1. Simultaneously targeting nitrocellulose and antibody by a dual-headed protein.
- Author
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Tran-Nguyen TS, Ngo-Luong DT, Nguyen-Phuoc KH, Tran TL, and Tran-Van H
- Subjects
- Cloning, Molecular, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Immobilized Proteins genetics, Immobilized Proteins immunology, Immunoglobulin Fc Fragments genetics, Immunoglobulin Fc Fragments immunology, Immunoglobulin G genetics, Immunoglobulin G immunology, Protein Binding, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Staphylococcal Protein A genetics, Staphylococcal Protein A immunology, Chromatography, Affinity methods, Collodion chemistry, Immobilized Proteins chemistry, Immunoglobulin Fc Fragments chemistry, Immunoglobulin G chemistry, Staphylococcal Protein A chemistry
- Abstract
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., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2021
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