Library Construction And Screen Of Phage-based Humanized Fab Antibody Against Enterovirus71

Objective To obtain human monoclone antibodies which wasneutralizing antibody and was specific for EV71.To provide alternative solutions inclinical prevention for HFMD caused by EV71. Methods The in directimmunofluorescence assay (IFA) and the neutralization assay were used to detect theantibody against EV71existed in peripheral blood specimens from16adultvolunteers. The lymphocytes from a volunteer, whose anti-EV71neutralizingantibody titrated as1:128, were isolated. The total RNA was extracted from theisolated lymphocytes and the cDNAs was synthesized. The genes of Fd fragment andlight chain of antibodies were amplified by a set of human IgG Fab antibody primersand then were cloned into phagmid vector pComb3.After successful insertion of lightand heavy chains, the insertion rate of light and heavy chains was verified. Helperphage VCSM13was used to infect recombinant bacterial XL1-Blue for package ofFab phage library. Based on the phage antibody library constructed as above, the Fabwas screened by panning and enrichment by using SP70, a polypeptide indentifiedpreviously as the linear neutralization epitopes on the VP1capsid protein of the EV71.After three rounds of the panning and enrichment, the recombinant phage infection ofE.coli XL1-Blue was induced and expressed. The supernatants from induced bacteriawere screened by SP70coated on a high adsorption96-well plate. Result (1) Aphage antibody library with size108was constructed. Insertion rate of light and heavychain inserts were nearly100%, indicating that it has successfully constructed ahumanized anti-EV71virus Fab phage antibody libraries.(2) The51human Fabclones were selected with reactivity to SP70and anti-Fab antibody by ELISA.Referring to Internet V-Base database and IMGT/V-QUEST database，sequenceanalysis reveals that51Fab clones could catograted into three unique clones. ELISAexperiments showed that these antibodies are specific for SP70. Conclusion Insummary, by using phage display technology, we successfully constructed ahumanized anti-EV71virus Fab phage antibody library and screened out three humanFab antibodies with specific reactivity to SP70, the linear neutralization epitope on theVP1capsid protein of the EV71.