severe edge effects

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HPLC1
HPLC1's picture
severe edge effects

I have encountered very severe edge effects when performing mouse cytokine ELISAs. Wells in the upper right corners generally have much lower readings, sometimes toward background levels, even if they contain high concentrations of the standards. As I understand, edge effects due to uneven temperature usually are not this bad. Advice is greatly appreciated.

ronpbl
ronpbl's picture
temperature, humidity and

temperature, humidity and incubation time can cause edge effect. Temp effects can be minimized by equlibrating reagents to the same temp (e.g RT). Low humidity can result in evaporation. First, make sure that the plate is sealed properly during each incubation step. Also, try placing the plate(s) in a humidified chamber or in simple form, a plastic container with some wet paper towels in the bottom. The longer the incubation time, the greater risk of edge effect. Therefore, you might try shortening the longest incubation step. Or perhaps performing at 4C instead of RT or 37. Shortening the time might limit the lower end sensitivity, so it depends on what levels you are anticipating in your samples whether this last choice is a good one. Also, you can choose to leave the outer wells blank. There are other ways to address this, but at the kit development level, not the user level (is this a commercial kit?). Hope this helps.

HPLC1
HPLC1's picture
These suggestions are really

These suggestions are really valuable.
We bought antibodies from BD Pharmingen or E-bioscience and used Nunc MaxiSorp plates. The uneven reading problem is so bad that sometimes the values in the upper right wells that contained solutions of the highest concentration could be almost 0.
Besides the temperature, humidity and incubation time, is it possible that the low readings in the upper right wells can also be caused either too much or too little volume of the buffer retained in these wells? For your information, we dont use an automatic washer for ELISA; we simply soak the plates in a container with PBS-Tween20 several times between each incubation steps. Thanks.

Typer
Typer's picture
Retained volume of wash

Retained volume of wash buffer could be a problem, though I would think that would cause more random well failures. You can try tapping the plates several times against a stack of paper towels to drive off remaining wash buffer. Given your washing method you're probably already doing this...

Wells can dry out after washing, though again I would think it would be more random wells rather than an "edge effect". Still, you never know. You can try having the next reagent(s) prepared in advance, so that immediately post-wash you can add it (them).

We find that doing all heated incubations in a water bath minimizes well-to-well variation. We stand our plates on a platform in the bath so that the well bottoms are barely in the water.

Hope this helps.

Sami Tuomivaara
Sami Tuomivaara's picture
HPLC1,I agree that

HPLC1,I agree that temperature effect seems unlikely. Also, since you are not using washer robot, uneven washing maybe unlikely too. Maybe your microplate reader has a problem. Try to read the plate in different orientation and see if the problem persists.Cheers,