Isolation and characterization of single-chain Fv genes encoding antibodies specific for Drosophila Poxn protein

• Published in: FEBS letters 1998-10, Vol.437 (1), p.75-80
• Main Authors: Hassanzadeh Gh, Gholamreza, De Silva, Kumudu S.K., Dambly-Chaudière, Christine, Brys, Lea, Ghysen, Alain, Hamers, Raymond, Muyldermans, Serge, De Baetselier, Patrick
• Format: Article
• Language: English
• Subjects: ABTS, 2′,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium; Animals; Antibodies, Monoclonal - isolation & purification; Antibody Specificity; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; BSA, bovine serum albumin; cDNA, complementary DNA; CDR, complementarity determining region; Cloning, Molecular; Drosophila; Drosophila - chemistry; Drosophila Proteins; EDTA, ethylene diamine tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; Escherichia coli - genetics; Evaluation Studies as Topic; Genes, Immunoglobulin; globH5, globular domain of histone H5; HRP, horseradish peroxidase; Immunoglobulin Variable Region - genetics; Intrabody; IPTG, isopropylthio-β-d-galactoside; mAb, monoclonal antibody; Mice; Mice, Inbred NZB; Mice, Inbred Strains; NBT, nitrotetrazolium chloride blue; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - immunology; Paired Box Transcription Factors; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; Poxn; Recombinant Proteins - isolation & purification; scFv, single chain variable fragment; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Single-chain variable fragment; Transcription Factors; VH, heavy chain variable domain; VL, light chain variable domain
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)01204-6

The usefulness of intrabodies as specific inhibitors of gene function has been extensively demonstrated in cell culture assays. However, very few experiments have been conducted with intrabodies expressed in whole organisms. To evaluate the intrabody technology in Drosophila, we focused on poxn protein, since its effects can be easily studied. We purified the recombinant poxn protein. We next isolated three single-chain variable fragments (scFv) which specifically recognize poxn protein. Two scFvs, designated α-Poxn2 and α-Poxn4, react with both denatured and native Poxn with half maximal inhibition values of 100 nM and 40 nM, respectively. The α-Poxn5 scFv also recognizes denatured Poxn but either does not recognize native Poxn or its half maximal inhibition value for native Poxn is high.