Gulig P A, Hansen E J
Infect Immun. 1985 Sep;49(3):819-27. doi: 10.1128/iai.49.3.819-827.1985.
The major outer membrane protein of Haemophilus influenzae type b (Hib) with an apparent molecular weight of 39,000 (39K) was purified from three different Hib strains and was shown to be free from detectable contamination with other proteins. However, these purified 39K protein preparations were found to contain Hib lipopolysaccharide (LPS). Immunization of rats with these 39K protein preparations resulted in the production of antisera containing both 39K protein-directed and LPS-directed antibodies, as determined by Western blot analysis. The reactivity pattern of the LPS-directed serum antibodies with different Hib strains was identical to the reactivity of these Hib strains with a set of monoclonal antibodies (mabs) previously shown to immunoprecipitate the 39K protein in a radioimmunoprecipitation (RIP) system. Examination of the antigenic specificities of the 39K protein-immunoprecipitating mabs by using Western blot analysis showed that these mabs were actually directed against Hib LPS. RIP analysis of 125I-labeled Hib cells and 32P-labeled Hib cells revealed that the 39K protein and LPS existed as a complex in a RIP system, which resulted in the coprecipitation of both antigens by LPS-directed mabs. The interaction between LPS and the 39K protein was highly selective for this protein and did not involve other outer membrane proteins. The LPS/39K protein complex could be reconstituted by mixing purified LPS and purified 39K protein; it could also be reconstituted with 39K protein from one Hib strain and LPS from another Hib strain. These findings have necessitated the reinterpretation of previous studies involving the 39K protein-immunoprecipitating mabs. Of primary importance is the fact that the demonstrated immunoprotective ability of a 39K protein-immunoprecipitating mab (E. J. Hansen, S. M. Robertson, P. A. Gulig, C. F. Frisch, and E. J. Haanes, Lancet i:366-368, 1982) must now be regarded as evidence that antibody directed against Hib LPS can be protective against experimental Hib disease.
从三株不同的b型流感嗜血杆菌（Hib）中纯化出了表观分子量为39,000（39K）的主要外膜蛋白，且证明其未被其他蛋白质污染。然而，这些纯化的39K蛋白制剂被发现含有Hib脂多糖（LPS）。用这些39K蛋白制剂免疫大鼠，通过蛋白质印迹分析确定，产生的抗血清中既含有针对39K蛋白的抗体，也含有针对LPS的抗体。针对LPS的血清抗体与不同Hib菌株的反应模式，与这些Hib菌株与一组先前已证明在放射免疫沉淀（RIP）系统中能免疫沉淀39K蛋白的单克隆抗体（mab）的反应性相同。通过蛋白质印迹分析检测39K蛋白免疫沉淀单克隆抗体的抗原特异性，结果表明这些单克隆抗体实际上是针对Hib LPS的。对125I标记的Hib细胞和32P标记的Hib细胞进行RIP分析发现，在RIP系统中39K蛋白和LPS以复合物形式存在，这导致针对LPS的单克隆抗体能使两种抗原共沉淀。LPS与39K蛋白之间的相互作用对该蛋白具有高度选择性，且不涉及其他外膜蛋白。LPS/39K蛋白复合物可以通过混合纯化的LPS和纯化的39K蛋白来重建；也可以用一株Hib菌株的39K蛋白和另一株Hib菌株的LPS来重建。这些发现使得有必要对先前涉及39K蛋白免疫沉淀单克隆抗体的研究进行重新解释。最重要的是，现在必须将一种39K蛋白免疫沉淀单克隆抗体（E. J. 汉森、S. M. 罗伯逊、P. A. 古利格、C. F. 弗里施和E. J. 哈内斯，《柳叶刀》i:366 - 368，1982年）所证明的免疫保护能力视为针对Hib LPS的抗体可对实验性Hib疾病具有保护作用的证据。
