We infused IL-1b mAb (10mg/kg) to the sheep fetus, and took blood samples at 1, 2, 4, 24 hr after infusion. Then, we tried to measure the IL-1b cytokine level in the plasma using Sandwich ELISA to prove if IL-1b was neutralized by IL-1b mAb.
Before starting the experiments, we optimized the experiments protocols e.g. blocking agents, dilution factors, incubation time... We detected nice IL-1b signals from sham and baseline (before infusion) samples, however we found also same strong signal from mAb-treated samples?!
We did also in vitro experiments to mimic the in vivo situation. We found the IL-1b mAb neutralized the IL-1 very well!
But why we still have signals from plasma samples? What should I do for the next step? Is the antigen-mAb complex broken by the Frozon-Difrozen process?
By the way, our mAb is mouse IgG1 against sheep.
Thank you very much for help in advance!