| Dairy cow mastit is the most important disease which affect the development of dairy industry and cause great economic losses in the world. Staphylococcus aureus(S.aureus) is one of the main pathogenic bacteria of this diseases.S.aureus is a normal bacteria in nature, in clinic, there are many capsular polysaccharide(CP) existence on the surface of S.aureus which separate form sick animals. Until now, CP can be classified 11 different serotypes, in these serotypes, CP5 and CP8 occupied more than 80%, they are not only virulence factor but also protective antigen to increase the virulence of Staphylococcus aureus. It is difficult to diagnosis and treatment the S.aureus disease because there so many different serotypes of CP. So it is necessary to establish a rapid and accurate diagnostic methods to identification the serotypes of CP. The purpose of this study is produce a secrecting CP5 Monoclonal antibody(McAb) and use this McAb establish of ELISA detection methods. This method have rapid, accurate, and efficient characteristics.Choose the Colombia liquid medium for culture of Staphylococcus aureus, this medium can increase the production of S.aureus.Totally, 120L medium was be used. Then, purified CP5 was prepared by process consisting of extraction with cetrimide(CTAB) and phenol, precipitation by ethanol, centrifugation, and so on, 157.47mg CP5 was acquired. These CP5 was be certified by spectrophotometer because there was the largest absorption peak in the 206nm. Through the phenol - sulfuric acid method, the purity of CP5 is 72.48%.There are no structures in CP5 that can produce antibodies. The molecular weight of CP5 is not big enough. In order to produce antibodies to it, the hapten must be immunogenic by coupling it to a carrier protein. We obtain complete antigen CP5-BSA through couple with protein BSA by using carbodiimide(EDC). The conjugates CP5-BSA was analyzed by UV absorbance method, the molecule conjugate ratio of CP5 to BSA was 2.88:1. The Balb/C mice were immunized with the man-made antigen CP5-BSA, and screened it with amination ELISA plate. Splenocytes from immunized mice were fused with SP2/0 myeloma cells. Two hybridomas of stable secrecting CP5-McAb were selected and were named 2F3and 5E8. The subclasses of McAb 2F3 was IgG1, 5E8 was IgG2a. The supernatant titer were all above 1:500, the ELISA titers of purification ascites was 1:105. Relative affinity showed2F3>5E8. According to competitive principle, an ic-ELISA method was established for the quantitative detection of CP5 and its optimized reaction condition as follow: The CP5 was diluted to microtiter plates at a concentration of 1:400 and incubated overnight at 37℃,then blocking it with 10% goat blood serum incubated 1h at 37℃, Then goat anti-mouse IgG-HRP(1:2000) was added and incubated 1h at 37℃. OPD was chosen to be enzyme reaction substrate. Followed by the addition of stopping solution(2M H2SO4) after 25min of incubated in the dark at 37℃. Absorbance at 490nm was determined by an enzyme immunoassay reader. Established the ic-ELISA method A linear dose-response standard curve was prepared by plotting log[CP5] versus percent inhibiting. The regression equation of standard curve was y=-1.6629x+4.9697, the correlation coeffecient R2=0.9901, the lower limit detection was 62.5ng/ml, rang from 62.5～4000ng/ml. We can accurate determine the serotype of CP as well as do epidemiological survey.
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