Borrelli S, Jansson P E, Lindberg A A
Clinical Research Centre, NOVUM, Karolinska Institute, Huddinge Hospital, Sweden.
Microb Pathog. 1996 Nov;21(5):307-18. doi: 10.1006/mpat.1996.0064.
Mouse monoclonal antibodies MAHI 4 and MAHI 10 reactive with Haemophilus influenzae lipopolysaccharide (LPS), were generated by fusing mouse myeloma cells with spleen cells of mice immunized with H. influenzae strain RM.7004-XP-1. The antibody MAHI 4 reacted in whole-cell enzyme immunoassay (EIA) and colony-dot-immunoblotting with 20 of 123 H. influenzae strains and to a few other human Haemophilus spp. and Neisseria spp., but not to any Bordetella pertussis, B. parapertussis, Aeromonas spp. or Moraxella catarrhalis strains tested. This suggests a specific epitope accessible to recognition in just a few strains. This conclusion was supported by the data on binding of MAHI 4 to only three of 18 H. influenzae LPSs tested, but not to any Haemophilus ducreyi or enterobacterial LPSs. The antibody MAHI 10 bound to 80 of 123 strains of H. influenzae and to a few strains of Neisseria spp. and M. catarrhalis as evaluated by EIA and colony-dot-immunoblotting, which suggests an epitope accessible to recognition in 65% of the H. influenzae strains tested. The antibody MAHI 10 reacted with 10 of 18 H. influenzae LPSs as determined by EIA. By using polysaccharides, obtained after both mild acidic hydrolysis, strong alkali treatment, and dephosphorylation, as inhibitors of the antibodies binding to H. influenzae LPS antigens it was shown that phosphate groups were essential for the binding of MAHI 10 to LPS but they did not affect antigenic recognition by MAHI 4. None of the monoclonal antibodies bound to isolated lipid A, but the aggregation caused by the fatty acids of lipid A was essential for optimum epitope recognition. Enzymatic treatment of homologous LPSs with galactose-oxidase led to products which were between 20 to 30 times less effective as inhibitors of the binding of the MAHI 4 than the native LPSs. Taken together the results indicate that MAHI 4 has the following pentasaccharide as the epitope Gal beta 1-->2 Hep alpha 1-->2Hep alpha 1-->3Hep alpha 1--> Kdo(P). These results emphasize the importance of the terminal beta-Gal residue in the definition of the MAHI 4 specificity, and of the terminal phosphorylated saccharide residues of some of the Haemophilus LPSs for the MAHI 10 specificity.
通过将小鼠骨髓瘤细胞与用流感嗜血杆菌RM.7004-XP-1菌株免疫的小鼠脾细胞融合，产生了与流感嗜血杆菌脂多糖（LPS）反应的小鼠单克隆抗体MAHI 4和MAHI 10。在全细胞酶免疫测定（EIA）和集落斑点免疫印迹中，抗体MAHI 4与123株流感嗜血杆菌中的20株以及其他一些人嗜血杆菌属和奈瑟菌属反应，但与所测试的任何百日咳博德特氏菌、副百日咳博德特氏菌、气单胞菌属或卡他莫拉菌菌株均无反应。这表明在少数菌株中存在可被识别的特定表位。这一结论得到了MAHI 4仅与所测试的18种流感嗜血杆菌LPS中的3种结合的数据支持，而与任何杜克雷嗜血杆菌或肠杆菌LPS均无结合。通过EIA和集落斑点免疫印迹评估，抗体MAHI 10与123株流感嗜血杆菌中的80株以及少数奈瑟菌属和卡他莫拉菌菌株结合，这表明在所测试的65%的流感嗜血杆菌菌株中存在可被识别的表位。通过EIA测定，抗体MAHI 10与18种流感嗜血杆菌LPS中的10种反应。通过使用轻度酸性水解、强碱处理和去磷酸化后获得的多糖作为抗体与流感嗜血杆菌LPS抗原结合的抑制剂，结果表明磷酸基团对于MAHI 10与LPS的结合至关重要，但它们不影响MAHI 4的抗原识别。没有一种单克隆抗体与分离的脂质A结合，但脂质A脂肪酸引起的聚集对于最佳表位识别至关重要。用半乳糖氧化酶对同源LPS进行酶处理后得到的产物作为MAHI 4结合抑制剂的效果比天然LPS低20至30倍。综合结果表明，MAHI 4具有以下五糖作为表位：Galβ1→2Hepα1→2Hepα1→3Hepα1→Kdo(P)。这些结果强调了末端β-Gal残基在定义MAHI 4特异性中的重要性，以及一些流感嗜血杆菌LPS的末端磷酸化糖残基对MAHI 10特异性的重要性。
