Isolation of human antibodies against hepatitis E virus from phage display library by immobilized metal affinity chromatography |
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Authors: | Chen Ying-Wei Luo Wen-Xin Wang Ming-Qiao Wang Jin Li Li-Feng Yuan Quan Zhang Jun Xia Ning-Shao |
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Institution: | National Institute of Diagnostic and Vaccine Development for infectious diseases, The Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian, China. |
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Abstract: | Objective 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). Methods 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. Results 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. Conclusion 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. |
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Keywords: | HEV antibodies Phage antibody library Affinity chromatography Panning |
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