New immunofluorescence assays for detection of human herpesvirus 8-specific antibodies
New immunofluorescence assays for detection of human herpesvirus 8-specific antibodies
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DOI：
10.1128/cdli.7.3.427-435.2000
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发表时间：
2000-05-01
期刊：
影响因子：
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通讯作者：
Pellett, PE
中科院分区：
文献类型：
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作者：
Inoue, N;Mar, EC;Pellett, PE
已开发了几种检测人类疱疹病毒8型(HHV-8)免疫球蛋白G抗体的方法，包括免疫荧光分析法(IFA)和酶联免疫吸附分析法(ELISA)，但由于缺乏“金标准”，这些方法的特异性和敏感性尚未完全确定。尽管以原发渗出性淋巴瘤(PEL)细胞系为基础的间接免疫吸附试验被广泛使用，但由于针对细胞成分的非特异性反应以及与其他疱疹病毒抗体的潜在交叉反应，这些检测方法可能会被混淆。为了提供更可靠的IFA，我们建立了表达HHV-8特异性蛋白ORF73和K8.1的重组塞姆利基森林病毒(RSFV)，并使用感染这些rSFV的BHK-21细胞(ORF73-IFA和K8.1-IFA)，通过rSFV系统高水平表达HHV-8特异性蛋白，使得IFA评分容易，从而提高了特异性，rSFV系统还允许检测针对K8.1糖基化依赖表位的抗体。基于rSFV的IFA和基于PEL的IFA测定的效价相关性很好(相关系数为0.9)，基于rSFV和基于PEL的IFA的血清反应性一致性为>；97%(kappa>；0.93)。K8.1-IFA比ORF73-IFA或多肽ELISA更敏感。以PEL裂解IFA为参照，K8.1-IFA的敏感性和特异性分别为94%和100%。经K8.1-IFA检测，HHV-8在人类免疫缺陷病毒(HIV)阳性(HIV+)卡波西肉瘤(KS)患者、HIV+KS患者和健康对照组中的感染率分别为100%、65%和6.7%，与以往的报道一致。因此，我们基于rSFV的IFA可能为流行病学研究提供一种特异而敏感的方法。此外，它们还将为进一步开发HHV-8感染的诊断试验提供基础。
Several assays have been developed for detection of immunoglobulin G antibodies to Human herpesvirus 8 (HHV-8), including immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs), However, the specificity and sensitivity of these assays are not completely defined due to the lack of a "gold standard." Although IFAs based on primary effusion lymphoma (PEL) cell lines are used widely, the assays can be confounded by nonspecific reactions against cellular components and potential cross-reaction with anti bodies against other herpesviruses. To provide more reliable IFAs, we established recombinant Semliki Forest viruses (rSFVs) expressing the HHV-8-specific proteins ORF73 and K8.1 and used BHK-21 cells infected with these rSFVs For IFA (ORF73-IFA and K8.1-IFA), Expression of the HHV-8-specific proteins at very high levels by the rSFV system allowed easy scoring for IFA and thereby increased specificity, The rSFV system also allowed detection of antibodies against glycosylation-dependent epitopes of K8.1. Titers measured by rSFV-based IFAs and PEL-based IFAs correlated well (correlation coefficients of >0.9), and concordances of seroreactivities between rSFV-based and PEL-based IFAs were >97% (kappa > 0.93). K8.1-IFA was more sensitive than either ORF73-IFA or peptide ELISAs. Using PEL-based lytic IFA as a reference assay, the sensitivity and specificity of K8.1-IFA were estimated to be 94 and 100%, respectively. HHV-8 prevalences determined by K8.1-IFA among the human immunodeficiency virus (HIV)-positive (HIV+) Kaposi's sarcoma (KS) patients, HIV+ KS- patients, and healthy controls were 100, 65, and 6.7%, respectively, which were consistent with prior reports. Therefore, our rSFV based IFAs may provide a specific and sensitive method for use in epidemiology studies. in addition, they will provide a basis for further development of diagnostic tests for HHV-8 infection.