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Prolonging sample incubation time to reduce plate drift?

Prolonging sample incubation time to reduce plate drift? - ELISA Assay Forum

Prolonging sample incubation time to reduce plate drift? - 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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  #11  
Old 04-25-2013, 09:17 AM
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Default Re: Prolonging sample incubation time to reduce plate drift?

Sounds very frustrating issue, trouble shooting ELISA is a bit tricky because it is difficult to look at one step at a time. If you have not tried this yet i would change the orientation of the plate during the assay, if you used to start at well A1 start at H12 insted and add reagents in the revers order than previously. If there is something in the instumentation that is causing the drift the drift will still go in the same direction as before, if it is the manual steps of adding the reagents and drying of wells then the drift will go in the opposite direction as before.

This probably is of little help to you but just wanted to say that i found out what my problem was.
I did an experiment where I used one pooled serum sample and compared how readings were effected by one hour benchtop time before incubation. I found that there was no difference between sample if they were on the plate for one hour or just one minute before incubation, if the incubation was 3hrs or over night at 4C. If the incubation was only two hours then there samples added first had higher readings. The manufacturers protocoll was 2hours but we will extend this to three as we saw no negative effect on controlls or std curve.
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Old 04-25-2013, 12:27 PM
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Default Re: Prolonging sample incubation time to reduce plate drift?

Could you explain a bit more, I don't quite understand what the issue was and how you solved it. What is "benchtop time"? Please be as detailed as possible.
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Old 04-26-2013, 12:33 AM
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Default Re: Prolonging sample incubation time to reduce plate drift?

Ok, I ran two plates today and both have very low drift. Here's what I did:

- Washed really vigorously and used about 1.5 times the volume of wash buffer I usually use. Just washed the crap out of the plates basically.
- Actually pat-dried a bit more vigorously than usual to make sure I got rid of all the residual wash buffer, but also made sure I pipetted very quickly right after.
- Incubated samples for 2h15m instead of my usual 1h30m (unintentional - got stuck in class...)
- Equilibrated TMB substrate to room temp for 1 hour before use.

I think what probably made the most difference was washing extremely well and using room temp TMB instead of cold TMB. I do worry that this won't last long and sooner or later I'll start seeing drift again though...
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Old 04-26-2013, 01:21 AM
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Default Re: Prolonging sample incubation time to reduce plate drift?

Peroxid: glad to see you fixed your problem

Msymeonides: sorry to see you are still having a problem with this.

If everything is well hydrated, then that's not the problem.

Most of our assays are TMB based and we target a 10 to 15 min development at RT to get a 650 nm OD of 1.0 at the high standard before stopping which usually results in the stopped high standard being at about 2.5 450-650 units. Less than 5 minutes' development gets tricky to control. This guideline is based on at least 100 different assays running 5000 plates annually and we don't get drift. What do you mean by cold TMB?

If development is too fast in method development, we typically cut the conjugate concentration as this doesn't affect the kinetics of the capture reaction.
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Old 04-26-2013, 12:00 PM
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Default Re: Prolonging sample incubation time to reduce plate drift?

Quote:
Originally Posted by msymeonides View Post
Could you explain a bit more, I don't quite understand what the issue was and how you solved it. What is "benchtop time"? Please be as detailed as possible.
The issue was that I have to add samples one by one and this takes 25minutes so the first sample is on the plate for 25minutes before I start the incubation. This 25 minutes difference between adding the first and last sample affected the results. When I extended the imcubation time from 2 till 3 hours I was able to reduce the effect between the first and last sample. The longer the incubation time the closer to equlibrium the ab-ag binding will be the eq and the eq is the same regardless of the start time.

The time the plate is on the benchtop while you prepare it I call bench top time. It is not in the protokoll but you can not avoid having some bench top time. In my case it is normaly 25 minutes when adding samples, as explained earlier. For washing and adding substrates it is only a minute or so.
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Old 04-26-2013, 01:42 PM
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Default Re: Prolonging sample incubation time to reduce plate drift?

To get around the issue of taking 25 minutes from the first to the last sample, you can use an "intermediate plate". Just take a regular 96-well PCR plate or tissue culture plate, and while your ELISA plate is still incubating in the blocking buffer, carry out your sample prep into the intermediate plate. Make sure the volumes in the plate are at least 130 ul per well so you can confidently pipette 100 ul out without bubbles. Once your samples are prepped you basically have an identical clone of the ELISA plate that you can simply transfer onto the ELISA plate itself quickly using a multichannel pipette. Basically, don't wash out the blocking buffer until your intermediate sample plate is done so there are no delays.

I would also suggest placing one column of your standard curve on the left edge of the plate and one column on the right edge, instead of two columns side by side. This way you'll be able to compare the ODs from the left to the right edge and get an idea of whether you are having sample drift.
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Old 04-28-2013, 11:36 AM
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Default Re: Prolonging sample incubation time to reduce plate drift?

I posted a response but my post hasn't been "approved by a moderator" yet...
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Old 05-06-2013, 10:40 AM
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Default Re: Prolonging sample incubation time to reduce plate drift?

Quote:
Originally Posted by msymeonides View Post
To get around the issue of taking 25 minutes from the first to the last sample, you can use an "intermediate plate". Just take a regular 96-well PCR plate or tissue culture plate, and while your ELISA plate is still incubating in the blocking buffer, carry out your sample prep into the intermediate plate. Make sure the volumes in the plate are at least 130 ul per well so you can confidently pipette 100 ul out without bubbles. Once your samples are prepped you basically have an identical clone of the ELISA plate that you can simply transfer onto the ELISA plate itself quickly using a multichannel pipette. Basically, don't wash out the blocking buffer until your intermediate sample plate is done so there are no delays.

I would also suggest placing one column of your standard curve on the left edge of the plate and one column on the right edge, instead of two columns side by side. This way you'll be able to compare the ODs from the left to the right edge and get an idea of whether you are having sample drift.
Thanks that is good advise, we run std curves on left and right to monitor drift. Using an intermedia plate would solve the problem but as you said it is difficult to pipett if you do not have 20-30microliter extra. So it is a balance if it is worth diluting the sampel, maybe it is.
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