双抗体夹心ELISA法测定猪α-干扰素的浓度

Development of double antibody sandwich ELISA for determining the concentration of porcine α-interferon( PoIFN-α)

为了能够准确测定猪血清中猪α-干扰素（Po IFN-α）含量及猪α-干扰素制品的浓度,本研究以制备并经过亲和层析提纯的兔抗猪α-干扰素Ig G多克隆抗体为包被抗体,以制备的鼠抗猪α-干扰素单克隆纯化抗体作为二抗,建立了测定猪α-干扰素浓度的双抗体夹心ELISA法（DAS-ELISA）。通过条件优化,最佳兔抗猪α-干扰素Ig G多克隆纯化抗体包被浓度为12.5μg·m L^-1,最佳鼠抗猪α-干扰素单克隆纯化抗体作用浓度为6.25μg·m L^-1,利用本实验室制备的已知浓度猪α-干扰素标准蛋白建立标准曲线,确定该方法检测猪α-干扰素浓度范围为0.0781-2.50μg·m L^-1。与Lowry法、冻干称量法测定猪α-干扰素浓度相比较,3种方法检测结果无显著差异,但所建立的双抗体夹心ELISA法能够检测血清中猪α-干扰素浓度,且方法简便。

In order to accurately measure the concentration of porcine u-interferon （PoIFN-α） in swine serum and PoIFN-α product. Double antibody sandwich ELISA （DAS-ELISA） method was developed by using purified rabbit anti PoIFN-α IgG as capture antibody, and purified mouse anti PoIFN-α IgG as detection antibody. The result indicated that the best package concentration of rabbit anti porcine PoIFN-α IgG was 12. 5 μg·mL^- 1, and mouse anti PoIFN-α IgG was 6. 25 μg·mL ^- 1. The detection range of the DAS-ELISA was between 0. 078 1 - 2. 50 μg· mL^-1 by standard curve established using the known concentration of standard protein of PoIFN-α. Compared with the lowry method and freeze-drying weighing method, there was no significant difference between the three methods. And this established DAS-ELISA method was simple and could detect concentration of PoIFN-α in serum.