Generation and oriented evaluation of nitrocellulosebinding achor protein fused protein A and its application

Abstract

Point-of-care testing diagnostics are current interest in research and development. Among them, Lateral Flow Test (LFT) is an on-site test strip with wide range of applications. Therefore, optimizing the sensitivity of rapid test strip is a necessary requirement. Immobilizing antibodies onto the nitrocellulose membrane is an important step to increase the sensitivity of the LFT strip for detecting pathogenic antigens. In this 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 specifically binds to the Fc tail of IgG antibody was generated. This fusion protein was expected to help IgG antibodies strongly binding onto the nitrocellulose membrane and orientating the Fab of immobilized antibodies outwardly, thereby increasing the ability to capture the target antigen. The recombinant vector pET22b-proA and pET22b-proA-3-helix encoded for protein A and A-3-helix, respectively were subsequently cloned. Protein A and A-3-helix was induced to express using IPTG, and confirmed by SDS-PAGE and Western blot. These proteins were purified by immobilized metal affinity chromatography with the purity of more than 90%. The purified proteins were used to evaluate orientation binding on nitrocellulose membranes by the lateral flow test and antibidy capture as well. Results showed that protein A 3 helix bound to nitrocellulose membrane better than that of protein A. The former proteins increased the antibody binding and stereochemical immobilizing onto nitrocellulose membrane compared to its protein A counterpart. The present results laid the foundation for the application of fusion proteins in the directed immobilization of antibodies in rapid test strips.