半抗原直接包被的四环素酶联免疫吸附分析法的建立

Establishment of an Enzyme⁃Linked Immunosorbent Assay Employing Direct Coating of Hapten for Tetracycline

为建立更优的四环素(tetracycline,TC)酶联免疫吸附分析法(enzyme-linked immunosorbent assay,ELISA),将TC与蛋白质偶联制备人工完全抗原免疫BALB/c小鼠,获得抗TC的单克隆抗体(monoclonal antibody,MAb)。采用酸性高锰酸钾羧化酶标板,将TC直接包被在酶标板上,并对ELISA反应体系条件进行优化,构建一种半抗原直接包被的TC ELISA检测方法。所制备的MAb特异性良好,交叉反应率低。所构建的检测方法最低检测限IC_(10)值为0.306 ng/mL,半数抑制率IC_(50)值为9.26 ng/mL,牛奶和水中加标回收率分别为96.46%~101.89%、96.84%~102.50%。与人工完全抗原包被的检测方法相比(IC_(10)值:0.624 ng/mL,IC_(50)值:28.24 ng/mL,牛奶和水中加标回收率分别为93.86%~105.12%、94.04%~105.56%),检测灵敏度有明显提高,并可用于实际样品中TC含量的检测,具备良好的应用前景。

An improved enzyme⁃linked immunosorbent assay(ELISA)for the detection of tetracycline(TC)was established.BALB/C mice were immunized with the artificial complete antigen conjugating TC and pro⁃tein,and anti⁃TC monoclonal antibody(MAb)was then prepared.Acidic potassium permanganate was used to carboxylate the ELISA plate,and TC was coated directly on the plate.The ELISA reaction system was opti⁃mized to establish an ELISA method employing direct coating of hapten for the detection of TC.The prepared MAb had good specificity and low cross⁃reaction rate.The established method showed the lowest detection limit IC_(10)of 0.306 ng/mL,the half inhibition rate IC_(50)of 9.26 ng/mL,and the recoveries of tetracycline being 96.46%-101.89%and 96.84%-102.50%,respectively,in milk and water.Compared with the method with the coating of artificial antigen(IC_(10):0.624 ng/mL,IC_(50):28.24 ng/mL,tetracycline recoveries of 93.86%-105.12%and 94.04%-105.56%in milk and water,respectively),the established method showed signifi⁃cantly improved detection sensitivity and can be used for the determination of TC content in actual samples,demonstrating a promising application prospect.