I'm trying to understand a figure in a paper. ELISA was used to determine how two drugs bind to different cell lines that express or do not express the target at two temperatures (20 C and 4 C). Nothing was said in the paper about why two temperatures would be used to determine binding, other than drug uptake into the cell by endosomes would be inhibited at 4 C. The methods section doesn't describe the details of the experiment adequately and refers to a paper that does not describe the method at all. All that is said in the results section is that both drugs bind better to expressing cells than to non-expressing cells, but the intensity of the wells is obviously different for the two drugs, so I used NIH imaging software to measure the intensities and found that there are things going on that need to be better explained. Other data in the paper support that drug #1 is taken up more slowly than drug #2. So it would make sense that drug #1 should have more surface binding at 20 C than at 4 C since ELISA using SA-HRP and ABTS doesn't get into the cell to detect intracellular binding (unless it does?). Drug #2 in the paper should have less intensity at 20 C than at 4 C since other data in the paper show that it is taken up rapidly into cells. Yet the opposite results are found. That is, for drug #1 for which uptake is very slow, 32% less well intensity is observed at 20 C than at 4 C after 1 hour incubation. For drug #2 for which uptake is rapid, 25% more intensity is observed at 20 C than at 4 C. It just doesn't make sense. Maybe I'm missing something? Any help in understanding these results would be greatly appreciated.