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Major ELISA Issue


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#1 matty2431

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Posted 19 August 2009 - 12:15 PM

hello

i am having trouble with my sandwich elisa with streptavidin hrp, 1 step tmb substrate.

6/10 plates i have run have presented the same issue and I have been observed by a collegue who confirmed its not my trchnique causing the odd results.

So, my plate develops perfect except for rows A,B, and C down the entire plate including std curve (500ng). its not a plate washer issue as we rotate our plates 180 and perform a second wash each step to overcome washer variability. all my incubations are at 37 for one hour. originally i thought the issue was stemming from stacked plates but I stopped that habit and results were the same.

so again, everything to the right of row B/C develops perfect but everything to the left doesnt develop at all. any ideas?

Edited by matty2431, 19 August 2009 - 12:16 PM.


#2 swanny

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Posted 19 August 2009 - 05:29 PM

hello

i am having trouble with my sandwich elisa with streptavidin hrp, 1 step tmb substrate.

6/10 plates i have run have presented the same issue and I have been observed by a collegue who confirmed its not my trchnique causing the odd results.

So, my plate develops perfect except for rows A,B, and C down the entire plate including std curve (500ng). its not a plate washer issue as we rotate our plates 180 and perform a second wash each step to overcome washer variability. all my incubations are at 37 for one hour. originally i thought the issue was stemming from stacked plates but I stopped that habit and results were the same.

so again, everything to the right of row B/C develops perfect but everything to the left doesnt develop at all. any ideas?

It sounds like you are not coating the plate in those three rows. Do you coat the plates yourself? Manual antigen coating or do you use an automated system? If so, you could have a blockage in the coating tubing; try the assay with the plate rotated 180 degrees from the start. If your coating has failed, your failed rows should change.
Other failures, such as blocking, would cause high background. The same for poor washing.

The problem is that with each reagent addition step, it should be obvious if you have not added the reagent...
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#3 matty2431

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Posted 19 August 2009 - 07:10 PM

No all the wells are coated, and all reagents are added. I mean we are talking about well A1 which probably is the easiest well to make sure you have all the reagents added to. Say for the standard curve, obviously antigen had to be added to well A1 for well A2-12 to be diluted/show any development at all. Same for the samples. I am getting down the dilution gradient development, just not in the first couple of columns. Its really odd, but I seem to be really good at reproducing the same results. Of the plates that have developed correctly, the standard curve had a HUGE hook effect. We are starting to think maybe the std conc is too high.. but that doesn't explain the samples acting the same.




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