I'd like to pick the brains of our esteemed group here with a question concerning microspheres. First - I would like to say thanks to all who responded a couple of months ago about my request for manufacturers of beads - Bangs Labs definitely make some very nice beads, and their protocols for conjugating proteins to beads work very well. 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? Thanks! -Doug Reed Douglas S. Reed, Ph.D. Microbiologist Department of Aerobiology and Product Evaluation Division of Toxinology and Aerobiology U.S. Army Medical Research Institute of Infectious Disease 1425 Porter St. Ft. Detrick Frederick, MD 21702-5011 301-619-6728 301-619-2541 fax Doug.Reed@det.amedd.army.mil
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