title: Polyclonal antibodies to glutathione S-transferase--verotoxin subunit a fusion proteins neutralize verotoxins.
abstract: The A1 subunits of verotoxin-1 (VT1) and VT2 genes were cloned into pGEX-4T-2 for the expression of glutathione S-transferase (GST) fusion proteins. The N-terminal and the transmembrane regions of the A1 subunits were excluded from the constructs in order to increase the product yields. Polyclonal anti-VT1A1 and anti-VT2A1 antibodies were produced by immunizing rabbits with GST-VT1A1 and GST-VT2A1 fusion proteins, respectively. The antibodies were tested for their ability to neutralize active toxins from 45 VT-producing Escherichia coli (VTEC) strains. The antibodies had significantly high neutralizing activities against their homologous toxins. The average percentages of neutralization of VT1 by anti-GST-VT1A1 and anti-GST-VT2A1 were 76.7% +/- 7.9% and 3.6% +/- 2.3%, respectively, and those of VT2 were 1.7% +/- 2.3% and 82.5% +/- 13.9%, respectively. VT2 variant toxin was neutralized by anti-GST-VT2A1, with cross neutralization being a possible consequence of sequence homology between VT2 and a VT2 variant. To our knowledge, this is the first report on the production of polyclonal antibodies from GST-VT fusion proteins. The antibodies were shown to exhibit specific toxin neutralizing activities and may be useful for immunological diagnosis of VTEC infections.
authors: Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Special Administrative Region, People's Republic of China.
authors: P H M
authors: PH
authors: Leung
authors: J S M
authors: JS
authors: Peiris
authors: W W S
authors: WW
authors: Ng
authors: W C
authors: WC
authors: Yam
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003601:Cytotoxicity Tests, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005982:Glutathione Transferase / Q000235:genetics; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D022622:Shiga Toxin 1 / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D022641:Shiga Toxin 2 / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D011993:Recombinant Fusion Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D005982:Glutathione Transferase