Fard A H Mohagheghi, Pam J Vallely
Department of Virology, School of Medicine, Zahedan Medical University, P.O. Box 98135-795, Zahedan, Iran.
Pak J Biol Sci. 2007 Oct 1;10(19):3306-13. doi: 10.3923/pjbs.2007.3306.3313.
Herpesviruses are ubiquitous in nature and both humans and animals harbour different species of this diverse family. However, only 8 species have been so far recovered from human beings. Different techniques (molecular and non-molecular) have been employed for rapid and efficient diagnosis of herpesviruses especially in case ofimmunocompromised hosts who are considered as high risk individuals. An indirect in-house ELISA was standardized to measure IgG antibody against Varicella-Zoster Virus (VZV) in sera from different groups of individuals. In the process of optimization of in-house ELISA, optimal dilutions of antigen, serum, conjugate and monoclonal IgG antibody along with selection of efficient blocking buffer and washing steps of microtitre plates were studied by the help of checkerboarding. The results were calculated according to Specific Binding Ratio (SBR) and cut-off procedures. The efficiency of newly developed indirect in-house ELISA was attempted and results were compared with data previously obtained by the agency of commercial kits or other serological techniques. The latter helped to investigate different technical aspects of in-house ELISA. When accuracy of ELISA was confirmed the protocol was applied in screening of the sera from immunocompetent and immunocompromised hosts that facilitated examination of the clinical aspects of the hosts. Diethylamine (DEA) in 20, 35 and 70 mM concentrations were used in order to assay IgG antibody avidity. The IgG avidity index was calculated by dividing OD of each sera obtained with denaturant to OD of the same specimen without application of denaturants. Avidity indices proved to be an important tool to differentiate between primary and recurrent infections and between seropositivity and seronegativity.
疱疹病毒在自然界中广泛存在，人类和动物体内都携带着这个多样病毒家族的不同种类。然而，迄今为止，仅从人类身上发现了8种疱疹病毒。人们采用了不同的技术（分子技术和非分子技术）来快速、高效地诊断疱疹病毒，尤其是对于被视为高危个体的免疫功能低下宿主。一种间接的内部酶联免疫吸附测定（ELISA）方法被标准化，用于检测不同个体组血清中针对水痘带状疱疹病毒（VZV）的IgG抗体。在优化内部ELISA的过程中，借助棋盘滴定法研究了抗原、血清、结合物和单克隆IgG抗体的最佳稀释度，以及高效封闭缓冲液的选择和微量滴定板的洗涤步骤。根据特异性结合率（SBR）和临界值程序计算结果。对新开发的间接内部ELISA的效率进行了测试，并将结果与之前通过商业试剂盒或其他血清学技术获得的数据进行比较。后者有助于研究内部ELISA的不同技术方面。当ELISA的准确性得到证实时，该方案被应用于免疫功能正常和免疫功能低下宿主血清的筛查，这有助于检查宿主的临床情况。使用浓度为20、35和70 mM的二乙胺（DEA）来检测IgG抗体亲和力。IgG亲和力指数通过将每个用变性剂处理后的血清的光密度（OD）除以未使用变性剂的同一样本的OD来计算。亲和力指数被证明是区分原发性感染和复发性感染以及血清阳性和血清阴性的重要工具。
