title: Isolation of human antibodies against hepatitis E virus from phage display library by immobilized metal affinity chromatography.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Ying-Wei
authors.initials: YW
authors.lastname: Chen
authors.forename: Wen-Xin
authors.initials: WX
authors.lastname: Luo
authors.forename: Ming-Qiao
authors.initials: MQ
authors.lastname: Wang
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Li-Feng
authors.initials: LF
authors.lastname: Li
authors.forename: Quan
authors.initials: Q
authors.lastname: Yuan
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Ning-Shao
authors.initials: NS
authors.lastname: Xia
mesh_terms: D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001435:Bacteriophages / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D016752:Hepatitis E virus / Q000276:immunology*; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D008670:Metals; D008969:Molecular Sequence Data
chemical_list: D000914:Antibodies, Viral; D007093:Imidazoles; D008670:Metals