Immobilizing antibodies on the nitrocellulose membrane is an important step to increase the sensitivity of the Lateral Flow Test strip for detecting pathogenic antigen. In our research, the fusion protein between nitrocellulose-binding anchor protein 3-Helix – a protein that has a strong affinity to nitrocellulose membrane and protein A – a protein that can bind to the Fc tail of IgG antibody was generated. This fusion protein was expected to help IgG antibodies to be more strongly binding and oriented immobilized onto the nitrocellulose membrane. The recombinant vector pET22b-proA and pET22b-proA-3-Helix coded for protein A and protein A-3-Helix were cloned. These proteins were overexpressed in BL21 and purified by immobilized metal affinity chromatography with purity above 90%. The purified protein was used to evaluate the orientation binding on nitrocellulose membranes by lateral flow challenge. Results showed that protein A-3-Helix binding to nitrocellulose membrane was better than that of protein A. The former protein increased antibody binding and stereochemical immobilizing onto nitrocellulose membrane compared to its protein A counterpart. In summary, we have succeeded in cloning, purifying, and characterizing a dual-head recombinant protein A and protein A-3-Helix. The results show the potential application of protein A-3-Helix in the immobilizing antibody on the test strip.

将抗体固定在硝酸纤维素膜上是提高侧向流动检测条检测病原性抗原灵敏度的重要步骤。在我们的研究中，产生了硝酸纤维素结合锚蛋白3-Helix（一种对硝酸纤维素膜具有很强亲和力的蛋白）与A蛋白（一种可以与IgG抗体的Fc尾部结合的蛋白）之间的融合蛋白。预期该融合蛋白可帮助IgG抗体更牢固地结合并定向固定在硝酸纤维素膜上。重组载体pET22b - proA和pET22b -proA-3-Helix编码蛋白质A的蛋白质，并克隆蛋白质A-3-Helix。这些蛋白质在BL21中过表达，并通过固定化金属亲和色谱纯化，纯度超过90％。纯化的蛋白质用于通过侧向流动挑战评估硝酸纤维素膜上的方向结合。结果表明，蛋白A-3-Helix与硝酸纤维素膜的结合优于蛋白A。与蛋白A对应物相比，前一种蛋白增强了抗体的结合，并将立体化学固定在硝酸纤维素膜上。总之，我们已经成功地克隆，纯化和鉴定了双头重组蛋白A和蛋白A-3-Helix。结果显示蛋白A-3-Helix在测试条上的固定抗体中的潜在应用。