A single chain Fv specific against Western equine encephalitis virus.
A recombinant single chain Fv (scFv) specific against Western equine encephalitis virus (WEE) was developed and characterized. The scFv was generated from 11D2 hybridoma producing anti-WEE antibody reactive to E1 component of viral envelope glycoprotein. V(L) and V(H) gene segments of 11D2 scFv were generated and joined together with a (gly4ser)3 linker by polymerase chain reaction (PCR). The resulting scFv was successfully expressed in P. pastoris expression system. Fifteen individual plasmids were tested and six of them were shown to drive scFv expression. DNA sequence analysis from three productive plasmids showed that they all carried the same VL and V(H) gene segments with a few base differences. Comparison of 11D2 scFv DNA sequence to the Kabat database showed that VH of 11D2 antibody belonged to subgroup IIID and subfamily XIV, while VL domain did not belong to any known subgroup or subfamily. Western blot analysis of 11D2 scFv using anti-c-myc antibody for detection showed different band pattern among clones derived from different plasmids. This was thought to be due to the different glycosylation where amino acid substitution occurred. Successful purification of 11D2 scFv could be done by immobilized metal affinity chromatography with an unoptimized yield of 700 microg/L. Functional studies showed that 11D2 scFv could bind to its respective WEE antigen as demonstrated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). The binding affinity of 11D2 scFv is reasonably good compared to the parental 11D2 bivalent monoclonal antibody (MAb). Thus, 11D2 scFv and its derivatives have a potential use as immunotherapeutic and immunodiagnostic agents of WEE infections.
Xu
B
B
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada.
Kriangkum
J
J
Nagata
L P
LP
Fulton
R E
RE
Suresh
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000276:immunology; D001483:Base Sequence; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D004667:Encephalitis Virus, Western Equine / Q000276:immunology*; D015870:Gene Expression / Q000276:immunology; D005803:Genes, Immunoglobulin / Q000235:genetics / Q000276:immunology; D006825:Hybridomas / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D013552:Swine; D014759:Viral Envelope Proteins / Q000276:immunology
D000914:Antibodies, Viral; D000956:Antigens, Viral; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D014759:Viral Envelope Proteins