Characterization of Monoclonal Immunoglobulin A and G Against Shiga Toxin Binding Subunits Produced by Intranasal Immunization

Abstract

Immunoglobulin A (IgA) is considered to play a major role in protection of the mucosal surface. However, its immunological and biological properties have not been extensively studied because the production of IgA class monoclonal antibodies (mAbs) is difficult. We compared the properties of IgA and IgG mAbs against Shiga toxin B subunits (Stx1B). These mAbs were secreted from hybridomas that had been produced from mice after intranasal immunization with recombinant Stx1B and cholera toxin. The dose response curves for the binding of the IgA (clone G2G7) and IgG (clone D11C6) mAbs to immobilized Stx1B were similar, as revealed on ELISA. The majority of the IgA mAb formed dimers while the IgG mAb was monomeric, as judged by immunoblot analysis. The IgG mAb completely inhibited the binding of Stx1B to Burkitt’s lymphoma cell line Ramos, while the inhibition by the IgA mAb was only partial. The IgG mAb was able to neutralize the cytotoxicity of Stx1 holotoxin towards Vero cells, whereas the IgA mAb was not. The binding affinity of each binding site was compared by means of surface plasmon resonance analysis involving a capture method, with which the binding of soluble Stx1B to immobilized mAb was detected. The association rate was similar but the dissociation rate was twofold faster in the case of the IgA mAb, resulting in twofold higher affinity of the IgG mAb. These results suggest that one can obtain high affinity IgA mAb but toxin neutralization is another challenge as to therapeutic antibodies of the IgA class.

Stable Expression and Characterization of Monomeric and Dimeric Recombinant Hybrid-IgG/IgA Immunoglobulins Specific for Shiga Toxin

“…9,10) We also produced an IgG1 mAb against Stx1B, termed D11C6. 11) On testing of the binding of Stx1B to Burkitt's lymphoma Ramos cells, we found that the IgG1 mAb but not the IgA one completely inhibited the binding of Stx1B to the target cells.…”

“…11,13) Stx1B-specific mouse mAb G2G7 (IgA, κ) and mouse mAb D11C6 (IgG1, κ) were prepared as described previously. 10,11) The cDNAs encoding the Stx1B-specific hybrid-IgG/IgA H chain, IgG1-associated L chain and J chain were prepared by subcloning into the pcDNA3.1(+), pcDNA3.1(+)/Hyg and pcDNA3.1(+) Zeo expression vec-tors (Life Technologies; Carlsbad, CA, U.S.A.), respectively, as described previously. 12 Gel Filtration The resulting cloned-CHO-K1 cells that produced recombinant antibodies were cultured in 10% FBS-DMEM/F12 in the presence of appropriate antibiotics.…”

“…10,11) The cDNAs encoding the Stx1B-specific hybrid-IgG/IgA H chain, IgG1-associated L chain and J chain were prepared by subcloning into the pcDNA3.1(+), pcDNA3.1(+)/Hyg and pcDNA3.1(+) Zeo expression vec-…”

Phagocytic Entry of <i>Legionella pneumophila</i> into Macrophages through Phosphatidylinositol 3,4,5-Trisphosphate-Independent Pathway

“…12) Mouse monoclonal antibodies against L. pneumophila were produced according to the methods described previously. 13) Hybridoma cells secreting specific antibodies were cloned and re-cloned by the limiting dilution method. For hybridoma screening and assessment of antibody binding to L. pneumophila, L. pneumophila-specific immunoglobulin G (IgG) antibodies in samples were detected by enzyme-linked immunosorbent assay (ELISA).…”

Strategies to avoid shiga toxin effects

“…Imai et al 22 found that Stx1B has low immunogenicity, not enough to induce an specific IgA response efficiently in mice. Tanikawa et al 23 produce an IgA m Ab (G2G7) specific to Stx 1B that was not efficient in neutralizing the toxicity of the Stx1 holotoxin, it yes was neutralized by IgG mAb D11C6. Belonging to the same research group, Tobisawa et al 24 produced a recombinant hybrid IgG/IgA, in which variable regions came from IgG mAb, while the heavy chain constant region was from IgA mAb.…”