Simultaneously targeting nitrocellulose and antibody by a dual-headed protein
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...
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Veröffentlicht in: | Protein expression and purification 2021-01, Vol.177, p.105764-105764, Article 105764 |
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Sprache: | eng |
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Zusammenfassung: | 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.
•A fusion protein A-3-Helix was purified, and characterized.•rA-3-Helix helped antibody stereochemically immobilize onto nitrocellulose membrane.•This dual-headed protein maximized the exposure of Fab region of capturing antibodies.•rA-3-Helix laid the groundwork for regionally orienting antibody on the test strip. |
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ISSN: | 1046-5928 1096-0279 |
DOI: | 10.1016/j.pep.2020.105764 |