title: Shiga toxin-induced apoptosis is more efficiently inhibited by dimeric recombinant hybrid-IgG/IgA immunoglobulins than by the parental IgG monoclonal antibodies.
abstract: Shiga toxin 1 (Stx1) is a virulence factor of enterohaemorrhagic Escherichia coli strains such as O157:H7 and Shigella dysenteriae. To prevent entry of Stx1 from the mucosal surface, an immunoglobulin A (IgA) specific for Stx1 would be useful. Due to the difficulty of producing IgA monoclonal antibodies (mAb) against the binding subunit of Stx1 (Stx1B) in mice, we took advantage of recombinant technology that combines the heavy chain variable region from Stx1B-specific IgG1 mAb and the Fc region from IgA. The resulting hybrid IgG/IgA was stably expressed in Chinese hamster ovary cells as a dimeric hybrid IgG/IgA. We separated the dimeric hybrid IgG/IgA from the monomeric one by size-exclusion chromatography. The dimer fraction, confirmed by immunoblot analyses, was used for toxin neutralization assays. The dimeric IgG/IgA was shown to neutralize Stx1 toxicity toward Vero cells by assaying their viability. To compare the relative effectiveness of the dimeric hybrid IgG/IgA and parental IgG1 mAb, Stx1-induced apoptosis was examined using 2 different cell lines, Ramos and Vero cells. The hybrid IgG/IgA inhibited apoptosis more efficiently than the parental IgG1 mAb in both cases. The results indicated that the use of high affinity binding sites as variable regions of IgA would increase the utility of IgA specific for virulence factors.
authors.affiliation: a Laboratory of Microbiology and Immunology; School of Pharmaceutical Sciences ; University of Shizuoka ; Shizuoka City , Shizuoka , Japan.
authors.forename: Kohta
authors.initials: K
authors.lastname: Kurohane
authors.forename: Kyoko
authors.initials: K
authors.lastname: Nagano
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Nakanishi
authors.forename: Koki
authors.initials: K
authors.lastname: Iwata
authors.forename: Masaki
authors.initials: M
authors.lastname: Miyake
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Imai
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D017209:Apoptosis; D016466:CHO Cells; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D006825:Hybridomas; D007070:Immunoglobulin A / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D051379:Mice; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000276:immunology; D022621:Shiga Toxin / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000633:toxicity; D014709:Vero Cells
chemical_list: D000911:Antibodies, Monoclonal; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D022621:Shiga Toxin
keywords: CHO, Chinese hamster ovary; J chain, joining chain; SIgA, secretory IgA; Shiga toxin; Stx1, Shiga toxin 1; Stx1B, B subunit of Stx1; annexin V; apoptosis; caspase-3; dimer; immunoglobulin A; recombinant antibody