A faster immunofluorescence assay for tracking infection progress of human cytomegalovirus.
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 μl 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 ∼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.
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, China.
Yingliang
Y
Duan
Lingfeng
L
Miao
Hanqing
H
Ye
Cuiqing
C
Yang
Bishi
B
Fu
Philip H
PH
Schwartz
Simon
S
Rayner
Elizabeth A
EA
Fortunato
Min-Hua
MH
Luo
D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003362:Cost-Benefit Analysis; D003587:Cytomegalovirus / Q000254:growth & development / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000821:virology; D005455:Fluorescent Antibody Technique / Q000191:economics / Q000379:methods*; D005909:Glioblastoma / Q000473:pathology / Q000821:virology; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000196:embryology; D008856:Microscopy, Fluorescence; D058953:Neural Stem Cells / Q000821:virology; D010750:Phosphoproteins / Q000378:metabolism; D015203:Reproducibility of Results; D013997:Time Factors; D014763:Viral Matrix Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*; D053586:Virus Internalization
D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C065418:cytomegalovirus matrix protein 65kDa