Borrelli S, Altmann K, Jansson P E, Lindberg A A
Department of Immunology, Microbiology, Pathology and Infectious Diseases, Karolinska Institute, NOVUM, Huddinge Hospital, Sweden.
Microb Pathog. 1995 Sep;19(3):139-57. doi: 10.1006/mpat.1995.0053.
Four murine monoclonal antibodies (MAbs) reactive with the outer-core region of the lipopolysaccharide (LPS) from Haemophilus influenzae were generated after immunization with azide-killed H. influenzae RM.7004 AH1-2 and their epitope specificities studied. The monoclonal antibodies: MAHI 6 (IgM), MAHI 5 (IgG2a), MAHI 8 (IgG3), and MAHI 11 (IgG2b) bound to synthetic glycoconjugates or glycolipids with terminal galabiosyl (Gal alpha 1-->4Gal beta 1-) or globotriaosyl (Gal alpha 1-->4Gal beta 1 1-->4GLc) residues as evaluated in enzyme immunoassays (EIA). Glycoconjugates or glycolipids with internally placed galabiose elements were not active, indicating selectively of the MAbs for recognition of the epitope. Nine LPSs from H. influenzae inhibited the binding of the four MAbs. The presence of the galabiosyl disaccharide element in these nine LPSs was evidence by the binding of 125I-labeled Shiga toxin isolated from the bacterium Shigella dysenteriae type 1, reported to have as receptor the Gal alpha 1-->4Gal beta disaccharide (Lindberg et al., J Biol Chem, 1987, 262: 1779-85). Structural studies of these H. influenzae LPSs were also in accord with the presence of the galabiosyl disaccharide, in addition 1H-NMR spectroscopy showed the presence of O-acetyl groups in the RM.7004 AH1-2 LPS. However, differential binding specificities of the MAbs to modified RM.7004 AH1-2 LPSs were observed. MAHI 6 and MAHI 11 bound equally well to LPS, polysaccharides obtained after mild acidic treatment, and dephosphorylated LPS samples as shown in inhibition EIA. In contrast, both dephosphorylated LPS samples and polysaccharides were poorer inhibitors of the binding of MAHI 5 and MAHI 8 to native RM.7004 AH1-2 LPS. Neither the de-O-acylated nor the de-O,N-acylated LPSs were effective inhibitors of any of the four MAbs. These results suggest that the MAbs recognition involves Gal alpha 1-->4Gal and O-acetyl and other saccharide residue(s) from the oligosaccharide moiety of the LPS. The epitopes are also expressed and accessible to recognition in clinical isolates coming from different sources of Neisseria spp., Haemophilus spp., and Moraxella catarrhalis, but not in Bordetella spp., Aeromonas spp. or Enterobacteriaceae as evaluated by whole-bacteria EIA and colony-dot-immunoblotting.
用叠氮化物灭活的流感嗜血杆菌RM.7004 AH1-2免疫小鼠后，产生了四种与流感嗜血杆菌脂多糖（LPS）外核心区域反应的鼠单克隆抗体（MAb），并对其表位特异性进行了研究。单克隆抗体：MAHI 6（IgM）、MAHI 5（IgG2a）、MAHI 8（IgG3）和MAHI 11（IgG2b），在酶免疫分析（EIA）中评估发现，它们与具有末端半乳糖二糖基（Galα1→4Galβ1-）或球三糖基（Galα1→4Galβ1 1→4Glc）残基的合成糖缀合物或糖脂结合。含有内部半乳糖二糖元件的糖缀合物或糖脂没有活性，表明这些单克隆抗体对表位识别具有选择性。来自流感嗜血杆菌的九种LPS抑制了这四种单克隆抗体的结合。从痢疾志贺氏菌1型分离的125I标记志贺毒素的结合证明了这九种LPS中存在半乳糖二糖，据报道该毒素以Galα1→4Galβ二糖为受体（Lindberg等人，《生物化学杂志》，1987年，262：1779 - 1785）。对这些流感嗜血杆菌LPS的结构研究也与半乳糖二糖的存在一致，此外1H - NMR光谱显示RM.7004 AH1 - 2 LPS中存在O - 乙酰基。然而，观察到单克隆抗体对修饰的RM.7004 AH1 - 2 LPS的结合特异性存在差异。如抑制EIA所示，MAHI 6和MAHI 11与LPS、轻度酸性处理后获得的多糖以及去磷酸化LPS样品的结合效果相同。相比之下，去磷酸化LPS样品和多糖对MAHI 5和MAHI 8与天然RM.7004 AH1 - 2 LPS结合的抑制作用较差。去O - 酰化和去O，N - 酰化的LPS都不是这四种单克隆抗体的有效抑制剂。这些结果表明，单克隆抗体的识别涉及Galα1→4Gal以及LPS寡糖部分的O - 乙酰基和其他糖残基。通过全细菌EIA和菌落斑点免疫印迹评估，这些表位在来自不同来源的奈瑟菌属、嗜血杆菌属和卡他莫拉菌的临床分离株中也有表达且可被识别，但在博德特菌属、气单胞菌属或肠杆菌科中则不然。
