ELISA test help

[Stentor]

Hello,

 

I am using the ELISA immunoassay for allergen testing. What is the proper way to manually wash the microtiter plates with PBS-T? Is it ok fill the wells with PBS-T with a squirt bottle (not overflowing), and then quickly invert the plate and tap it? Or will doing it this way cause cross-contamination between the wells?

 

Any help would be greatfully appreciated.

[ecoli]

I haven't done this particular assay, but I've done a similar thing with biofilm assay. As far as I know, cross contamination shouldn't occur if you're careful. Don't quote me on that though.

[CharonY]

As each incubation step is long enough to ensure specific binding, cross-contamination is usually not an issue. There are different flavours of ELISAs but for instance in sandwich ELISA you usually use a high enough concentration of the first antibody to saturate the binding capacity of the respective wells. So even if you use different antibody on the same plate one would expect that small overflows would not add to the already saturated neighbouring plates.

Later one you block the free antibodies using some kind of blocking reagent and so on. There may be specific variations where more care must be given, but the standard ELISA protocols usually ensure that cross contaminations as such do not occur (or are below the detection limit).