Molecular Characterization of Antibody Epitopes of Ehrlichia chaffeensis Ankyrin Protein 200 and Tandem Repeat Protein 47 and Evaluation of Synthetic Immunodeterminants for Serodiagnosis of Human Monocytotropic Ehrlichiosis
Molecular Characterization of Antibody Epitopes of Ehrlichia chaffeensis Ankyrin Protein 200 and Tandem Repeat Protein 47 and Evaluation of Synthetic Immunodeterminants for Serodiagnosis of Human Monocytotropic Ehrlichiosis
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DOI：
10.1128/cvi.00331-09
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发表时间：
2010-01-01
影响因子：
--
通讯作者：
McBride, Jere W.
中科院分区：
文献类型：
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作者：
Luo, Tian;Zhang, Xiaofeng;McBride, Jere W.
最近，查菲埃里希体的三种免疫反应性串联重复蛋白 (TRP) TRP32、TRP47 和 TRP120 中的主要物种特异性抗体表位已在串联重复 (TR) 区域内得到鉴定和分子表征。在这项研究中，我们绘制了查菲埃里希菌 200-kDa 锚蛋白 (Ank200) 的主要免疫决定簇以及查菲埃里希菌 TRP47 N 端和 C 端区域的次要免疫决定簇。 Ank200 的主要抗体表位位于末端酸性结构域的四个多肽区域（分别为 18 聚体、20 聚体、20 聚体和 21 聚体），这些区域与人类单核埃利希体病 (HME) 患者和查菲埃利希体感染的狗的血清中的抗体发生反应。在 TRP47 的 N 末端和 C 末端鉴定出两个含有次要表位的区域。通过酶联免疫吸附测定 (ELISA) 测定了代表恰菲埃里希体的这些和其他明确的主要免疫决定簇的合成肽的敏感性和特异性。通过间接荧光抗体测定 (IFA) 检测了 31 份 HME 患者血清样本，这些样本含有可检测到的查菲埃里希菌抗体（效价从 64 到 8,192）。所有 31 份血清样品均与至少一种恰菲埃里希菌肽发生反应，其中 30 份 (96.8%) 与 TRP120 肽发生反应，27 份 (87.1%) 与 TRP32 肽发生反应，24 份 (77.4%) 与 TRP47 肽发生反应，19 份 (61.3%) 与 Ank200 肽发生反应，28 份 (90.3%) 与重组 TRP120-TR 蛋白发生反应。与单独使用 TRP120 相比，TRP120 和 TRP32 两种最敏感肽的混合物并未提供增强的分析灵敏度。我们的结果表明，TRP120 肽可用于开发 HME 的标准化敏感现场护理和参考实验室免疫诊断。这是第一项比较分子定义的主要抗体表位分析与 IFA 诊断 HME 的研究。
Recently, major species-specific antibody epitopes in three immunoreactive tandem repeat proteins (TRPs) of Ehrlichia chaffeensis, TRP32, TRP47, and TRP120, have been identified and molecularly characterized within tandem repeat (TR) regions. In this study, we mapped the major immunodeterminants of the E. chaffeensis 200-kDa ankyrin protein (Ank200) and the minor immunodeterminants in the N-and C-terminal regions of E. chaffeensis TRP47. Major antibody epitopes of Ank200 were localized to four polypeptide regions (18-mer, 20-mer, 20-mer, and 21-mer, respectively) in terminal acidic domains, which reacted with antibodies in sera from human monocytotropic ehrlichiosis (HME) patients and an E. chaffeensis-infected dog. Two minor epitope-containing regions were identified in the N terminus and the C terminus of TRP47. The sensitivities and specificities of synthetic peptides representing these and other well-defined major immunodeterminants of E. chaffeensis were determined by enzyme-linked immunosorbent assay (ELISA). Thirty-one HME patient serum samples that had detectable E. chaffeensis antibodies (titers from 64 to 8,192) by indirect fluorescent-antibody assay (IFA) were tested. All 31 serum samples reacted with at least one E. chaffeensis peptide, 30 (96.8%) with TRP120 peptides, 27 (87.1%) with TRP32 peptides, 24 (77.4%) with TRP47 peptides, 19 (61.3%) with Ank200 peptides, and 28 (90.3%) with recombinant TRP120-TR protein. A mixture of the two most sensitive peptides from TRP120 and TRP32 did not provide enhanced analytical sensitivity compared to that provided by TRP120 alone. Our results demonstrate that the TRP120 peptide can be utilized for development of standardized sensitive point-of-care and reference laboratory immunodiagnostics for HME. This is the first study to compare analysis of molecularly defined major antibody epitopes with IFA for diagnosis of HME.