Luo Tian, Zhang Xiaofeng, Nicholson William L, Zhu Bing, McBride Jere W
Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, USA.
Clin Vaccine Immunol. 2010 Jan;17(1):87-97. doi: 10.1128/CVI.00331-09. Epub 2009 Dec 2.
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.
最近，已在恰菲埃立克体（Ehrlichia chaffeensis）的三种免疫反应性串联重复蛋白（TRP），即TRP32、TRP47和TRP120中鉴定出主要的物种特异性抗体表位，并在串联重复（TR）区域内进行了分子特征分析。在本研究中，我们绘制了恰菲埃立克体200 kDa锚蛋白（Ank200）的主要免疫决定簇以及恰菲埃立克体TRP47 N端和C端区域的次要免疫决定簇。Ank200的主要抗体表位定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细胞埃立克体病（HME）患者血清和一只感染恰菲埃立克体的犬血清中的抗体发生反应。在TRP47的N端和C端鉴定出两个含次要表位的区域。通过酶联免疫吸附测定（ELISA）确定了代表这些以及恰菲埃立克体其他明确的主要免疫决定簇的合成肽的敏感性和特异性。对31份通过间接荧光抗体测定（IFA）检测到恰菲埃立克体抗体（滴度为64至8192）的HME患者血清样本进行了检测。所有31份血清样本均与至少一种恰菲埃立克体肽发生反应，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，来自TRP120和TRP32的两种最敏感肽的混合物并未提供更高的分析灵敏度。我们的结果表明，TRP120肽可用于开发针对HME的标准化敏感即时检测和参考实验室免疫诊断方法。这是第一项比较分子定义的主要抗体表位分析与IFA用于诊断HME的研究。
