基于α_s-酪蛋白特异性的牛乳总蛋白质ELISA定量检测方法的建立

Establishment of a quantitative ELISA method for detection of total milk protein based on the specificity of α_s-casein

本研究旨在建立牛乳中总蛋白质的ELISA定量检测方法,并评价该方法在牛乳掺假辨识中的应用效果。首先用αs-酪蛋白标准品免疫新西兰白兔,制备αs-酪蛋白特异性抗血清,并建立αs-酪蛋白的间接ELISA检测方法,结果显示血清效价为1∶640,αs-酪蛋白检测的线性范围是25-600 ng/ml,R2=0.999 5,回收率是96.6%-105.2%。再应用乳成分分析仪,凯氏定氮法和所建立的ELISA法测定8份来源不同的原料乳中总蛋白和αs-酪蛋白含量,结果显示不同牛乳中总蛋白质含量在0.029 2-0.029 8 g/ml,αs-酪蛋白含量在0.008 4-0.009 2 g/ml,占牛乳总蛋白质的比例恒定,平均为0.3,并用该系数建立牛乳中总蛋白质的定量方法。最后对5份人为兑水稀释、添加尿素、BSA和三聚氰胺的模拟掺杂牛乳样品进行检测,测定结果表明基于αs-酪蛋白的ELISA方法比乳成分分析仪法和凯氏定氮法具有更高的灵敏性和特异性,含氮添加物对测定结果无显著影响（P〉0.05）,更适合于作为原料乳及含乳产品中牛乳蛋白质的定量分析。

This study aims to establish a ELISA method to quantify the total protein（ TP） in milk and to evaluate its application in identification of the adulterated milk. Here,αs-casein（ αs-CN） standard sample was used to immunize New Zealand white rabbits for preparing anti-αs-CN polyclonal antibody（ Pc Ab）. The result showed that the titer of Pc Ab was 1∶640,and the linear range of the detection of αs-CN was 25 ~ 600 ng / ml with correlation coefficient of 0. 999 5 and the recovery rate between 96. 6% and 105. 2%. The contents ofαs-CN and TP in milk from 8 different resources ranged from 0. 008 4 to 0. 009 2 g / ml and from 0. 029 2 to 0. 029 8g / ml detected by milk analyzer and kjeldah method. The percentage of αs-CN to TP stayed constant at 0. 3,which led to the successful development of αs-CN-based ELISA for detection of TP in milk. The ELISA method showed better sensitivity and specificity than the other two approaches in four milk samples which were diluted with water and artificially added with urea,BSA and melamine,respectively. The content of αs-CN was independent of nitrogen additives. In conclusion,the established ELISA method was suitable for analyzing the TP content in the milk and milk products.