For cell stimulation with Silica I am measuring IL-1B with ELISA. To control for possible absorption of IL-1B by the silica I measure the effect of different concentrations of silica on the IL-1B concentration. This is done by combining the silica with our ELISA IL-1B standard at a concentration of 500 pg/mL. This is done in DMEM and after incubating at 37°C overnight it is frozen and thawed (this was more out of convenience then actually wanting to incorporate it). When measuring through ELISA our control for this experiment (500pg/mL IL-1B in DMEM without silica) is around 5pg/mL.

The only difference with the control for this experiment and our ELISA standard used on the day itself is that the medium is DMEM instead of PBS + 1% BSA, the overnight incubation and freeze thawing. Does anyone have experience with this and knows which of these steps might cause the IL-1B to breakdown or otherwise become almost immeasurable?