ELISA edge effect - (Oct/08/2013 )
What is the most effective approach to minimize edge effect in a 96well elisa? All reagents & plates are brought to room temperature before using. Plates are covered during incubations, stacking no more than 4 high. Coating takes place at 37C O/N in a humidified container. All other incubations take place at RT (all 1 hour incs.), but not in a humidified container, just on the benchtop with lids on the plates. Room temperature is consistant. Substrate is diethanolamine, not incubated in the dark. Plates are medium bind Greiner. Edge effect happening is a larger than expected drop from the top row to the second row in a 2-fold serial dilution down the plate, ends up more like 2.5-3 fold, other dilutions below the 2nd well come down as expected compared to the well above it.
1- Can coating at a shorter incubation(5 hours) period at 37 help or keep O/N inc with 4C instead? ODs may not be high enough, this is a lower binding assay to begin with, many time only getting 1 or 2 usable ODs for a sample-which makes it unreliable to quantitate when the edge effect happens.
2- Can using a high bind plate help, so that incubations might be shorter?
3- Can using a rotator for the plates during incubations help?
Edge effect is a big problem. There will be no difference between medium binding and high binding. By using a rotator, there will be a litlle change.
If you see gradient, check humidity during the washes or the step of filling the chromogen. If there is classic edge effect, the problem is mainly the coating step. Probably, there is the deed of increasement of the coating reagent concentration.
Shaking the plates is a good idea. It will add consistency in your results and help to normalize the wells. How many plates are you running at one time?
Typically about 12 plates at a time. They are all timed in separate batches of 4 plates, so inc times are not an issue. Reagent is added almost immediately after washing each plate to minimize drying out.
I tried a comparison of stacking only 2 plates high & added the humidified chamber (plastic box w/ wet paper towels) for all of the incubations & it still happened, I think slightly worse if any change at all.
We don't have a rotator, but heard it helps with consistancy.
Maybe I'll try 4C coating, decreasing 37C coating time &/or increasing Ag conc. for the coat.