We are using BD OptEIA ELISA set for TGFB1, and in the original protocol the incubation time is 2 hours at RT (http://www.bdbiosciences.com/external_files/Doc_Recon_2.0/pm/tds/559119.pdf). Does anyone knows if incubating samples overnight at 4ºC increases assay sensitivity, as occurs with the Ready-set-go ELISA kit from E-biosciences (http://www.ebioscience.com/media/pdf/tds/88/88-8350.pdf)?
Yes,
Overnight incubations have better senstivity than the 2h room temp. This doesnt matter if the cytokine is released in high amounts, but for lowly produced cytokines it might be important. So it all depends on the type of the experiment and the time-point in which your examining cytokine production. I would recommend the overnight incubation because it works for all situations, whereas the 2h incubation may not detect lowly expressed cytokines.
Not sure whether it makes a big difference, because the capture mAb is always in abundance?I would agree with coating pltes overnight at 4C
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