为获得效价高、特异性好的氟苯尼考的小鼠多抗血清,建立氟苯尼考的免疫学检测方法。选用氟苯尼考琥珀酸钠为靶标,用活化酯法将其分别与牛血清蛋白(albumin from bovine serum,BSA)和卵清蛋白(ovalbumin,OVA)进行偶联,制备F-S-Na-BSA人...为获得效价高、特异性好的氟苯尼考的小鼠多抗血清,建立氟苯尼考的免疫学检测方法。选用氟苯尼考琥珀酸钠为靶标,用活化酯法将其分别与牛血清蛋白(albumin from bovine serum,BSA)和卵清蛋白(ovalbumin,OVA)进行偶联,制备F-S-Na-BSA人工免疫原和F-S-Na-OVA包被原,并用紫外扫描(UV)和SDS-PAGE法鉴定。用50μg/只剂量免疫8周龄的BALB/c小白鼠,制备小鼠多抗血清(pAb),用间接ELISA测定多抗血清(pAb)的效价和特异性。结果:220~350 nm内,F-S-Na-BSA和F-S-Na及BSA的最大吸收峰发生了位移;在SDS-PAGE凝胶电泳中,F-S-Na-BSA的迁移量比BSA略小,与预期的结果相符合。免疫的63只小鼠效价均达到1∶6 400以上;效价最高的1号小鼠IC_(50)≈10μg/m L。结论:F-S-Na-BSA偶联成功,注射小鼠获得了良好的免疫效果,但该多抗血清特异性较差。展开更多
The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to...The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.展开更多
基金Supported by Joint Funds of the NSFC and Henan Province(U1204327)Henan Provincial Key Laboratory Construction(122300413217)
文摘The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.
