I am trying to standardise the OX40 assay in order to use it to measure antigen specific CD4 cells. I have confirmed that it is indeed a lot more sensitive than other assays, however I have the trouble that each time I run the same donor on different days with the same protocol and antigen, I get very varied results. In essence inter assay variability is poor is my hands. Has anyone done this assay using the same or different antigens and found similar results or is it just my hands? Any tips in resolving this would be very helpful even if it is borrowed from other flow based activation marker readout assays. 

Many thanks

Ane