition to E2, without cross-reaction for the major steroids, including estrone (El), estriol (E3), dihydrotestosterone (DHT), androstenedione, and testosterone (T), which was commonly found in human serum. The addition of sodium trichloracetate (Na-TCA) in the enzyme buffer as a blocking agent contributed to the realization of direct analysis of E2 in human serum without extraction. The proposed method had a detection limit of 2.51 pg/mL with a larger working range of 15-1000 pg/mL. The inter-assay and intra-assay CV were both less than 15%. The average recoveries of three different spiked concentration samples were 93.3,106 and 101%, respectively. The method has been successfully applied to the determination of E2 in 105 human sera and showed a good correlation compared with the commercial RIA kit with a correlative coefficient of 0.9892.Chapter 4 This method was based on the competitive reaction of HRP labeled antigen and E2 with a limited amount of polyclonal antibody, with donkey anti-rabbit IgG as the secondary antibody coating micro-plates, and the HRP-luminol-H2O2 chemiluminescent system with high sensitivity was chosen as the detection system. The CLIA with secondary antibody can be applied to determine E2 with good precision at concentrations as low as 1.48 pg/mL. The proposed method has been successfully applied to the determination of E2 in 97 human sera and showed a good correlation compared with the commercially RIA kit with a correlative coefficient of 0.9881. This method has exhibited great potential in the fabrication of diagnostic kit and could be used in the clinical analysis of E2 in human serum.Chapter 5 The MPs coated with secondary antibody were used as dispersed solid phase for the immunoassay, and HRP-luminol-H2O2 chemiluminescent system with high sensitivity was chosen as the detection system. At optimal experimental conditions, the proposed method had a detection limit of 0.2 ng/mL with a larger working range of 0～100 ng/mL, and the inter-assay and intra-assay CV were both less than 11%. The method has been successfully applied to the determination of E2 in 70 human sera with the recoveries of 82～112%, and showed a good correlation compared with the commercial RIA kit with a correlative coefficient of 0.9897.