Construction and characterization of a novel recombinant single-chain variable fragment antibody against Western equine encephalitis virus.
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.
Long
M C
MC
Medical Countermeasures Section, Defence Research Establishment Suffield, Medicine Hat, Alberta, Canada.
Jager
S
S
Mah
D C
DC
Jebailey
L
L
Mah
M A
MA
Masri
S A
SA
Nagata
L P
LP
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000956:Antigens, Viral / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D004667:Encephalitis Virus, Western Equine / Q000276:immunology*; D006801:Humans; D006825:Hybridomas; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
D000914:Antibodies, Viral; D000956:Antigens, Viral; D007141:Immunoglobulin Fc Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins