ELISA plate washer - (Dec/23/2009 )

Hello,

I have a problem with my sandwish ELISA.
Since I use an ELISA plate washer, my assay crash down.
It's a real problem because if I do not use it, my bacground and control are not homogenous.
I try to decrease wash strenght but it's increase my background...
I really need to use the ELISA plate washer because we plan to do some screening on this assay and it's impossible to wash manually all the time.

Usually, I wash 4 times with PBS+ Tween 0.05%, soaking time 1min, for all the different steps.
Strenght wash: 700ul/sec

Maybe someone of you have some experience with this kind of material and can help me. I think I miss a critical step (maybe too more wash or something...)

Thanks a lot for your answers.

On first sight i see no problem but what do you mean with:
"It's a real problem because if I do not use it, my bacground and control are not homogenous. "

I want to say I have significative variation on my background and control values when I don't use ELISA plate washer.

Clearly, I really think there is something wrong with the washer. During the test before, we buy it, all was ok. But with the new one, my results are really poor. Maybe I did something wrong during programmation (wash depth, wash strenght...). If someone use this kind of equipment (Biotek ELx50), what do you usually use for your washing program?

Thanks a lot for your answer.

I don't have a washer but it would not only be your background and controls that would show variation...it would be all results (curve and samples as well).

There may not be one universal program. Suggest you start with one parameter at a time. First just run curve in triplicate and calculate %CVs of each level. The variation is most likely due to fluid remaining in the wells.

Do 4 washes. 400 ul/ per or less avoid overfill. Allow wash to remain 15 sec. then complete aspiration. If there is a force/speed setting i would use average.