Iwata Koki, Kurohane Kohta, Nakanishi Katsuhiro, Miyake Masaki, Imai Yasuyuki
Laboratory of Microbiology and Immunology, University of Shizuoka School of Pharmaceutical Sciences.
Biol Pharm Bull. 2014;37(9):1510-5. doi: 10.1248/bpb.b14-00323. Epub 2014 Jul 1.
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.
抗原特异性免疫球蛋白A（IgA）通过在黏膜表面进行被动免疫，可能对预防传染病有用。我们之前建立了针对志贺毒素1结合亚基（Stx1B）的小鼠IgA和IgG单克隆抗体（mAb）。我们还开发了一种重组杂交IgG/IgA，其中存在来自IgG mAb的可变区。通过瞬时表达验证了重组杂交IgG/IgA的结合活性。为了实现持续供应，我们建立了稳定表达单体或二聚体杂交IgG/IgA的中国仓鼠卵巢细胞。编码重链和轻链的cDNA共同表达以产生单体杂交IgG/IgA，而编码重链、轻链和连接链的cDNA共同表达以产生二聚体杂交IgG/IgA。对克隆转染子的无血清培养上清液进行尺寸排阻色谱分析。洗脱模式表明，与固定化Stx1B的结合和组装免疫球蛋白的免疫印迹信号相关。在二聚体杂交IgG/IgA的转染子中，观察到了单体和二聚体。尺寸排阻色谱使我们能够制备不含单体的二聚体杂交IgG/IgA样品。从各自的转染子中制备单体和二聚体形式的杂交IgG/IgA，以检测其对Stx1的中和作用。用单体或二聚体杂交IgG/IgA预处理后，Stx1对Vero细胞的细胞毒性被消除。此外，就毒素中和而言，二聚体形式比单体形式有效10倍以上。这些结果表明，二聚体杂交IgG/IgA结合位点的四价特征有助于毒素中和的效果。
