间接ELISA检测奶牛宫颈黏液ASA免疫不孕

Detection of Immune Infertile Dairy Cows with ASA in Cervical Mucus by Indirect ELISA

为建立检测奶牛宫颈黏液ASA免疫不孕的间接ELISA方法,以精子膜蛋白为包被抗原,进行奶牛宫颈黏液ASA的间接ELISA的优化试验。对27头ASA阳性不孕和29头ASA阳性可孕奶牛进行了间接ELISA检测标准的确定试验;对137头奶牛的宫颈黏液ASA免疫不孕进行间接ELISA临床检测。奶牛宫颈黏液ASA的间接ELISA优化条件为:抗原包被量为5μg/mL,宫颈黏液稀释度为1:5,宫颈黏液反应时间1h,二抗反应时间1.5h;奶牛宫颈黏液ASA免疫不孕检测的判定标准为:当样品OD490nm值大于0.513时判定为ASA阳性免疫不孕,当样品OD490nm值小于0.410时可孕,当样品OD490nm值介于0.410~0.513时为ASA阳性疑似,重复试验的变异系数均小于10%。对临床137份宫颈黏液样品进行检测,发现13头ASA阳性免疫不孕奶牛都不能受孕,而98头阴性结果牛中有89头怀孕。结果表明,间接ELISA可以有效检测奶牛宫颈黏液ASA免疫不孕。

In order to establish an indirect ELISA method for detection of cervical mucus ASA immune infertility in dairy cows, this study was conducted using sperm membrane protein as coating antigen, and the optimization of indirect ELISA for dairy ASA of cervical mucus was tested. Totally 27 ASA positive infertile cows and 29 ASA positive pregnant cows were tested to determine indirect ELISA detection standard. The cervical mucus ASA from the 137 cows was detected using the indirect ELISA respectively. It was the indirect ELISA optimization condition for cervical mucus ASA in dairy cows that antigen coated amount was 5μg/mL, cervical mucus dilution was 1:5, cervical mucus reaction time was 1h, and reaction time of the enzyme-labeled second antibody was 1.5h. The criteria for cervical mucus ASA immune infertility in dairy cows was that the OD490nm values higher than 0.513 were determined as ASA positive immune infertility for the indirect ELISA and lower than 0.410 as negative fertility, and the OD490nmvaluesbetween0.410and0.513weredeterminedasASApositivesuspectedcases.Thecoefifcient of variation for the method was less than 10% in the repeated trials. The results of the 137 clinical samples of cervical mucus showed that the 13 ASA positive immune infertile cows could not conceive, and there were the 89 pregnancies among the 98 dairy cows with negative results. It can be concluded that the indirect ELISA was an effective method on investigation of ASA in cervical mucus of immune infertility dairy cows.