A faster immunofluorescence assay for tracking infection progress of human cytomegalovirus
A faster immunofluorescence assay for tracking infection progress of human cytomegalovirus
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用于追踪人类巨细胞病毒感染进展的更快免疫荧光测定
DOI：
10.1093/abbs/gms041
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发表时间：
2012-07-01
影响因子：
3.7
通讯作者：
Luo, Min-Hua
中科院分区：
文献类型：
--
作者：
Duan, Yingliang;Miao, Lingfeng;Luo, Min-Hua
免疫荧光分析(IFA)是生物科学和临床诊断中最常用的方法之一，但价格昂贵，耗时长。为了克服这些局限性，我们通过修改标准的IFA，开发了一种更快、更经济的IFA(f-IFA)，并将该方法应用于跟踪不同细胞中人巨细胞病毒(HCMV)感染的进展。我们研制的f-IFA不仅节省了时间，而且大大减少了抗体(Ab)的数量，这将有利于IFA在临床诊断中的应用。F-IFA封闭只需要15分钟，每个原代和次级抗体孵育10分钟，然后在每次孵育后进行1分钟的广泛清洗。在一次和二次抗体孵育步骤中，每张盖片只需要25 L稀释的抗体溶液。此外，所有步骤都是在室温下进行的。该f-IFA已成功应用于跟踪病毒粒子进入(Pp65)和病毒基因(IE1、UL44和pp65)的表达，以跟踪HCMV感染过程的细节。F-IFA检测发现，约0.5个HCMV感染的T98G细胞可形成多个微核(IE1和细胞核染色)，并有病毒脱落(pp65染色)，这是传统IFA无法检测到的。我们的结果表明，f-IFA是一种敏感、方便、快速和经济的方法，可用于研究病毒感染过程中的细节，特别是HCMV感染。F-IFA具有快速、经济、灵敏度和特异度高的特点，在临床诊断中具有潜在的应用前景。
Immunofluorescence assay (IFA) is one of the most frequently used methods in the biological sciences and clinic diagnosis, but it is expensive and time-consuming. To overcome these limitations, we developed a faster and more cost-effective IFA (f-IFA) by modifying the standard IFA, and applied this method to track the progression of human cytomegalovirus (HCMV) infection in different cells. The f-IFA that we developed not only saves time, but also dramatically reduces the quantity of antibody (Ab), which will facilitate the application of IFA in clinic diagnosis. f-IFA requires only 15 min for blocking, 10 min incubation for each primary and secondary Abs, followed by 1 min extensive wash after each incubation. Only 25 l of diluted Ab solution was needed for each coverslip at the primary and secondary Ab incubation steps. In addition, all steps were performed at room temperature. This f-IFA has been applied successfully to follow virion entry (pp65) and expression of viral genes (IE1, UL44, and pp65) in order to track the details of HCMV infection process. We found that approximate to 0.5 HCMV-infected T98G cells formed multiple-micronuclei (IE1 and nucleus staining) and had virus shedding (pp65 staining) by f-IFA, which could not be detected by the traditional IFA. Our results indicated that f-IFA is a sensitive, convenient, fast, and cost-effective method for investigating the details of virus infection progress, especially HCMV infection. The faster and cost-effective feature with higher sensitivity and specificity implies that f-IFA has potential applications in clinical diagnosis.