Hi Stevan,

Tommaso and I seem to be in minority opinion about this matter. Why would you want the extent of antigen/antibody complex formation on the plate walls to be diffusion limited? If your incubation steps are < 2-3 hrs, shaking should increase the chance of productive collisions on the solid phase. Gentle continuous agitation on a back-forth plate shaker (such as ThermoLab Systems Wellmix, designed for this purpose) with up to 0.2 ml solution/well in a 96-well plate should not risk cross-contamination. Once substrate is added, it is critical to apply vigorous shaking to distribute product homogenously in the well just before reading. This non-proteinacous, detergent-free solution will not spill over to neighboring wells due to the strong meniscus effect.

Hi Stevan,

we never shake our ELISA plates (we do DAS-ELISA).

Cheers, Nadine

Hi Stevan,

I'm in agreement with Nadine. We generally don't use agitation during any of the steps. You can increase the number of washes if necessary (and make sure you are using enough wash buffer/well. We use 200 uL/well for 96-well plates). Our results are quite consistent in the absence of shaking/agitation, so we've never tried shaking during washes or incubations.

Hi Steven

I agree with Nadia & Tina.

Thanks! I appreciate all of your feedback. I'm doing sandwich ELISAs and have NOT been shaking so far (and getting fairly consistent results). After browsing online I noticed a few protocols recommended shaking so I was wondering if I could improve my assay. But after your answers I will continue to leave my assay as is, without any shaking.

don't shake in ELISA, repeated pipetting may help you to mix the solutions well.

We don't repeat the pipetting steps! We mix our solutions before we pipette it inside the wells. If you do repeated pipetting steps inside the wells you may destroy the coated wells!

In fact as has been observed by colleagues, shaking neither adds to the quality of results nor is it practical, but the worst is that it may end up causing serious contamination in adjacent wells.

Hello, usually i put the plate in gently agitation during the stages of incubation with the antibodies but not during the wash cycles. I'm in agreement with Nadine to don't pipetting inside the wells.

Hi Stevan,

Tommaso and I seem to be in minority opinion about this matter. Why would you want the extent of antigen/antibody complex formation on the plate walls to be diffusion limited? If your incubation steps are < 2-3 hrs, shaking should increase the chance of productive collisions on the solid phase. Gentle continuous agitation on a back-forth plate shaker (such as ThermoLab Systems Wellmix, designed for this purpose) with up to 0.2 ml solution/well in a 96-well plate should not risk cross-contamination. Once substrate is added, it is critical to apply vigorous shaking to distribute product homogenously in the well just before reading. This non-proteinacous, detergent-free solution will not spill over to neighboring wells due to the strong meniscus effect.

I also have the the same question i.e whether Shaking is required  for antigen coating, blocking, antigen antibody interaction step and washing while doing ELISA.

I am doing it for the first time. My logic was shaking will enhance the antigen antibody to interact more and hence did all the steps with shaking. But did not get any positive result. So wondering whether shaking is necessary or absolutely not necessary.

Just a short lab note: I've compared shaking (up to 1000 rpm) vs. non-shaking conditions at various temperatures and incubation times in Sandwich ELISAs using premixed antigen and secondary HRP:AB conjugate. My experience is that you may see enhanced signals with shaking. However, the differences are not overwhelming. At most I could see a 1.5 to 2-fold improvement in terms of signal and estimated LOD. This comes, as mentioned here before, with an increased risk of cross-contamination between neighbouring wells.