Characterization of monoclonal immunoglobulin a and g against shiga toxin binding subunits produced by intranasal immunization.
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.
Tanikawa
T
T
Laboratory of Microbiology and Immunology and the Global COE Program, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka-shi, Shizuoka, Japan.
Ishikawa
T
T
Maekawa
T
T
Kuronane
K
K
Imai
Y
Y
D000281:Administration, Intranasal; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology*; D015153:Blotting, Western; D002772:Cholera Toxin / Q000008:administration & dosage / Q000276:immunology; D053938:DNA Fragmentation; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006825:Hybridomas; D007070:Immunoglobulin A / Q000737:chemistry / Q000276:immunology*; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D021122:Protein Subunits / Q000276:immunology; D022622:Shiga Toxin 1 / Q000008:administration & dosage / Q000276:immunology*; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D021122:Protein Subunits; D022622:Shiga Toxin 1; D002772:Cholera Toxin