title: Functional enhancement of a partially active single-chain variable fragment antibody to Venezuelan equine encephalitis virus.
abstract: Previously cloned recombinant A116 single chain fragment variable (scFv) antibody gene has been re-engineered for enhanced reactivity to Venezuelan equine encephalitis virus (VEE) successfully. A PCR-based site-directed mutagenesis approach was adopted to re-introduce the three single-base deletions in the 5' region of the V(L) gene of A116, corresponding to the framework-1 region. The mutagenized A116 was designated as MA116. The introduction of these three bases corrected a localized frame-shift to a consensus framework-1 amino acid sequence. Four MA116 clones (MA116-4, MA116-14, MA116-15, and MA116-16) have been analysed in detail for their reactivity to VEE antigen, and all showed varying degrees of reactivity to VEE antigen. ScFv antibody expressed by MA116-14, MA116-15, and MA116-16 clones showed three to five-fold enhanced enzyme-linked immunosorbant assay reactivity to VEE antigen over the parental A116 clone, while scFv antibody from MA116-4 was less reactive than A116 clone. MA116-15 purified scFv protein showed comparable reactivity to the parental 1A4A-1 monoclonal antibody in recognizing VEE antigen. Sequence analysis revealed that only MA116-15 had incorporated the three intended base insertions. The varying degrees of reactivity of MA116 clones are discussed in light of their molecular changes.
authors.affiliation: AFAM ALVI Researchers and Consultants, Medicine Hat, Canada.
authors.forename: Azhar Z
authors.initials: AZ
authors.lastname: Alvi
authors.forename: Wei-Gang
authors.initials: WG
authors.lastname: Hu
authors.forename: R Elaine
authors.initials: RE
authors.lastname: Fulton
authors.forename: Jeffrey E
authors.initials: JE
authors.lastname: Coles
authors.forename: Melissa C
authors.initials: MC
authors.lastname: Long
authors.forename: Les P
authors.initials: LP
authors.lastname: Nagata
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000914:Antibodies, Viral / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D004666:Encephalitis Virus, Venezuelan Equine / Q000276:immunology*; D005803:Genes, Immunoglobulin; D005818:Genetic Engineering / Q000379:methods; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017422:Sequence Analysis, DNA
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region