Development of Sensitive Probes for Deaminated Neuraminic Acid (KDN) to Search for KDN-Glycoconjugates

1986年，我们首次报道了鱼卵糖蛋白中天然存在的脱氨基神经氨酸（2-酮-3-脱氧-d-甘油-d-半乳糖醛酸，KDN）。从那时起，越来越多的含KDN的糖缀合物被报道。我们怀疑，开发更灵敏的检测方法可能会证明在许多其他动物类型的组织和细胞中存在KDN-糖缀合物。本研究的目的是建立用于检测KDN-糖缀合物的化学和免疫化学探针。(1)检测和表征的寡/聚（唾液酸）和寡/聚（KDN）单位的糖缀合物：-我们开发了新的GLC和HPLC程序的鉴定-定量的nonulosonates和低聚物的NeuAc，NeuGc，和KDN。我们研究并比较了使用Mono-Q HR 5/5树脂对^3 H标记的寡糖醇（NeuAc）、寡糖醇（NeuGc）和寡糖醇（KDN）进行高效阴离子交换色谱分离的方法。我们还检查了一个M ...更多信息 本发明涉及一种用于分离和纯化聚唾液酸和聚（KDN）糖蛋白的低聚物和聚合物的选择性和定量微沉淀方法。(2)用于KD-聚糖链的免疫化学探针的产生和使用：-KD-糖缀合物的可用性促使我们产生针对在KD-神经节苷脂和KD-糖蛋白中发现的KD-聚糖链的单克隆抗体。我们首先制备了特异性结合（KDN）GM 3但不结合（NeuAc）GM 3的单克隆抗体IgG 3亚类（mAb.kdn3G）。用KDN-gp免疫BALB/c小鼠，取其脾细胞与P3 U1骨髓瘤细胞融合，制备了单克隆抗体IgM（mAb.kdn8kdn）。检测这些单克隆抗体mAb.kdn3G和mAb.kdn8kdn的特异性，并确定表位结构分别为KDNalpha 2 * 3Galbeta 1 * 和KDNalpha 2 * 8 KDNalpha 2 * 结构。这些单克隆抗体的潜在效用是解决在寻找含有这些表位结构的KDN-糖缀合物。少

In 1986 we first reported the natural occurrence of deaminated neuraminic acid (2-keto-3-deoxy-d-glycero-d-galactonononic acid, KDN) in fish egg glycoprotein. Since then, an increasing number of KDN-containing glycoconjugates have been reported. We suspected that development of more sensitive assay procedures might demonstrate KDN-glycoconjugates in tissues and cells of many additional animal types. The present investigation has, for its objective, the establishment of chemical and immunochemical probes for the detection of KDN-glycoconjugates.(1) Detection and characterization of oligo/poly(sialic acid) and oligo/poly(KDN) units in glycoconjugates:- We developed new GLC and HPLC procedures for the identification-quantitation of nonulosonates and lower oligomers of NeuAc, NeuGc, and KDN. We examined and compared high-performance anion-exchange chromatographic separation of ^3H-labeled oligo(NeuAc), oligo(NeuGc), and oligo(KDN) alditols by using Mono-Q HR 5/5 resin. We also examined a m … More ethod of selective and quantitative microprecipitation for separation and purification of oligomers and polymers of polysialo- and poly(KDN)-glycoproteins.(2) Generation and use of immunochemical probes for KDN-glycan chains:- The availability of KDN-glycoconjugates prompted us to generate monoclonal antibodies against the KDN-glycan chains found in KDN-ganglioside and KDN-glycoprotein. We first produced a monoclonal antibody IgG3 subclass (mAb.kdn3G) that specifically bound to (KDN)GM3, but not to (NeuAc)GM3. We also generated another monoclonal antibody IgM (mAb.kdn8kdn) by fusion of spleen cells from a BALB/c mouse immunized with KDN-gp and P3U1 mouse myeloma cells. The specificity of these monoclonal antibodies, mAb.kdn3G and mAb.kdn8kdn, was examined and the epitope structures were determined to be KDNalpha2*3Galbeta1* and KDNalpha2*8KDNalpha2* structures, respectively. The potential utility of these monoclonal antibodies is addressed in searching for KDN-glycoconjugates which contain these epitope structures. Less