Back again with real problems this time using microspheres to quantitate virus. 1) binding of YOYO-1 to the microsphere in the absence of any virus. Currently we incubated with YOYO-1 for an hour and then wash twice - reducing the incubation time reduces the binding in the beads-only control but also reduces the staining of particles with virus bound to them. Any thoughts on how we could reduce this? We'd like to maximize our signal over background staining so we can quantitate very low numbers of virus. 2) a high degree of variability from sample to sample, and a large amount of non-specific binding in biological samples that contain things other than virus. We've tried a solution of PBS with 1% BSA and 0.05% Tween-20, but this does little to reduce the non-specific binding. Has anyone tried any other 'blockers' with any success? My wife ( a technician worth 10 times her weight in gold) has suggested nonfat dry milk instead of BSA. I've also seen Molecular Probe's BlockAid to prevent nonspecific binding to microspheres - has anyone tried it? Thanks! -Doug 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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