在本视频中，我们演示一种利用免疫过氧化物酶检测法这一酶联抗原检测技术来检测病毒并测定其滴度的替代方法。我们将向您展示如何采集病毒样本、制备用于检测的细胞，最后通过系列稀释进行免疫过氧化物酶检测，以确定病毒滴度。
本文内容
勘误通知
Important: There has been an erratum issued for this article. View Erratum Notice
摘要
摘要
测定感染性病毒滴度是病毒学研究中一项基础且关键的实验方法。对于不引起明显细胞病变效应的病毒，传统的噬斑测定法无法适用，人类冠状病毒（HCoV）的原型株229E和OC43即属于此类情况。因此，本研究建立了一种替代性的间接免疫过氧化物酶测定法（IPA），用于检测和滴定此类病毒，并在此详细描述。将含有病毒的样品进行系列对数稀释后，接种于96孔板中的易感细胞。病毒增殖后，通过IPA检测病毒，获得以“50%组织培养感染剂量”（TCID50）表示的感染性病毒滴度。该数值代表在一系列实验孔中，有一半孔内含有具有复制能力的感染性病毒时的样品稀释倍数。该技术为在细胞、组织及体液等生物样品中滴定HCoV-229E和HCoV-OC43提供了可靠的方法。本文内容基于首次发表于《分子生物学方法》（Methods in Molecular Biology，2008年，第454卷，第93–102页）的研究工作。
关键词：感染性病毒滴度，间接免疫过氧化物酶测定法，IPA，TCID50，HCoV-229E，HCoV-OC43，人类冠状病毒，病毒滴定，生物样品
方案
本实验方法的完整文字方案可在 Springer Protocols 中获取。
披露
作者无任何利益冲突需要披露。
重印与许可
勘误
Formal Correction: Erratum: Titration of Human Coronaviruses Using an Immunoperoxidase Assay
Posted by JoVE Editors on 4/01/2012. Citeable Link.
A correction was made to: Titration of Human Coronaviruses Using an Immunoperoxidase Assay. A revised abstract was republished due to a publisher error.
Revised Abstract:
Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.
Original Abstract:
Calculation of infectious viral titers represents a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for strains 229E and OC43 of human coronavirus (HCoV). An alternative indirect immunoperoxidase assay (IPA) is herein described for the detection and titration of these viruses. Susceptible cells are inoculated with serial logarithmic dilutions of samples in a 96-well plate. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as "Tissue Culture Infectious Dose" (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain replicating virus. This technique is a reliable method for the titration of HCoV in biological samples (cells, tissues or fluids).
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