ELISA troubles
I am attempting to develop my own ELISA (coating the antigen overnight at 4C in carbonate-bicarbonate buffer, wash, blocking at room temperature for 2 hours with PBST + BSA, wash, adding the sample from mouse sera diluted in PBS + BSA, wash, adding the secondary antibody diluted in PBS, wash, then adding TMB + stop). However, my plate shows great variability. Each row is a single sample with different dilutions (1/4, 1/8, 1/16... all the way to column 9). The dilutions do not look good. This is the second time I have tried this assay. I need to scale up to 3 plates. When I try with one plate, the signal is higher and the dilutions look good. However, when I scale up, I get no signal in the highest dilutions and variable signal across the other dilutions. Any ideas?
