| Chemiluminescence immunoassay applicated widely in life and environmental science fields for its sensitivity, specific, and simple. It combines the advantages of the immunoassay and chemiluminescent analysis. Our work carried out a series study of chemiluminescence immunoassay for the determination of free in human serum. It is momentous practice and academic significance for the screening and detection of numbers clinic samples and the development of the in-vitro diagnostic reagents.First, The principle of chemiluminescent immunoassay were reviewed. The categories, sources and functions of thyroid hormone were introduced. Then, various detection methods for free thyroxine were summarized.Secondly, a simple method to the quality identification of the small molecule substance enzyme markers was proposed. The experiment showed that the proposed method could be used in selecting the experimental raw material to ensure the accuracy of the future experiment research.Thirdly, a magnetic particles-based chemiluminescence enzyme immunoassay with high sensitivity, specificity, rapidity, and reproducibility was proposed for the determination of free thyroxine in human serum. A competitive assay has been proposed with horseradish peroxidase labeled thyroxine analog. The immunomagnetic particles coated with anti-fluorescein isothiocyanate antibody was used as dispersed solid phase and separation means for the immunoassay. Experimental conditions, such as temperature, the volume of magnetic particles and substrate, incubation time, dilution ratio and other relevant variables upon the immunoassay have been examined and optimized. The proposed method exhibited high performance which the linear range was 0-122 pmol L-1 and the detection limit was 0.25 pmol L-1. A coefficient of variance of less than 14.6% was obtained for both intra-assay and inter-assay precision. The present method has been successfully applied to the analysis of free thyroxine in human serum. The diagnostic accordance rate of the method for normal serum, hyperthyroidism and hypothyroidism are satisfactory. Good correlations were obtained between the results by the proposed method and the commercial radioimmunoassay kit.The proposed method exhibits good potential in the fabrication of FT4 diagnostic kits which could be used in the clinical analysis and facilitated the development of automated operation systems in the clinical practice.Finally, a solid phase CLEIA for the determination of free thyroxine in human serum was established, with the highly sensitive HRP-luminol-H2O2 system as chemiluminescence detection system, donkey anti-goat IgG as secondary antibody. The donkey anti-goat IgG was indirectly immobilized on the 96-wells polystyrene microplate, the gaot anti-thyroxine through the immunoreaction with secondary antibody to prepare the solid phase antibody, a competitive assay has been proposed with horseradish peroxidase labeled thyroxine analog. After the immune complex formed, added the the substrate solution, detected the chemiluminescence intensity for the quantitative detection. The effect of several experimental conditions, such as temperature, the volume of magnetic particles and substrate, incubation time, dilution ratio and other relevant variables upon the immunoassay were examined and optimized. The assay was evaluated and every methodological index can meet the demand of quantitative analysis. This method has been successfully applied to the evaluation of free thyroxine in 119 human serum and the results showed a good correlation (r2=0.8121) with the commercially available RIA kit, which shown that it can be used in the clinical practice. |
PDF Full Text Request