Borrelli S, Roggen E L, Hendriksen D, Jonasson J, Ahmed H J, Piot P, Jansson P E, Lindberg A A
Department of Immunology, Microbiology, Pathology and Infectious Diseases, Karolinska Institute, NOVUM, Huddinge, Sweden.
Infect Immun. 1995 Jul;63(7):2665-73. doi: 10.1128/iai.63.7.2665-2673.1995.
Mouse monoclonal antibodies (MAbs) DP8 [immunoglobulin G1(kappa)] and DH24 [immunoglobulin M(kappa)], which are specific for Haemophilus ducreyi lipopolysaccharide (LPS), were generated by fusing mouse myeloma NS0 cells with spleen cells of BALB/c mice immunized with a total membrane preparation of H. ducreyi. MAb DP8 reacted in whole-cell enzyme immunoassay (EIA) and colony dot immunoblotting with all 50 strains of H. ducreyi but not with any other bacteria tested, which suggests an exposed and species-specific epitope on the H. ducreyi cell surface. This conclusion was supported by the finding that DP8 bound to all six H. ducreyi LPSs tested but not to any of the Haemophilus influenzae or enterobacterial LPSs or synthetic glycoconjugates. The MAb DH24 bound to 43 of 50 strains of H. ducreyi and to few strains of H. influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis, as evaluated by whole-cell EIA and colony dot immunoblotting. The MAb DH24 reacted with five of the six H. ducreyi LPSs tested and with the lacto-N-neotetraose (Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc) series of synthetic glycoconjugates, as determined by EIA. By using polysaccharides obtained after both mild acidic hydrolysis and strong alkali treatment and dephosphorylated samples as inhibitors of the MAbs binding to H. ducreyi LPS antigens, it could be shown that phosphate groups were essential for the binding of DP8 to LPS but that they did not affect antigenic recognition by DH24. None of the MAbs bound to isolated lipid A, but aggregation caused by the fatty acids of lipid A was essential for epitope recognition.
通过将小鼠骨髓瘤NS0细胞与用杜氏嗜血杆菌全细胞膜制剂免疫的BALB/c小鼠的脾细胞融合，产生了对杜氏嗜血杆菌脂多糖（LPS）具有特异性的小鼠单克隆抗体（MAb）DP8 [免疫球蛋白G1（κ）]和DH24 [免疫球蛋白M（κ）]。MAb DP8在全细胞酶免疫测定（EIA）和菌落斑点免疫印迹中与所有50株杜氏嗜血杆菌发生反应，但与测试的任何其他细菌均无反应，这表明杜氏嗜血杆菌细胞表面存在一个暴露的、物种特异性的表位。这一结论得到以下发现的支持：DP8与测试的所有六种杜氏嗜血杆菌LPS结合，但不与任何流感嗜血杆菌或肠杆菌LPS或合成糖缀合物结合。通过全细胞EIA和菌落斑点免疫印迹评估，MAb DH24与50株杜氏嗜血杆菌中的43株以及少数流感嗜血杆菌、淋病奈瑟菌和脑膜炎奈瑟菌菌株结合。通过EIA测定，MAb DH24与测试的六种杜氏嗜血杆菌LPS中的五种以及乳糖-N-新四糖（Galβ1→4GlcNAcβ1→3Galβ1→4Glc）系列的合成糖缀合物发生反应。通过使用轻度酸性水解和强碱处理后获得的多糖以及去磷酸化样品作为MAb与杜氏嗜血杆菌LPS抗原结合的抑制剂，可以表明磷酸基团对于DP8与LPS的结合至关重要，但它们不影响DH24的抗原识别。没有一种MAb与分离的脂质A结合，但脂质A脂肪酸引起的聚集对于表位识别至关重要。
