抗羟基红花黄色素A抗血清的制备-经济学家.PDF

150 2009, Vol. 30, No. 03 食品科学 ※生物工程 侯世斌，吕 芳，苏幼红，张富春，李江伟﹡ (新疆大学生命科学与技术学院，新生物资源与基因工程重点实验室，新疆乌鲁木齐 830046) ：目的：通过人工抗原的构建，制备抗羟基红花黄色素A(HSYA) 的特异性抗血清。方法：采用直接合成 法将HSYA 与牛血清白蛋白(BSA)和卵清蛋白(OVA)的共价偶联，分别合成免疫抗原HSYA-BSA 和包被抗原HSYA- OVA ，并进行相关鉴定；用合成的人工抗原免疫家兔，制得抗羟基红花黄色素A 的特异性抗血清，采用酶联免 疫法鉴定。结果：根据建立的标准曲线方程计算得HSYA 和BSA 的结合比是50:1 ，HSYA 和OVA 的结合比是 112.8:1；理想的包被抗原的浓度为4 μg/ml ，抗血清和二抗的工作浓度分别为1:200 和1:2000；交叉反应实验表明， 该抗体与芦丁、金合欢素的反应率均接近于零；亲和性实验结果表明，在0.25～2 μg/ml 浓度范围内，抑制率与浓 度呈线性关系，线性回归方程为y= － 16.213lgx+102.47(r=0.9575)，抑制中浓度IC50 为1.5μg/ml ，最低检测限为 0.250 μg/ml 。结论：通过直接偶联法可以成功制备出具有抗羟基红花黄色素A 的特异性抗血清。 ：羟基红花黄色素A ；半抗原；抗血清；酶联免疫吸附测定 Preparation of Hydroxysafflor Yellow A Antiserum . . HOU Shi-bin ，LU Fang ，SU You-hong， ZHANG Fu-chun ，LI Jiang-wei* (Key Laboratory of Biological Resources and Genetic Engineering of Xinjiang, College of Life Science and Technology, Xinjiang University,Urumqi 830046,China) Abstract ：Objective: To construct artificial immunogen and prepare specific antiserum of hydroxysafflor yellow A (HSYA). Method Immunogen HSYA-BSA and coating antigen HSYA-OVA were prepared by immediate coupling method, and the characterizations of artificial antigen were conducted in color change, ultraviolet and infrared radiation spectrum shift.The polyclonal antiserum was produced from two rabbits immunized with conjugates HSYA-BSA, and then the antiserum was assayed with indirect ELISA method. Results: The ideal coating antigen concentration is 4μg/ml, the available anterium dilution 1:200, and the ideal IgG-HRP dilution is 1:2000. The affinity studies showed that the real detection limit of ELISA for standards is 250 ng/ml with an I50 value of 1.5μg/ml. The