Antibody reactivity - (May/14/2013 )
I'm doing my phagocytosis assay based on a research paper which stated "before commencing assay, cells were opsonized with 10ug/ml monoclonal antibody". That's pretty vague to me becaused I dont know for sure how many cells would be opsonized by that amount of antibody (ie 10^5 cells/ml opsonized by 10ug/ml or what?). Are there any ways that I can determine the exact (or relative) number of cells that were opsonized by a particular amount of antibody?
I'm rather new at these stuffs so any helps would be very much appreciated!
I don't have much experience with opsonization assays but do know that everything is antibody dependent. If you are using a different monoclonal antibody than the paper used, you would have determine the amount of antibody/cells no matter what. It is partially dependent upon the affinity of the antibody. My guess is that some titration work would be necessary.
Well, I used the same monoclonal antibody. It's gift from another collaborating group so it's not commercially available. Actually there is a paper on the characterization of this monoclonal antibody which showed its apparent affinity of 5x10^9 M^-1. As this antibody targets capsular polysaccharides of my pathogen of interest, I assume that this 5x10^9 M^-1affinity is somehow related to the capsular polysaccharides concentration used in the characterization experiments. If this is the case, how do I deduce the binding capacity of this mAb (ie 1ug sufficient to bind 10^10 or 10^9 or etc... bacteria cells???). I need to figure this out somehow because my next step is a phagocytosis assay. If I dont get the optimal opsonization prior to the phagocytosis assay then my phagocytosis uptake is very low.
I thinking about a setting up a new serum bacteriocidal assay (opsonizing using different concentrations of antibody and incubate with complements and observe complement killing capacity) but wonder if there are any simpler ways?