Establishment And Application Of Chemiluminescence Immunoassay Method Of Steroid Hormones

Comamonas testosteroni contains the key enzyme of degrading steroid nucleus—17β-hydroxysteroid dehydrogenase (17β-HSD).When steroidal compounds present, Comamonas testosteroni secretes17β-HSD enzym,and degrades steroidal compounds completely by complex metabolic pathway of degrading steroid nucleus of many enzymes.The subject cloned17β-HSD gene from the Comamonas testosteroni’s chromosomal DNA.and recombined it into plasmid pET-15b.in that we got recombinant plasmid pET-15b-17β-HSD. Then the recombinant plasmid was transformed into E. coli BL21, E. coli BL21expressed17β-HSD protein when1mMIPTG induced. We Collected expressed protein and purified it with nickel column.in conclusion we got the concentration of3.65mg/ml of17β-HSD protein.We immuned rabbit with17β-HSD protein,and collected rabbit serum, in that way polyclonal antibody were prepared. Then we determined the optimal working concentration of Polyclonal antibody by indirect ELIS A method.and it is4,000times diluted. We immuned mice with17β-HSD protein and then we merged Spleen cells and myeloma cells together by cell fusion technology, hybridoma cell strains were prepared about Highly expressing anti-17β-HSD monoclonal antibody. Then we prepared anti-17β-HSD monoclonal antibody,And purified monoclonal antibody with the bitterness-ammonium sulfate and G-protein affinity chromatograpic column. Finally, we labeled monoclonal antibody with horseradish peroxidase and got the HRP-antibody, we determined the optimal working concentration of HRP-antibody by indirect ELISA method,and it is8,000times diluted.We established a double antibody sandwich of chemiluminescent assay method about steroid hormone by using the two antibody we prepared, and for the initial application.We determined four samples of xinlicheng reservoir,yitong river,yinma river and Changchun tap water. Determined results are0.621ng/ml,9.332ng/ml.5.287ng/ml,0.321ng/ml.Results of HPLC determined conform to results of the establishment method.which proved double-antibody sandwich of chemiluminescent assay about steroid hormone is feasibility and correctness. The assay method is sensitive, and high-throughput assay of a large number of samples. It provides experimental For the assay of steroid hormone and supports a new-method for monitoring steroid hormones environment pollution,but also provides basis for the establishment of scientifically sound assay environmental hormone.