Generation of Monoclonal antibodies to Galß1-4Gal epitopes: a key tool in studies of species-specific glycans expressed in fish, amphibians, and birds.
Generation of Monoclonal antibodies to Galß1-4Gal epitopes: a key tool in studies of species-specific glycans expressed in fish, amphibians, and birds.
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针对 Galß1-4Gal 表位的单克隆抗体的生成：研究鱼类、两栖动物和鸟类中表达的物种特异性聚糖的关键工具。
DOI：
10.1093/glycob/cws129
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发表时间：
2013
期刊：
影响因子：
4.3
通讯作者：
Katsumura T,Oota H,Hanihara T,Oga A,Hirabayashi J,Yamamoto K.
中科院分区：
文献类型：
--
作者：
Suzuki N,Nawa D,Tateno H,Yasuda T,Oda S,Mitani H,Nishimaki T;Katsumura T,Oota H,Hanihara T,Oga A,Hirabayashi J,Yamamoto K.
尽管Galβ1-4Gal表位很少在哺乳动物聚糖中发现，但它已在各种非哺乳动物脊椎动物物种（如鱼类、两栖动物和鸟类）的聚糖中发现。虽然在这些脊椎动物中含有Galβ1-4Gal的聚糖通过使用质谱法和/或NMR光谱法对聚糖进行精确的结构分析来检测，但是没有方便的方法来检测来自各种样品的Galβ1-4Gal。为了系统分析Galβ1-4Gal在自然界中的分布，我们以青鳉卵提取物为免疫原，制备了Galβ1-4Gal的鼠源性单克隆抗体（mAb）。通过酶联免疫吸附试验筛选获得4株单克隆抗体（2株免疫球蛋白（IG）Ms和2株IgG 1）。用142种2-氨基吡啶（PA）衍生的寡糖进行正面亲和层析，评价这些mAb的特异性。虽然所有mAb均与非还原末端含（Galβ1-4Gal）寡糖相互作用，解离常数（Kd）范围为1.0 × 10− 5至2.8 × 10− 4 M，但未观察到与任何其他聚糖的明显相互作用。N-聚糖上含有Galβ1-4Gal的分支数对IgG 1亚类mAb的Kd无显著影响，但IgM mAb的Kd随分支数的增加而降低约1个数量级。使用单克隆抗体，我们确定Galβ1-4Gal也在非洲爪蛙的各种组织中的糖蛋白上表达。免疫组织化学染色显示，Galβ1-4Gal抗原表位在与外界环境或入侵生物直接接触的内皮、上皮和表皮中均有表达。因此，这些单克隆抗体是有用的，系统地研究物种特异性表达的聚糖，这可能会作为一个屏障，防止感染。
Whereas the Galβ1-4Gal epitope is rarely found in mammalian glycans, it has been found in glycans of various species of non-mammalian vertebrates, such as fish, amphibians and birds. Although glycans containing Galβ1-4Gal in these vertebrates were detected by precise structural analysis of the glycans using mass spectrometry and/or NMR spectrometry, there are no convenient methods to detect Galβ1-4Gal from various samples. To analyze systematically the distribution of Galβ1-4Gal in nature, we generated mouse monoclonal antibodies (mAbs) specific for Galβ1-4Gal using extracts of medaka eggs as an immunogen. Four mAbs (two immunoglobulin (Ig)Ms and two IgG1s) were obtained by enzyme-linked immunosorbent assay-based screening. The specificities of these mAbs were evaluated by frontal affinity chromatography using 142 kinds of 2-aminopyridine (PA)-derivatized oligosaccharides. While all mAbs interacted with (Galβ1-4Gal)-containing oligosaccharides at their non-reducing termini with dissociation constants (Kd) ranging from 1.0 × 10−5to 2.8 × 10−4M, no apparent interaction was observed with any other glycans. The number of branches containing Galβ1-4Gal onN-glycans did not significantly affectKdof mAbs of IgG1 subclasses, but those of IgM mAbs were decreased by ∼1 order of magnitude, in increments of the number of branches present. Using the mAbs, we established that Galβ1-4Gal is also expressed on glycoproteins in various tissues from the African clawed frog. Immunohistochemical staining of medaka sections revealed that Galβ1-4Gal epitopes were expressed in the endothelium, epithelium and epidermis, which directly contact the external environment or invading organisms. Thus, these mAbs are useful for systematically investigating the species-specific expression of glycans, which may act as a barrier against infection.