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-   -   Prolonging sample incubation time to reduce plate drift? (http://www.molecularstation.com/forum/elisa-assay-forum/87735-prolonging-sample-incubation-time-reduce-plate-drift.html)

per_oxid 03-20-2013 12:58 PM

Prolonging sample incubation time to reduce plate drift?
 
Hello!
I was very happy to see that there is a forum for ELISA discussions.

I have a problem with plate drift. I add samples by single channel pipett from microtubes(1.5ml). I do this since we have very low sample volumes (220myliters) and I wish to mix samples carefully and doing this using racks and multichannel pipett I found difficult. The drift is not very strong but is clearly seen when runing standard in collumn 1 and column 12, a value of 2.3 at first column is typicaly 2.0 at the last. The time difference from adding first to last sample is 20-25minutes. The incubation time is then 120min at 37C

Is there a some way to reduce this drift, exept from being quicker to add samples. I was thinking that i could change the incubation time from 120min at 37C to 18H at 4C to allow a steady state to establis.

Would appreciate any help
Regards
Per

luisillo 03-21-2013 02:38 PM

Re: Prolonging sample incubation time to reduce plate drift?
 
But you are not working at 37C. You adding your samples at room temperature which should oscilate between 20-25C, are you not? You state that the volumes vary from well to well when using a multichannel pipette (I don't trust them either) but, has the incubation time caused you any issue? If not, why modifying it?

per_oxid 03-21-2013 04:47 PM

Re: Prolonging sample incubation time to reduce plate drift?
 
Sorry if I was unclear.

I am working in roomtemperature 23C. The reason I dont use multichannel pipett is that I have 220 microliter of sample and use 2*100. It is difficult to pipett accurately with 8-channels with so little excess volume, transferring samples to a rack from tubes will cause loss of some volume as well.

I get different results at the end of the run than at the start because it takes me about 25minutes to add samples. Therefore the first sample is on the plate for a longer period of time than the last sample. The first samples have 25minutes of room temp plus 120minutes at 37C whereas the last sample only has 120 minutes at 37C to bind to AB in well. If all samples are added within a very short time the end of run effect is removed, I have tested this.

I was hoping that allowing the binding to approach equilibrium by extending the incubation time would make the time it takes to transfer samples to plate less critical.

luisillo 03-22-2013 04:15 PM

Re: Prolonging sample incubation time to reduce plate drift?
 
Ok, I understand. Then yes, you could modify the temp of incubation to 4C but you would still have that 25 min of extra inc time at room temp for your first samples.

You could also try to even the delay of single channel pipetting by discarding supernant (unbound Ag) by absorbing it with the pipette (single channel) well by well in the order you add them, instead of turning the plate upside down. This will allow you to work at 37C as you describe you usually do.

Whether which one you use is your choice.

Good luck

luisillo 03-22-2013 04:29 PM

Re: Prolonging sample incubation time to reduce plate drift?
 
By the way, if you try the second choice I gave above be sure of repeating it for each time you add any reagent that needs incubation using single channel pipette, for reproducibility and confidency of your results. Don't forget to tap the plate over paper towels after supernatant discarding. Also, I highly suggest you use a wash bottle or a multichannel pipettes for the wash solution washing steps.

Elisadeveloper 03-23-2013 02:14 AM

Re: Prolonging sample incubation time to reduce plate drift?
 
In my experience drift is the result of variable binding of sample/antibody to the immobilized phase due to variable hydration state of the immobilized surface (drying the immobilzed surface will result in conformational / binding changes)
Either keep the ELISA plate hydrated by not beating the plate after washing and immediately loading the sample from a 96 well polypropylene preload plate and a multi-channel pipette (taking less than 1 minute) or dry the plate completely before loading the samples. The first option is preferable and standard practice in my lab.

If you don't have enough volume for your pre-load plate load less or dilute.


If you are already working with a dry coated plate, don't underestimate the effect of evaporation of your sample in the first wells resulting in higher analyte concentration in those wells and also, don't forget that slow loading of secondary antibody steps (more than 1 minute) after excessive plate beating may also result in drift

good luck

Elisadeveloper 03-23-2013 02:17 AM

Re: Prolonging sample incubation time to reduce plate drift?
 
you may want to refer to previous post on 'smooth drift across plate'

per_oxid 03-25-2013 10:06 AM

Re: Prolonging sample incubation time to reduce plate drift?
 
Thank you for advice!
I will explore the options. The drying of plate effect is interesting but in this case I add samples to a dry plate. I am working with a commercial kit with pre-coated wells.
Have a nice day

Per

msymeonides 04-25-2013 12:30 AM

Re: Prolonging sample incubation time to reduce plate drift?
 
Hello, I'm the person who posted the "smooth drift" thread on a very similar issue. Amazingly, I am still having this problem and have not figured out how to get rid of it. It is incredibly annoying and I've resorted to simply finding ways to run my assays without the need of an ELISA.

All my loading steps take less than 1 minute and I never fully dry the plate. I wash using a squirt bottle. At the end of 4 washes I gently pat-dry the plate because I can still see a volume of wash in some wells, but everything is ready to go so I can load my next step immediately, so no well on the plate is really never left without some solution in it for more than 1 minute. I still see this very consistent gradient from left to right on every plate, and it can be as bad as a one third decline from column 1 to column 12 (e.g. 0.6 becomes 0.4).

I really suspect that the very last step (TMB) is where the gradient actually appears. I think it just develops too fast. I use it cold. I'm at wits' end with this.

msymeonides 04-25-2013 12:43 AM

Re: Prolonging sample incubation time to reduce plate drift?
 
It's interesting to see the suggestion of completely drying the plate before sample loading as an alternative to making sure it doesn't dry. This might be a better option for me even though my sample loading uses a multichannel and an intermediate plate, and takes less than 1 min. Perhaps the initial step of drying "damage" is extremely fast and if I actually let all the wells equilibrate to a dry state where the extent of "damage" between wells is fairly consistent, the gradient would disappear. I am trying this tomorrow. Could you suggest the best way to quickly and uniformly dry the plate? I could place it at 37 C, or freeze it.

Also, would you suggest skipping the washing step after the overnight capture Ab incubation and before adding block and just directly adding block to the plate while the capture Ab is still in it? This would mean basically that there would be no partial drying steps until after blocking for an hour, at which point the plate would be dried completely.


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