Hi everybody. I have a very important question. How to avoid RBCs agglutination on adding the monoclonal antibody to RBCs? On testing my sample, I've diluted the EDTA whole blood 1:200 (RBCs count was 5.3 millions/mL); stained with the monoclonal antibody using 5 uL (& 10 uL in a separate trial), added to 100uL of the diluted sample; did 4 washes after staining; mixing the sample by vortex prior to reading. I've tried adding one monoclonal antibody & adding two antibodies of two different dyes (FITC & PE) in the same tube. In all trials (using low & higher concentrations of the antibodies, or using each solely & combined with the other one), RBCs agglutination couldn't be stopped. Anything I did wrong or any other should be done? I really need the advice of anyone who practiced it. Thank you all.
Yes may be you're right, as washing solution itself may have some proteins that may cause agglutination. However, it has been advised to do washing more than once as it can help to decrease nonspecific binding of antibody and/or remove excess fluorochrome. It may also help decrease aggregation.
Thanks for the reply.
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