为准确检测奶牛乳房炎乳中结合珠蛋白(Hp)含量,本研究采用牛血红蛋白(Hb)包被酶标板捕获乳清中Hp,利用特异性抗血清和酶标抗体建立夹心ELISA方法,并对建立的方法进行优化,用于检测牛奶中Hp含量.结果表明,ELISA方法检测下限为0.08μg/mL,与其他几种常见乳房炎乳中急性期蛋白质无交叉反应,批内试验和批间试验变异系数分别为3.27%～4.98%和5.46%～8.31%.应用本实验建立的夹心ELISA方法和商品化试剂盒分别检测50份已知患乳房炎牛乳清中Hp含量,同时对检测结果做相关性分析.结果表明,夹心ELSIA方法与商品化试剂盒具有很好的相关性(R2=0.996 2),同时验证了乳清中体细胞数与Hp含量呈正相关.夹心ELISA方法可准确测定牛奶中Hp含量,并可为奶牛乳房炎的诊断提供一种简易有效的辅助手段.%To determinate the concentration of haptoglobin (Hp) in milk of cows with mastitis, a sandwich ELISA was established by coating with bovine-hemoglobin to capture Hp in whey and detecting with the anti-Hp-antibody and ap-conjugatedantibody. The Hp detection limit of ELISA was 0.08 μg/mL. The ELISA test had no cross-reaction with other acute phase proteins of cows with mastitis. The variant coefficients of ELISA intra- and inter-batches were from 3.27％ to 4.98％ and from 5.46％ to 8.31％, respectively. Correlation between analytical results of the ELISA test and the commercial kits test was similar (R2=0.996 2) based on detection of 50 whey samples. This method could provide an simple and effective approach for the diagnosis of mastitis.
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