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Smooth drift gradient across ELISA plate

Smooth drift gradient across ELISA plate - ELISA Assay Forum

Smooth drift gradient across ELISA plate - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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  #1  
Old 12-12-2011, 10:39 PM
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Default Smooth drift gradient across ELISA plate



I use a home-made sandwich ELISA (96-well format) to detect the HIV p24 protein in cell lysates and virus-containing supernatants. The setup is as follows:

96-well Immulon 2HB polystyrene microtiter plate, coat at 4C overnight with monoclonal mouse Ab
Wash 4x (PBS/0.2% Tween-20) then block with wash buffer/10% FBS, 60 min (everything from here on is at room temp)
Samples (kept on ice) are diluted in sample diluent (PBS/1% Triton X-100/10% FBS), transferred into a separate 96-well tissue culture plate, and serially diluted twice in the plate using a multichannel pipettor
Wash 4x and load samples using multichannel pipettor from tissue culture plate into ELISA plate, 90 min
Wash 4x, human primary Ab @1:8000 in sample diluent, 45 min
Wash 4x, anti-human-HRP secondary Ab @ 1:8000 in sample diluent, 45 min
Wash 4x, TMB substrate (Thermo), until standard curve has developed well, stop with H2SO4, read at 450nm, blank on sample diluent alone.



I have recently noticed that samples I load on the left side of the plate tend to have higher absorbance than samples loaded on the right side. In order to confirm this, I multichannel-loaded all 96 wells with the same sample out of a trough and looked for drift. I was able to detect a smooth gradient running from left-to-right, with samples at the right edge of the plate being estimated about 1.3 times lower than samples at the left edge (I have seen variability across the plate as high as 2-fold in other cases).

In parallel to that plate, I did the exact same thing on another plate except for loading the plate right-to-left (i.e. the opposite of what I normally do). The gradient in that case followed my direction of loading, and not the plate's inherent orientation. This suggests that the gradient is not due to the plate but due to some aspect of the handling.

I have been trying to troubleshoot this and have thought of the following possibilities:

- Pipetting inconcistencies: I use a multichannel pipettor for all the steps. I change tips and forward-pipette (i.e. aspirate from first stop, expel to second stop) when loading my samples. I use the same set of tips but reverse-pipette (i.e. aspirate from second stop, expel to first stop) out of a trough for coating, washing, blocking, antibody and reagent steps. I am confident that by reverse-pipetting, I should be pipetting the same volume in each well (I have weighed water pipetted onto parafilm and can very consistently get the correct weight when I reverse-pipette).

- Washing: Tiny inconsistencies during pipetting might be amplified due to the high number of washes in the same direction. I therefore now flip the plate over by 180 degrees for every other wash. This should eliminate any directionality from washing as an equal number of washes are done in each direction.

- Time it takes to load the plate: I have timed myself and am able to load a 96 well plate with reagent in 23 seconds. Considering that most of my reagents are incubated for an hour or more, 23 seconds should not contribute at all to any variability. I have even optimized my sample loading step by using the intermediate extra 96-well plate (instead of using individual tubes) so that when it comes to loading samples, it is also very quick (usually about 2 minutes per plate).

- Incomplete mixing of reagents: While this is a possibility, there is no reason I can think of for this to result in a consistent left-to-right gradient. It could contribute to variability in random positions across the plate, but not a consistent directional drift.

- The sample is sticking to the intermediate 96-well plate, or the trough (in the case where I loaded the whole plate with the same sample): Once again, I pipette fast enough that I really doubt that between pipetting the leftmost wells and the rightmost wells, 25% of the protein in the sample has stuck to the trough.

- Bad batch of plates: This should be addressed by the fact that I loaded two plates in parallel but in opposite directions and the gradient followed my loading direction, not the plate direction, so it is probably not the plates.



Sorry for the long post. I am at a loss right now and cannot afford to waste plates and reagent with wild stabs in the dark. I am hoping that someone out there has seen this issue before, or can think of something I haven't thought of. I am hoping that it is something really simple that is just staring me in the face. Thanks.
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  #2  
Old 12-21-2011, 02:23 AM
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Default Re: Smooth drift gradient across ELISA plate

I think you problem may lie in excessive attempts to dry the plate between washes and reagent application. In the first wells to be loaded the wells are relatively hydrated compared to the last wells to be loaded. I know it's only 23 seconds, but if each well contains a microlitre or so of residual wash buffer, by the end of 23 seconds, that microlitre of residual wash buffer may be gone or mostly gone, so the reagents captured in the well are exposed to increasing concentrations of denaturing salt and detergent from left to right. The rate of drying will be dependent upon the humidity in the lab which can vary seasonally. Don't be afraid of leaving a few microlitres of wash buffer in the well...the wash buffer should be compatible with all reagents so a little bit won't hurt. Wash the plate, with dispense at the end of the cycle so that you end up with a plate full of wash buffer, check that your pipette is set correctly, reagents ready and then dump plate contents, blot gently (don't beat) and apply next reagent without delay. An alternative, but in my opinion less desirable, would be to take this to the opposite extreme, and dry the plate fully between each reagent application.

One or more of your reagents may be susceptible to variable dehydration, and it is easy to check out which one by doing timed loadings for each column with each reagent.

The only other thing that I would be a little concerned about is the use of a 2HB for antibody coating, preferred would be 4HBX or maxisorp.

Let us know how you get on
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  #3  
Old 12-21-2011, 08:41 AM
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Default Re: Smooth drift gradient across ELISA plate

Thanks for replying! As I said, I alternate the orientation of the plate for every wash, so that should eliminate any directionality coming from washing or drying. For every washing session, I do an equal number of washes in each direction. I do tend to overdry the plate after each wash, but again if the drying efficiency follows the direction of the wash, that should cancel out the way I perform it.

Not exactly sure what you mean by variable dehydration and how that would contribute to the smooth gradient.

We have no issues with antibody coating with the 2HB, it has worked fine for years with antibody at 5 ug/ml.


I should mention that I've done one more set of testing since I wrote this post, but still have not found the specific cause of the front-to-back effect. In order to check whether TMB loading was introducing the effect, I tried simultaneously performing two assays in the same direction for every step except the TMB substate (and the H2SO4 stop solution), for which I loaded the plates in opposite direction. The front-to-back effect did not follow the TMB/H2SO4 direction, but followed the sample loading direction. Once again the sample loading is out of a trough with a multichannel and only takes a few seconds compared to the hour and a half incubation that follows it, so it is definitely not a simple end-of-run effect.

The last thing I tried was loading two different sample concentrations on the same plate, in order to see whether the effect was more pronounced with a higher analyte concentration. Although this wasn't true in every single case, there was some tendency for the higher-concentrated sample to have a more pronounced front-to-back effect across the row.




I would be most grateful if you had some more ideas for what I could try next (provided you agree that the effect cannot come from washing since the washing direction is alternated). Right now we are running low on capture Ab so I am growing up hybridoma cells to produce it, until then I cannot run any ELISAs unless I know for certain that I have identified the issue and can get around it somehow.
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  #4  
Old 12-26-2011, 06:40 PM
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Default Re: Smooth drift gradient across ELISA plate

I have seen the same issue on multiple assays and have eliminated it by avoiding any opportunity for the plate surface to dry duing the procedure. We routinely perform 2 x wash followed by 2x wash in the opposite direction to eliminate any plate washer performance issue. To be more specific, the wash cycle is aspirate 5 seconds, wash 300 uL, aspirate 5 seconds, dispense 300 uL. Rotate and repeat, so we end up with liquid in the well, which is dumped immediately before the next loading step. We do not beat plates to remove every drop of wash buffer, we blot gently and keep some moisture in the wells. In some problematic assays we have had to specify that the duration between the end of wash and end of loading is no more than 90 seconds. This is recorded by the analyst and we reject the assay if we exceed 90 seconds.

I agree that the loading time is insignificant relative to the 90 minute incubation duration.

By your experimentation you show that left to right loading results in lower signal on the right, and right to left results in lower signal on the left. So in the left to right, the wells on the right are exposed to air longer than those on the left and vice versa. This is the only variable. You have also demonstrated that the TMB loading is not a problem, so once the HRP is in the well, it retains full activity and is not susceptible to this problem.

I don't know whether my previous suggestions were clear. One of the reagents is becoming denatured upon drying and dehydration. So in the hydrated state, it is able to function optimally, and after a period of dehydration, it functions with reduced efficiency. So immediately after removal of the liquid from the final wash cycle, binding is at 100%, and after beating the plates dry binding is at 60% (for example) loading the first wells is at 50% and last wells at 40% resulting in a 20% drop from the first to the last wells to be loaded. If the lab is humid, the effect will take longer, if it is dry (40% or lower) the effect will be rapid.

You can identify which is the susceptible reagent by breaking the assay down step by step as you did for the TMB experiement.

I suspect that the coating reagent is the problem and becomes less well orientated and less able to capture the analyte after a period of dehydration.

The solution is not to allow the plate to dry after washing, do not beat the plate, do not attempt to remove all the liquid from the wells before loading the next reagent or sample. The alternative is to fully dry the plate between each step in a controlled humidity environment.

4HBX or maxisorp are charge coated to orientate IgG with the Fc down, so are preferred for such assays. 2HB or polysorp are a plan 'B' for antigen capture assays where a different charge coating may be beneficial.

If you switch to maxisorp or 4HBX you may find you can drop coating to 1 ug/mL giving the same signal. Coating efficiency may be improved further by adding equal or 2 x mass of inert protein (BSA or Casein for example) to the coating antibody solution.

Good luck
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  #5  
Old 12-27-2011, 02:12 AM
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Default Re: Smooth drift gradient across ELISA plate

This is excellent advice, thank you so much. I understand the dehydration issue now and will try to avoid drying as much as possible, I have definitely been over-drying (banging plates etc.). Perhaps even during the sample loading step which is the slowest loading step (the only one where I change tips for each column and pipette out of a plate instead of a trough) I might leave the last wash in and pipette it out immediately before pipetting in the sample.

I will also talk about the 4HBX plates with my labmates and decide whether we should buy a batch to try them out.

Once again, thank you so much. I will get back to you once I have run another test and eliminated the drift, but I am pretty confident that dehydration is the issue as you've explained it.
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Old 12-31-2011, 07:46 PM
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Default Re: Smooth drift gradient across ELISA plate

I've got a nice big batch of brand new capture mAb produced, so I'm back on the ELISAs. I ran another test plate and did the following:

- I am now washing using a squirt bottle. I fill each well completely and overflow it so that the entire plate is flooded.
- I no longer dry the plate in between washes or before loading samples/reagents. I simply flick out the wash, lightly pat dry the surface of the plate, and very quickly pipette in the reagent.

Result: The drift effect is pretty much gone! There are still very slight hints of it but nothing remotely like what it used to be. I am confident that with even better technique and avoidance of drying, there will be no drift at all. I am immensely grateful for your advice on this!

One issue though: Since I do not have easy access to a plate washer, I will probably stick to squirt bottle washing since multichannel pipetting is too slow. However, I believe the squirt bottle method has really increased my CV, where it's 5-8% now, which is not really acceptable.

Have you ever used a squirt bottle for washing, and do you have any advice on how to do it to minimize intraplate variation?
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Old 01-01-2012, 01:43 PM
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Default Re: Smooth drift gradient across ELISA plate

I don't have too much experience with squirt bottle washing..although I'm not convinced that washing 'too hard' will remove bound reagents. Scratching the plate surface with a pipette tip might cause problems. We do manual wash on occasion, but use a multichannel repeater with 1.2 mL tips which allows us to dispense 4 columns with one charge. I am not really aware of any increase in the CVs with the manual wash procedure if it's done properly, it's just a convenience thing. Perhaps just take care that residual solution from wells containing high analyte concentration doesn't fall into wells with low analyte concentration on the first discard and that wash buffer from the first wash doesn't overflow into adjacent wells.
I would consider an 8% response CV (96 wells loaded with mid curve concentration) to be borderline resulting in 20% assay failure rate based on bioanalytical guidance acceptance criteria.
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Old 01-03-2012, 12:10 AM
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Default Re: Smooth drift gradient across ELISA plate

Can anyone identify the pipette that appears at 1:55 in this video:

(link in next post)

I would LOVE to use that pipette for washing. It looks like a combination of a bottle dispenser and a repeater pipette.
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Old 01-03-2012, 12:11 AM
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Default Re: Smooth drift gradient across ELISA plate

Here is the video:

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Old 01-05-2012, 03:12 PM
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Default Re: Smooth drift gradient across ELISA plate

(my post with the link to the video is still being approved)

I received an email back from the people who posted the video, this is the pipette:
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Looks great!
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