应用二抗技术建立灵敏检测奶中孕酮含量的酶联免疫分析法（EIA）  

DEVELOPMENT OF A SENSITIVE ENZYME IMMUNOASSAY(EIA)FOR DETERMINATION OF PROGESTERONE IN MILK BY A SECOND ANTIBODY TECHNIQUE

作　　者：曾宪垠[1] 郭大智[1] 石兴文 

机构地区：[1]四川农业大学原子能农业应用研究室,四川农业大学奶牛场

出　　处：《核农学报》1996年第1期11-15,共5页Journal of Nuclear Agricultural Sciences

摘　　要：本研究应用二抗技术建立了灵敏检测奶中孕酮含量的酶联免疫分析法（ＥＩＡ）。微量滴定板首先包被二抗（羊抗兔ＩｇＧ），４℃孵育过夜。免疫反应溶液中包括２μｌ的奶样（用分析缓冲液稀释至２０μｌ），５０μｌ酶标孕酮和５０μｌ抗体。标准曲线的测试范围为０．２～１２．５ｐｇ／孔，相当于０．１～６．２５ｎｇ／ｍｌ，最小可测量０．１５ｐｇ／孔，５０％结合率对应的孕酮浓度是０．９２ｐｇ／孔。样品测定的平均批内与批间变异系数分别是４．６８％和１１．１０％。样品测定的回收率为９２．６７±１０．７９％。应用该方法测定全奶样品所得结果与应用ＲＩＡ测定结果高度相关（ｒ＝０．９７，ｎ＝４８，ｐ＜０．００１）。结果表明：该方法的灵敏度是本室ＲＩＡ的８０倍，与直接包被孕酮抗体的ＥＩＡ相比较，可以节约珍贵的抗孕酮抗体和酶标孕酮。A sensitive enzyme immunoassay(EIA)for determination of progesterone in whole mllkusing a second antibody technique was developed in this study.The microtitration plates usedin the assay were first coated with sheep anti-rabbit IgG and incuhated at 4℃ overnight.Theimmunoreaction was performed by incubating a mixture of 2μl of milk sample(diluted to 20μlwith assay buffer),50μl of enzyme labelled progesterone and 50μl of antibody against pro-gesterone. The standard curve was sensitive in the range of 0.2pg/well～12.5pg/well,corre-sponding to 0.1ng/ml～6.25ng/ml.The detecting limit of the assay is 0.15pg/well and themild-point relative binding(50%relative binding)is at 0.92 pg/well. The intra-and inter-assay coefficients of variation were 4. 68%and 11.10% respectively. Recovery of the assayis 92.67±10.79%. Milk samples from dairy cows were tested by running parallel RIA andEIA. A good correation (r= 0.97,n=4.8,P<0.001 ) was obtained and the estimated valueswere similar in both techniques.The results show the EIA developed in this study not onlyprovides very reliable assay,with a sensitivity 80 times higher than the RIA previously vali-dated in the laboratory,but also reduces the amount of valuable antibody and enzyme label incomparlson to direct coating.

关 键 词：奶 孕酮 酶联免疫分析 二抗 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]