title: Expression and functional analysis of recombinant scFv and diabody fragments with specificity for human RhD.
abstract: In an attempt to generate recombinant anti-D reagents for possible diagnostic and therapeutic use we cloned the genes encoding the variable (V) domains of a human anti-D antibody secreted by the lymphoblastoid cell line BTSN4. A single-chain Fv (scFv) fragment was constructed using a 21 amino acid linker to join the genes encoding the variable domains of the BTSN4 heavy (VH) and light chains (VL). A diabody construct was also generated by reducing the length of the scFv linker from 21 to 10 residues. The scFv and diabody constructs were cloned into the pFLAG-CTS vector, expressed in E. coli host cells and the recombinant proteins were affinity-isolated from bacterial culture medium. Analysis of the recombinant proteins indicated that they retained the D antigen binding specificity of the parental BTSN4 IgG. Furthermore, both fragments mediated agglutination of papain-treated D positive erythrocytes in the absence of a cross-linking second antibody. While the agglutinating property of BTSN4 diabody was readily explained by the non-covalent association of this protein as a bivalent dimer, oligomeric forms of BTSN4 scFv were not detected when the protein was analysed by size exclusion chromatography. Thus, the agglutinating property of the scFv is not the result of the formation of non-covalently associated multimeric forms of the antibody fragment.
authors.affiliation: Immunobiology Unit, Department of Cell and Molecular Biology, University of Technology, Sydney, Broadway, NSW, Australia.
authors.forename: Parisa
authors.initials: P
authors.lastname: Asvadi
authors.forename: Anne
authors.initials: A
authors.lastname: Fletcher
authors.forename: Robert L
authors.initials: RL
authors.lastname: Raison
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D006386:Hemagglutination Tests; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007518:Isoantibodies / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012204:Rh-Hr Blood-Group System / Q000276:immunology*
chemical_list: D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D007518:Isoantibodies; C033242:RH-antibodies; D011994:Recombinant Proteins; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; C055966:immunoglobulin Fv