title: Stable expression and characterization of monomeric and dimeric recombinant hybrid-IgG/IgA immunoglobulins specific for Shiga toxin.
abstract: Antigen-specific immunoglobulin A (IgA) may be useful for preventing infectious diseases through passive immunization on the mucosal surface. We previously established mouse IgA and IgG monoclonal antibodies (mAbs) specific for the binding subunit of Shiga toxin 1 (Stx1B). We also developed a recombinant hybrid-IgG/IgA, in which variable regions from the IgG mAb were present. The binding activity of recombinant hybrid-IgG/IgA was verified by transient expression. Aiming at a constant supply, we established Chinese hamster ovary cells stably expressing monomeric or dimeric hybrid-IgG/IgA. The cDNAs encoding heavy and light chains were co-expressed for the monomeric hybrid-IgG/IgA, while those encoding heavy, light, and joining chains were co-expressed for the dimeric one. Serum-free culture supernatants of the cloned transfectants were subjected to size-exclusion chromatography. The elution patterns showed that the binding to immobilized Stx1B and the immunoblot signals of assembled immunoglobulins were correlated. In the transfectant for the dimeric hybrid-IgG/IgA, both monomers and dimers were observed. Size-exclusion chromatography enabled us to prepare a sample of the dimeric hybrid-IgG/IgA devoid of the monomeric one. The monomeric and dimeric forms of hybrid-IgG/IgA were prepared from the respective transfectants to examine the neutralization of Stx1. After pretreatment with monomeric or dimeric hybrid-IgG/IgA, the cytotoxicity of Stx1 toward Vero cells was abolished. Furthermore, the dimeric form was more than 10-fold more effective than the monomeric one in terms of toxin neutralization. These results suggest that the tetravalent feature of the binding sites of the dimeric hybrid-IgG/IgA contributes to the efficacy of toxin neutralization.
authors.affiliation: Laboratory of Microbiology and Immunology, University of Shizuoka School of Pharmaceutical Sciences.
authors.forename: Koki
authors.initials: K
authors.lastname: Iwata
authors.forename: Kohta
authors.initials: K
authors.lastname: Kurohane
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Nakanishi
authors.forename: Masaki
authors.initials: M
authors.lastname: Miyake
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Imai
mesh_terms: D000818:Animals; D016466:CHO Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D007070:Immunoglobulin A / Q000276:immunology* / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology* / Q000378:metabolism; D055503:Protein Multimerization; D021122:Protein Subunits / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D022622:Shiga Toxin 1 / Q000276:immunology* / Q000378:metabolism; D014709:Vero Cells
chemical_list: D007070:Immunoglobulin A; D007074:Immunoglobulin G; D021122:Protein Subunits; D011994:Recombinant Proteins; D022622:Shiga Toxin 1