Construction and Characterization of a Novel Recombinant Single-Chain Variable Fragment Antibody Against Western Equine Encephalitis Virus

Abstract

A novel recombinant single-chain fragment variable (scFv) antibody against Western equine encephalitis virus (WEE) was constructed and characterized. Using antibody phage display technology, a scFv was generated from the WEE specific hybridoma, 10B5 E7E2. The scFv was fused to a human heavy chain IgG1 constant region (CH1-CH3) and contained an intact 6 His tag and enterokinase recognition site (RS10B5huFc). The RS10B5huFc antibody was expressed in E. coli and purified by affinity chromatography as a 70-kDa protein. The RS10B5huFc antibody was functional in binding to WEE antigen in indirect enzyme-linked immunosorbent assays (ELISAs). Furthermore, the RS10B5huFc antibody was purified in proper conformation and formed multimers. The addition of the human heavy chain to the scFv replaced effector functions of the mouse antibody. The Fc domain was capable of binding to protein G and human complement. The above properties of the RS10B5huFc antibody make it an excellent candidate for immunodetection and immunotherapy studies.

Preparation of single chain variable fragment of MG7mAb by phage display technology

“…Colony count and restriction analysis were conducted for evaluating the volume and quality of MG7 recombinant antibody library. The large volume of MG7 recombinant antibody library (2×10 6 ) and high recombinant rate (72.7%) confirmed that MG 7 phage antibody library comprised sufficient repertoire of recombinant clones for further research. ELISA assay and SDS-PAGE showed that the soluble MG 7 ScFv had antigen-binding activity and was M r 31.…”

“…Additionally, the B lymphocyte isolated from PBMC of immunized animals with given antigen is an alternative material source [20] . Besides the B lymphocyte population from patients or immunized animals, many kinds of antibody-producing hybridomas are also suitable as a kind of material source for construction of the recombinant phage antibody library [6,19] . Owing to the unraveling of biological functions over many antigen recognized by antibody from hybridomas, hybridomas are more favorable as material source for construction of recombinant phage antibody library.…”

“…One of the efficient solutions to this problem is to remove the constant region of antibody which makes main contribution to the immunogenicity of the murine antibody to human being. It has been proved that antibody devoid of constant region still maintains its capacity of specific antigen-binding affinity [4][5][6][7][8][9][10] . Additionally, antibody without constant region, termed ScFv, is a small molecule and comprises 1/6 of its original antibody in molecular mass.…”

Encephalitic Development in Alphaviral Infection

“…However, prophylactic or early (within 24 h) administration provided passive protection, but not sterilizing immunity (Wyckoff & Tesar, 1939). Some mouse monoclonal antibodies were shown some of these antibodies were shown to provide protection from infection following passive transfer of antibody (Long, et al, 2000;Long, et al, 2001). Mice were protected from challenge with WEE when injected with antibodies against E1 and E2 in passive transfer studies (Hunt & Roehrig, 1985;Yamamoto, 1986).…”

Development of Human and Macaque Antibodies Using Antibody Phage Display for the Detection of Equine Encephalitis Viruses

“…To our knowledge no anti-WEEV antibody fragments derived from any kind of library is published. However, scFvs were generated from the murine IgGs 11D2 (Das et al, 2004;Xu et al, 1999) and 10B5 E7E2 (Long et al, 2000) for diagnostic purposes. The authors of this review generated scFvs against WEEV by phage display using a macaque immune antibody gene library (Rülker et al in preparation), both for diagnosis and therapeutic purposes.…”