Functional enhancement of a partially active single-chain variable fragment antibody to Venezuelan equine encephalitis virus.
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.
Alvi
Azhar Z
AZ
AFAM ALVI Researchers and Consultants, Medicine Hat, Canada.
Hu
Wei-Gang
WG
Fulton
R Elaine
RE
Coles
Jeffrey E
JE
Long
Melissa C
MC
Nagata
Les P
LP
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
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region