RESUMO
Previously, an mAb 10F7 was developed against H5N1 hemagglutinin, which was highly specific to 34 different H5N1 strains and showed good neutralizing activity. In the present study, the single-chain fragment of the antibody was cloned into a prokaryotic vector and then expressed in E. coli. The activity of the scFv was tested in hemagglution inhibition and neutralization experiment. Two H5N1 virus strains were inhibited to bind erythrocyte cells by the scFv while the H9 virus was not. Also, five H5N1 virus strains were neutralized during infecting MDCK cells. These results showed an approachable method for developing therapeutic antibody to H5N1 virus.
Assuntos
Animais , Sequência de Aminoácidos , Anticorpos Antivirais , Genética , Alergia e Imunologia , Metabolismo , Especificidade de Anticorpos , Alergia e Imunologia , Aves , Virologia , Linhagem Celular , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Virus da Influenza A Subtipo H5N1 , Alergia e Imunologia , Influenza Aviária , Virologia , Testes de Neutralização , Proteínas Recombinantes , Alergia e Imunologia , Metabolismo , Anticorpos de Cadeia Única , Genética , Alergia e Imunologia , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To isolate human antibodies against hepatitis E virus from phage display library by a new method of panning phage antibody library based on immobilized metal affinity chromatography (IMAC).</p><p><b>METHODS</b>Phage antibody library was allowed to mix with hex-His tagged expressed HEV specific antigen, NE2, in solution for adequate binding before affinity resin for hex-His was added. The non-specific phage antibodies were removed by extensive washing and the specific bound phage antibodies could then be eluted to infect TG1 or repeat the binding process for subsequent rounds of purification. The specificity of the selected human antibodies were tested by antigen competitive ELISA, human sera blocking ELISA, scFv expression, and sequence analysis.</p><p><b>RESULTS</b>His-NE2 specific recombinant phages were successfully enriched after panning procedure. Two individual phage clones, 126 and 138, showed 50% inhibition in NE2 antigen competition ELISA and obvious blocking effect by HEV positive serum in blocking ELISA. Soluble scFv of 126, 138 bound to NE2 specifically.</p><p><b>CONCLUSION</b>Two specific human phage antibodies against hepatitis E virus (HEV) from phage display library were isolated by immobilized metal affinity chromatography. The immobilized metal affinity chromatography applied to phage antibody selection was a helpful supplement to the selection in solution.</p>