Looking to try to explain why our ELISA results have skewed down since we started seeding our own wells with the DuoSet from R and D. It shows us low values for the literature and for what we see. We recently ran it side by side to the same result. The standards are about what is to be expected, but using differentiated THP-1 cells we only see around 300pg/mL of TNF after 4 hours compared to over 1000pg/mL with the other system (about lit value). Any insight?
If the standards are the same, and your values are within the linear range, then the cells are probably making less TNF than before. You may want to start growing up cells from a new stock.
Alternatively, the TNF could be bound up in receptors, and the Lifetechnologies kit has an Ab that recognizes unbound and bound TNF whereas the R and D ELISA Ab only measures the free cytokine.
My question to you is, does it really matter if the value is lower, as long as all your experiments are done consistently with one kit? I would ask R and D and Life technologies if their Abs measure only free TNF. Then you can decide which kit is best suited for your needs.
TNF-a ELISA is also giving us problems with Duoset. Quantikine's good but expensive
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