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首页> 外文期刊>The American Journal of Tropical Medicine and Hygiene >Characterization of monoclonal antibodies to an immunodominant protein of the etiologic agent of human granulocytic ehrlichiosis.
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Characterization of monoclonal antibodies to an immunodominant protein of the etiologic agent of human granulocytic ehrlichiosis.

机译：针对人粒细胞埃希氏菌病病原体免疫优势蛋白的单克隆抗体的表征。

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摘要

Immunodominant proteins in the range of 42-45 kD are important for the serodiagnosis of human granulocytic ehrlichiosis (HGE). Antigens from human isolates of the etiologic agent of HGE cultivated in HL-60 cells were used to immunize BALB/c mice and generate a panel of hybridomas secreting monoclonal antibodies. Using an enzyme immunoassay, an immunofluorescent assay (IFA), and Western blotting, we showed that culture supernatants and ascites of these hybridomas were reactive with human isolates of the etiologic agent of HGE, Ehrlichia equi and E. phagocytophila. Following screening and subcloning, we selected three stable hybridomas, R1B10, R5E4, and R5A9, which were determined to be of the isotypes IgG3, IgG1, and IgG2a, respectively. These results suggest that the epitopes of the 42-45-kD protein recognized by these three monoclonal antibodies are conserved among E. equi, E. phagocytophila, and the etiologic agent of HGE. Western blot analysis showed reactivity with the 44-kD protein of human isolates of the HGE agent. None of the monoclonal antibodies were reactive with HL-60 cells that were not infected with the HGE agent. No cross-reactivity with related intracellular pathogens could be detected when undiluted supernatants from hybridoma cultures were allowed to react by IFA with antigens from E. chaffeensis, E. risticii, E. platys, Rickettsia rickettsii, R. prowazekii, or Coxiella burnetii. The additivity index of two antibodies, R5E4 and R1B10 was near zero, suggesting that these two antibodies may compete for the same epitope of the 44-kD protein, while monoclonal antibody R5A9 appears to interact with a different epitope. The antibodies secreted by these hybridomas may be useful as immunologic agents in serodiagnostic, immunohistochemical, and other studies of the etiologic agent of HGE.
机译：42-45 kD范围内的免疫蛋白对人类粒细胞埃希氏菌病（HGE）的血清诊断非常重要。在HL-60细胞中培养的HGE病原体人类分离株的抗原用于免疫BALB / c小鼠并产生一组分泌单克隆抗体的杂交瘤。使用酶免疫测定，免疫荧光测定（IFA）和Western印迹，我们显示这些杂交瘤的培养上清液和腹水与人类分离的HGE，Ehrlichia equi和吞噬性大肠杆菌的病原体有反应。筛选和亚克隆后，我们选择了三种稳定的杂交瘤R1B10，R5E4和R5A9，它们分别被确定为同种型IgG3，IgG1和IgG2a。这些结果表明，被这三种单克隆抗体识别的42-45-kD蛋白的表位在马大肠杆菌，吞噬性大肠杆菌和HGE的病原体之间是保守的。蛋白质印迹分析显示与人类分离的HGE试剂的44 kD蛋白具有反应性。没有一种单克隆抗体可与未感染HGE试剂的HL-60细胞反应。当IFA使杂交瘤细胞培养物中未稀释的上清液与Chaffeensis，Risticii，R。platys，Rickettsia rickettsii，R。prowazekii或Coxiella burnetii的抗原反应时，未检测到与相关细胞内病原体的交叉反应。两种抗体R5E4和R1B10的可加性指数接近于零，表明这两种抗体可能竞争44-kD蛋白的相同表位，而单克隆抗体R5A9似乎与不同表位相互作用。这些杂交瘤分泌的抗体可用作血清学诊断，免疫组化和其他HGE病原学研究的免疫学试剂。

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