I'm in the process of developing a cytokine sandwich ELISA with monoclonals we have generated. I did a BIAcore experiment and found a few paired antibodies and am now struggling to increase sensitivity into the pg/ml range. The assay maxes out (>2.0AU) at concentrations ~50ng/ml and then sharply drops to 0.5AU at ~6.5ng/ml. In fact, the absorbance dropped exactly by half each dilution (as I'm diluting my antigen 1:2). I'm not quite sure what I can do to increase sensitivity 1000fold. Most of the commercial kits I've seen have very linear AU readings from 1000pg/ml to 6.25pg/ml, (from like 1.5AU to 0.2AU). Could it be that my antibodies just don't have the affinity to detect cytokine at such low levels? I'm currently running a checkerboard but I doubt it would increase sensitivity 5 fold, let alone 1000fold...
Any help or comments would be appreciated!
EDIT: Also my background appears to be quite high, which I suppose can be some reactivity of the detection antibody with the capture!
Edited by SebS, 09 February 2011 - 02:37 AM.