| Objective: (1) To prepare recombinant human sperm-specific lactate dehydro- genases(rhLDH-C4) antigen by prokaryotic expression system. (2) To provide a new method to detect antisperm antibodies(ASA) in sera for the diagnosis of immunological infertility. (3) To investigate the relationship between serum anti-LDH-C4-antibody(IgG) and infertility. Methods: (1) Escherichia coli BL21(DE3)plysS was transformed by pET-28a(+)- hLDH-C recombinant plasmid, which were constructed and characterized in our laboratory. Subsequently the transformed bacteria were characterized by PCR and enzyme digestion. The transformed bacteria containing pET-28a(+)-hLDH-C were treated with IPTG induction. By using of Ni+-NTA affinity chromatography, purified rhLDH-C4 protein was prepared. Enzymatic activity of rhLDH-C4 was detected by activity staining of lactate dehydrogenase isoenzymes. (2) One serum sample containing anti-LDH-C4-antibody was identified by Western blotting, and used as positive control for antoantibody detection in infertile sera. By detecting OD values of 25 serum samples of girls aged under 10-year-old , cut-off value was also determined. Thus indirect ELISA method was established. Other reactive parameters of ELISA were further optimized. (3) OD values of 74 serum samples from fertile individuals(fertile group) and 177 serum samples from infertile patients with unknown cause (infertile group) were detected by this method.Results: (1) The total quantity of 1.5mg purified rhLDH-C4 protein could be obtained from 1000ml bacteria culture medium with IPTG induction.Only single band appeared on SDS-PAGE when 5μg purified rhLDH-C4 was loaded.The result of activity staining showed that the rhLDH-C4 appeared as a band near LDH-4. (2) Determination of ELISA detection parameters: Optimal concentration of coated rhLDH-C4 antigen was 1μg/ml. Cut-off value was 0.120. (3) ELISA results of serum samples: Positive percentage of anti-LDH-C4-...
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