检测牛乳κ-酪蛋白的间接竞争ELISA法与间接ELISA法比较

试验旨在建立一种快速简单检测牛乳κ-酪蛋白（κ-CN）含量的酶联免疫吸附（ELISA）方法,为解决牛乳蛋白掺假问题提供一定的技术支持。以牛乳κ-CN为包被抗原,以酶标抗体（HRP-IgG）为检测抗体分别建立间接竞争ELISA法和间接ELISA法,并对两种检测方法进行分析比较。结果表明：对于间接竞争ELISA法,κ-CN抗原包被浓度为2.5 μg/mL,线性范围为62.5~1 000.0 ng/mL,变异系数< 2%,回收率在98.46%~101.68%;对于间接ELISA法,κ-CN抗原包被浓度为1.56 μg/mL,线性范围为0.098~3.125 μg/mL,变异系数< 1%,回收率在99.10%~101.06%。比较两种检测方法,间接竞争ELISA法检测耗时较短,抗体应用量较少,而间接法不需要包被成本较高的目标标准蛋白,相关系数也较高,但其检测体系组成成分较多,且需包被待测样品,较难组成ELISA试剂盒体系,不宜用于现场检测。因此,大规模制备ELISA试剂盒体系用于快速检测蛋白含量时可采用间接竞争法,不仅抗体应用量少、节约成本且快速、简单,可更好地用于科研实践。

Comparison Between Indirect Competitive ELISA and Indirect ELISA in Detecting Bovine κ-casein

The study was aimed to develop a rapid and simple enzyme-linked immunosorbent assay (ELISA) method for the detection of milk κ-casein (κ-CN) content and provide technical services for solving the problem of adulterated milk protein.The indirect competition ELISA method and the indirect ELISA method were respectively established by taking the milk κ-CN as the envelope antigen and the enzyme labeled antibody (HRP-IgG) as the detection antibody,and the two detecting methods were analyzed and compared.The results showed that the κ-CN antigen concentration was 2.5 μg/mL,the linear range was 62.5 to 1 000.0 ng/mL,the coefficient of variation was less than 2%,the recovery rate was 98.46% to 101.68% when using the indirect competitive ELISA method; The κ-CN antigen concentration was 1.56 μg/mL,the linear range was 0.098 to 3.125 μg/mL,the coefficient of variation was less than 1%,the recovery rate was 99.10% to 101.06% for the indirect ELISA method.Comparing these two methods,the indirect competitive ELISA method took shorter time and the amount used of antibody was less.The indirect ELISA method did not need to cover the higher cost of protein,and the correlation coefficient was higher.The latter was difficult to form ELISA kit system because of its more components and samples need to be coated,so it was not suitable for field testing.The indirect competition method could be used for the large-scale preparation of ELISA kit system for rapid detection of protein content,because of its low dosage of antibodies,cost savings,fast and simple operation,was better for practical applications.