ELISA检测奶牛乳房炎疫苗效果方法的建立

建立一种检测抗乳房炎疫苗抗体的酶联免疫吸附试验(ELISA)方法,用于免疫后具有保护性效果的抗体水平。通过对细胞抗原制备、最佳抗原包被浓度的确定、抗原包被和封闭最佳条件确定、酶标抗体的最佳工作浓度及作用时间等反应体系的筛选和确定,建立了检测抗乳房炎疫苗抗体的间接ELISA方法。结果表明,金黄色葡萄球菌(Staphylococcus aureus)、无乳链球菌(Streptococcus agalactiae)、停乳链球菌(Streptococcus dysgalactiae)抗原最佳包被质量浓度分别为5 mg.L-1、10 mg.L-1、10 mg.L-1;包被条件为4℃过夜;用2%人血白蛋白作为封闭剂最佳封闭条件是37℃封闭30 min;酶标抗体最佳作用时间为30 min。结论攻毒试验表明,疫苗免疫后,血清中抗体滴度达到或超过免疫前2倍时,奶牛即具有较好的乳腺保护效力。这一指标可作为疫苗效力检验的标准。

Development of Indirect ELISA for Detecting Cow Mastitis Vaccine

An indirect ELISA was developed by optimizing the concentraction of the coating antigen,and the incubating time of the serum and the sheep-against-cow IgG labeled by HRP.The developed indirect ELISA was primarily applied for detecting the antibody against mastitis.The results showed that the optimal coating concentration was 5 μg·mL-1 for Staphylococcus aureus,10μg/·mL-1 for Streptococcus agalactiae and 10μg·mL-1 for Streptococcus dysgalactiae.The optimal coating time was 12h at 4℃.The optimal reaction time of the 2% human albumin and the sheep against cow IgG was 30min.The results also indicated that the cow mastitis vaccine had protection effect when the antibody titer was double after cow being immune.