Human interferon-beta ELISA - (Oct/17/2007 )

Hello
Has anyone here done any human IFN-b ELISA before?
I infected human bronchial epithelial cell lines with rhinoviruses, and poly I:C 100ug as my positive control, and i collected the supernatant at 6h, 12h, 24h, and 48h, then tried to measure the amount of IFN-b released with human IFN-b Elisa kit from interferonsource (PBL), all the samples were negative, even though polyIC are supposed to be a good inducer for IFN-b. The standards worked really well, and the blank are low.


I froze the samples in -80 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-b binding site. cuz i checked the IFN-b sequence that it can be bound to Zn. might be able to bind to metal residues.

any suggestion??

many thanks

Alan

PS. any clues as to why IFN-b ELISA kit are so expensive??

Hello
Has anyone here done any human IFN-b ELISA before?
I infected human bronchial epithelial cell lines with rhinoviruses, and poly I:C 100ug as my positive control, and i collected the supernatant at 6h, 12h, 24h, and 48h, then tried to measure the amount of IFN-b released with human IFN-b Elisa kit from interferonsource (PBL), all the samples were negative, even though polyIC are supposed to be a good inducer for IFN-b. The standards worked really well, and the blank are low.


I froze the samples in -80 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-b binding site. cuz i checked the IFN-b sequence that it can be bound to Zn. might be able to bind to metal residues.

any suggestion??

many thanks

Alan

PS. any clues as to why IFN-b ELISA kit are so expensive??