I infected human bronchial epithelial cell lines with Influenza NS1 clones, and poly I:C 5ug/ml as my positive control, and I collected the supernatant at 0, 2h, 4h, 6h, 12h, 24h, and 48h, then tried to measure the amount of IFN-beta released with human IFN-beta Elisa kit from interferonsource (PBL), all the samples were negative, even though poly I:C are supposed to be a good inducer for IFN-beta. The standards worked really well, and the blank are low.
I froze the samples in -20 freezer and thaw it just before ElISA, could this freeze-thaw step disrupt the structural integrity or antibody binding site? or could the Mg and Ca in the media bound to the IFN-beta binding site. Because checked the IFN-beta sequence that it can be bound to Zn. might be able to bind to metal residues.
I am Using FDMEM medium without Antibiotic and L-glutamin for transfection.
Please give me suggestion? I have almost wasted 5 kits in this way.