Alternatively, another cell density could be required, based on the cell line required for viral growth. Gently rock plates back and forth and from side to side so that cells are distributed evenly. Once cells have been seeded, allow the cells to grow all-night.
Make a series of dilutions at of the original virus sample. Fill the first tube in series with 2. Repeat to make a serial dilution of the virus, e. Label lid of well dish by drawing grid lines to define quadruplicates and number each grid to correspond to the virus sample and label the rows of the plate for the dilution that will be plated. Include four negative wells on each plate that will not be infected. Carefully remove all but 0.
Mildly add 0. The endpoint is determined when the CPE or immunofluorescence assay IFA read-out appear the same per dilution for 3 separate readings. The titer is calculated using the method of Muench and Reed. Corresponding author. Received Mar 10; Accepted Apr This article has been cited by other articles in PMC. Supplementary material 2 XLSX 39 kb. Dear Editor The most important property of a virus is its infectivity. Reed—Muench Method Table 1 shows typical data for titration of virus stocks available in the literature.
Open in a separate window. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary material 1 PDF kb K, pdf. Compliance with Ethics Standards Conflict of interest The authors declare that they have no conflict of interest. Human and Animal Rights Statement This article does not contain any studies with human or animal subjects performed by any of the authors.
References Bliss CI. The determination of the dose-the proportion responding curve from small numbers. Q J Pharma Pharmacol. Virology: a laboratory manual. London: Academic Press; Probit analysis. Cambridge: Cambridge University Press; Virus isolation and quantitation.
Virology methods manual. Beitrag zur kollektiven behandlung pharmakologiseher reihenversuche. Arch Exp Path Pharmacol. Virus quantification is of great importance for commercial and academic laboratories involved in research or production of viral vaccines, recombinant proteins, viral antigens, or antiviral agents. While more modern methods, including quantitative real-time PCR qPCR and enzyme-linked immunosorbent assays ELISA are becoming more frequently used, there remain significant drawbacks associated with all of these assays.
For example, plaque titer assays require between 4 to 10 days to provide a measure of infectious counts. Likewise, while qPCR may be able to provide results within a single day, it is laborious, requires a skilled operator, and is sensitive to contamination. In general, there is still a need for new analytical methods that can rapidly quantify viral concentration to reduce costs and alleviate bottlenecks associated with current assays. This instrument and assay represent a time and cost-effective alternative for the determination of virus concentrations.
The assay uses two dyes—one specific for proteins and one specific for nucleic acids—to stain virus samples. The larger upper window shows a 3 ms segment of data and the smaller rectangle box in the lower portion of Figure 1 shows 75 ms of elapsed data. The upper display includes the nucleic acid and protein threshold values indicated by the overlapping horizontal lines used to discriminate virus events from background noise.
The Virus Counter has been used to quantify a wide range of viruses, matrix purity levels, and virus concentrations. Figure 1. The raw PMT signals for the nucleic acid blue and protein red channels can be seen in both the large window that displays 3 ms of data and the small window at the bottom, which represents 75 ms of data. In this study we choose several of these most common virus applications for which commercial sources of well-characterized viruses were available to demonstrate the relationship between Virus Counter results with those from other assays.
Sample details, including suppliers and information provided with each sample, are provided in the Table. Virus genomes ranged from 10— kilobases kb and represent a broad range of single- and double-stranded RNA and DNA viruses.
0コメント