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Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay
Published on: September 7, 2018
Production of secretory immunoglobulin A against Shiga toxin-binding subunits in mice by mucosal immunization
Yasuyuki Imai1, Rio Nagai, Yousuke Ono
1Department of Microbiology and COE Program in the 21st Century, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka 422-8526, Japan. imai@u-shizuoka-ken.ac.jp
The toxicity of Shiga toxins (Stx) depends on the binding of their B subunits to carbohydrate ligands on host cells. The production of antibodies against B subunits, especially immunoglobulin A (IgA) secreted on the mucosal surface, should contribute to host defense. One of the major problems in attempts to produce IgA against Stx was the poor immunogenicity of B subunits. We were able to produce serum IgA as well as IgG against Stx1B in mice of the H-2d haplotype by means of intranasal immunization with recombinant B subunits of Stx (Stx1B) together with cholera toxin as a mucosal adjuvant. Secretory IgA (S-IgA) was detected in nasal washes but not in feces. We prepared chemically cross-linked Stx1B for use as an immunogen, and the formation of stable oligomers was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. When the cross-linked Stx1B was used together with cholera toxin for the intranasal immunization of BALB/c mice, strong enhancement of the immune response was observed. The S-IgA titers in nasal washes were 16- to more than 64-fold higher than those in mice immunized with native Stx1B plus cholera toxin. Furthermore, fecal IgA was detectable when the cross-linked Stx1B was used. The use of cholera toxin was necessary for the induction of high titers of S-IgA in the nasal washes. However, the effect of cross-linking was dependent on the major histocompatibility complex haplotype; that is, no enhancement of IgA production was observed in C57BL/6 mice. The present results provide a practical means of producing IgA against Stx1B in BALB/c mice.
The toxicity of Shiga toxins (Stx) depends on the binding of their B subunits to carbohydrate ligands on host cells. The production of antibodies against B subunits, especially immunoglobulin A (IgA) secreted on the mucosal surface, should contribute to host defense. One of the major problems in attempts to produce IgA against Stx was the poor immunogenicity of B subunits. We were able to produce serum IgA as well as IgG against Stx1B in mice of the H-2d haplotype by means of intranasal immunization with recombinant B subunits of Stx (Stx1B) together with cholera toxin as a mucosal adjuvant. Secretory IgA (S-IgA) was detected in nasal washes but not in feces. We prepared chemically cross-linked Stx1B for use as an immunogen, and the formation of stable oligomers was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. When the cross-linked Stx1B was used together with cholera toxin for the intranasal immunization of BALB/c mice, strong enhancement of the immune response was observed. The S-IgA titers in nasal washes were 16- to more than 64-fold higher than those in mice immunized with native Stx1B plus cholera toxin. Furthermore, fecal IgA was detectable when the cross-linked Stx1B was used. The use of cholera toxin was necessary for the induction of high titers of S-IgA in the nasal washes. However, the effect of cross-linking was dependent on the major histocompatibility complex haplotype; that is, no enhancement of IgA production was observed in C57BL/6 mice. The present results provide a practical means of producing IgA against Stx1B in BALB/c mice.
