Detection The CD58 Content In Goat Serum

In this study, the surfactant protein CD58 of sheep red blood cells (SRBC) was prepared with enzymatic digestion and column chromatography. Rabbits were immunised with the methods of Freund's adjuvant and lymphocyte adsorption. The titer of anti-CD58 serum was tested with the establishment indirectly enzyme linked immnosorbent assay (ELISA). The optimised working concentrations of coated antigen and antibody were performed with phalanx titration. The inhibition of indirect competitive ELISA methods was established to detect the level of CD58 in goat serum, and used to detect the goat serum levels of CD58 estrous cycle and anestrus period. The results were as follows:1. The preparation of CD58 was tested with Rosette Inhibition Test, its inhibition rate was the percentage of 62.01±2.06. There were three kinds of proteins in the CD58 preparation from SDS-PAGE.2. Compared to Freund's adjuvant, the method of lymphocyte adsorption was obtained specific antibodies anti-CD58, antibody level was 1︰1×105, the method of using cell surface receptor and ligand to immune animals to prepare antibody is feasible.3. The indirect ELISA method, coated antigen 10.0μg·mL-1 CD58 dissolved in carbonate buffer solution (CBS, pH9.6, 0.05 mol·L-1), with HRP-labelled goat anti-rabbit IgG diluted by PBS (pH7.4, 0.1 mol·L-1) in l∶2 000 as a secondary antibody, 1.0% gelatinum as a blocking agent, was developed to detect antibody against CD58. The method's reproducibility was good results and its coefficient of variability (CV) was 2.48% ~ 7.78%.4. Establishment the indirect competitive ELISA to test the level of CD58 in goat serum, the linear range 20.0~500 000.0 ng·mL-1, regression equation Y = 0.207X-1.159 (R2 = 0.988), the lowest concentration of CD58 is 1.76 ng·mL-1, which can be detected theoretically. Statistical analysis indicated that the anestrus and the estrus period goat serum CD58 content is significant differences (P <0.05).In a word, the method of lymphocyte adsorption and indirect ELISA could be used to prepared specific antibodies anti-CD58 and test the content of CD58 in goat serum. The anestrus and the estrus period goat serum CD58 content was detected with indirect ELISA. It would be provided theoretical basis to study the different physiological goats in the further study.