Development of Chemiluminescent Immunoassay for Determination Sexual Steroid Hormones
|Keywords||Chemiluminescence immunoassay (CLIA) Magnetic microparticles ( MPS ) Estradiol (E2) Progesterone (P) Sodium trichloroacetate|
Immunoassay method has the advantages of high sensitivity, wide linear range, and can achieve full automation, and a wide range of applications in the life and environmental sciences. It mainly consists of radioimmunoassay, enzyme immunoassay, fluorescence immunoassay and chemiluminescence immunoassay and other methods. This thesis is focused on the chemiluminescence immunoassay technology, to carry out a series of studies of the determination of steroid sex hormones on the screening and testing of large numbers of samples is very important academic significance and application value. The text is divided into five chapters are as follows: Chapter types of introductory steroid sex hormones on the basis of the origin and role function, steroid sex hormones detection techniques and methods are reviewed. In addition, this chapter also provides an overview of the the chemiluminescence immunoassay basic principles, methods and research progress, and research prospects. The second chapter to establish a simple, rapid, high-sensitivity chemiluminescent enzyme immunoassay method for the determination of environmental water, 17β-estradiol. The second antibody coated magnetic particles as a solid phase separation agent of the immunoassay system. This Law to determine the actual samples, using solid phase extraction method for sample pre-treatment, in order to eliminate the interference of matrix effects. In addition, various experimental factors (such as the degree of dilution of the immunological reagents, the immune reaction conditions, the magnetic particles and the light-emitting volume of the substrate, the light emitting substrate reaction time and pH, etc.) were examined and optimized. Measured under optimal conditions, the linear range of E2 is 20 ~ 1200 pg / mL, and the detection limit of 2.0 pg / mL. System for the determination of the total time of the sample within 45 min, intra-and inter-assay coefficients of variation were less than 10%, adding an average recovery is 86.3 to 108% of the sample concentration. This method has been successfully used for the waste water, river water, and tap water of E2 measured and commercialization radioimmunoassay (RIA) kit compared to having a good correlation. Chapter on magnetic microparticles as a carrier, the establishment of a high-sensitivity, high specificity, and reproducibility of chemiluminescence immunoassay method, and applied serum estradiol (E2) Determination. The second antibody coated magnetic particles as the solid phase of the immunoassay system separating agent, catalytic H2O2-luminol chemiluminescence system select the high sensitivity of the horseradish peroxidase (horseradish peroxidase, HRP) as a detection system. This method showed high specificity, with the major steroid hormones: estrone (E1), estriol (E3), dihydro-testosterone (DHT), androstenedione and testosterone are not cross. Blocker, the introduction of the sodium trichloroacetate, does not need to be extracted to achieve the direct determination of human serum estradiol. The method estradiol detection limit of 2.51 pg / mL, the linear range was 15 ~ 1000 pg / mL, system of intra-and inter-assay variation were less than 15% under the best conditions. The average recovery added three different concentrations of samples and were 93.3,106,101%. The method has been successfully applied to the determination of 105 clinical human serum samples, with good correlation commercialization radioimmunoassay kit test results, a correlation coefficient of 0.9892. These results are expected to be applied in the development of domestic chemiluminescence diagnostic kits and automatic chemiluminescence immunoassay analyzer. Chapter IV Law according to the competitive reaction between the HRP markers and estradiol antigen with a certain amount of antibody, donkey anti-rabbit secondary antibody coated polystyrene plates a solid phase immunoassay system, select high sensitivity horseradish peroxidase catalyzed H2O2-luminol chemiluminescence system as a detection system. The detection limit of the method for determination of estradiol to 1.48 pg / mL, and successfully applied to the determination of 97 clinical human serum samples, and commercialization radioimmunoassay kit test results correlated well, a correlation coefficient of 0.9881 can meet the needs of the day-to-day clinical diagnosis. Chapter magnetic microparticles as a dispersed solid phase separation agent, HRP-catalyzed H2O2-luminol chemiluminescence system as a detection system. Introduced in the reaction system coupled goat anti-rabbit secondary antibody of the magnetic microparticles and, ultimately, the formation of immune complexes. The effect of repeatedly applying a magnetic field, there will be no free protein binding by washing with immune complexes separated treat Determination of measuring the antigen can be achieved. The method progesterone detection limit of 0.2 ng / mL linear range of 0 ~ 100 ng / mL, the system of intra-and inter-assay variation were less than 11% under the best conditions. Applied to the determination of 70 clinical serum samples, recoveries ranged from 82 to 112%, a good correlation of test results with the commercialization of imported chemiluminescence immunoassay kit, and a correlation coefficient of 0.9897.