Doug Reed wrote: >Okay, on to the question. We are trying to develop a new assay to quantitate >virus particles in aerosol samples and tissues using a technique published >in Biotechniques last year. Essentially, a monoclonal against the virus of >interest is bound to a bead (in the original paper adenovirus, in our case >vaccinia). You culture the beads with your sample of interest, wash, then >follow with a DNA stain (PI or TOTO-1 in the original paper, YOYO-1 in our >case). By using dilutions of a known concentration (pfu - plaque-forming >units) of virus, you build a standard curve to compare your unknown with. >The principle is that you only recognize intact (protein + nucleic acid) >virus particles since the mAb binds the outer coat but the DNA stain is your >signal. > >So far, so good. You can use the standard curve, but this relates the >fluorescence simply to pfu. > >The question we have is how many intact virus particles are in 1 pfu? > >Does anyone have any suggestions for quantitating the actual number of virus >particles bound to the bead? Is it possible to calculate the number of >fluorophores that bind to the DNA of the virus and then calculate the amount >of DNA bound, and hence the number of particles? Vaccinia are relatively large viruses. The assay you're using already established that you can stain them with dyes such as TOTO-1 and YOYO-1, and that there is at least one antibody binding site; one would expect, viruses being built as they are, that there are several binding sites, and that one could therefore stain an individual vaccinia virion with at least a few hundred molecules of YOYO-1 or a similar dye (Pico green might be even better) and a few dozen molecules of PE-antibody. The high-sensitivity, slow-flow cytometers first described by me and the Block group in the late 1970's, and since made and improved on at Los Alamos and CalTech, should be able to detect both scatter and the fluorescence signals from individual vaccinia viruses; the CalTech instrument could sort them as well. As it happens, I now have some funding to look into this, so I invite interested parties to get in touch. -Howard
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