Functional enhancement of a partially active single-chain variable fragment antibody to Venezuelan equine encephalitis virus. 2003

Azhar Z Alvi, and Wei-Gang Hu, and R Elaine Fulton, and Jeffrey E Coles, and Melissa C Long, and Les P Nagata
AFAM ALVI Researchers and Consultants, Medicine Hat, Canada.

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.

UI MeSH Term Description Entries
D007128 Immunoglobulin Fragments Partial immunoglobulin molecules resulting from selective cleavage by proteolytic enzymes or generated through PROTEIN ENGINEERING techniques. Antibody Fragment,Antibody Fragments,Ig Fragment,Ig Fragments,Immunoglobulin Fragment,Fragment, Antibody,Fragment, Ig,Fragment, Immunoglobulin,Fragments, Antibody,Fragments, Ig,Fragments, Immunoglobulin
D007135 Immunoglobulin Variable Region That region of the immunoglobulin molecule that varies in its amino acid sequence and composition, and comprises the binding site for a specific antigen. It is located at the N-terminus of the Fab fragment of the immunoglobulin. It includes hypervariable regions (COMPLEMENTARITY DETERMINING REGIONS) and framework regions. Variable Region, Ig,Variable Region, Immunoglobulin,Framework Region, Immunoglobulin,Fv Antibody Fragments,Fv Fragments,Ig Framework Region,Ig Variable Region,Immunoglobulin Framework Region,Immunoglobulin Fv Fragments,Immunoglobulin V,Antibody Fragment, Fv,Antibody Fragments, Fv,Fragment, Fv,Fragment, Fv Antibody,Fragment, Immunoglobulin Fv,Fragments, Fv,Fragments, Fv Antibody,Fragments, Immunoglobulin Fv,Framework Region, Ig,Framework Regions, Ig,Framework Regions, Immunoglobulin,Fv Antibody Fragment,Fv Fragment,Fv Fragment, Immunoglobulin,Fv Fragments, Immunoglobulin,Ig Framework Regions,Ig Variable Regions,Immunoglobulin Framework Regions,Immunoglobulin Fv Fragment,Immunoglobulin Variable Regions,Regions, Immunoglobulin Variable,Variable Regions, Ig,Variable Regions, Immunoglobulin
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004666 Encephalitis Virus, Venezuelan Equine A species of ALPHAVIRUS that is the etiologic agent of encephalomyelitis in humans and equines. It is seen most commonly in parts of Central and South America. Encephalomyelitis Virus, Venezuelan Equine,Encephalitis Viruses, Venezuelan Equine,Venezuelan Equine Encephalitis Viruses,Venezuelan equine encephalitis virus,Virus, Venezuelan Equine Encephalitis,Viruses, Venezuelan Equine Encephalitis
D005803 Genes, Immunoglobulin Genes encoding the different subunits of the IMMUNOGLOBULINS, for example the IMMUNOGLOBULIN LIGHT CHAIN GENES and the IMMUNOGLOBULIN HEAVY CHAIN GENES. The heavy and light immunoglobulin genes are present as gene segments in the germline cells. The completed genes are created when the segments are shuffled and assembled (B-LYMPHOCYTE GENE REARRANGEMENT) during B-LYMPHOCYTE maturation. The gene segments of the human light and heavy chain germline genes are symbolized V (variable), J (joining) and C (constant). The heavy chain germline genes have an additional segment D (diversity). Genes, Ig,Immunoglobulin Genes,Gene, Ig,Gene, Immunoglobulin,Ig Gene,Ig Genes,Immunoglobulin Gene
D005818 Genetic Engineering Directed modification of the gene complement of a living organism by such techniques as altering the DNA, substituting genetic material by means of a virus, transplanting whole nuclei, transplanting cell hybrids, etc. Genetic Intervention,Engineering, Genetic,Intervention, Genetic,Genetic Interventions,Interventions, Genetic
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D000911 Antibodies, Monoclonal Antibodies produced by a single clone of cells. Monoclonal Antibodies,Monoclonal Antibody,Antibody, Monoclonal

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