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Higher Titers on outside rows

Higher Titers on outside rows - ELISA Assay Forum

Higher Titers on outside rows - 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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Old 06-08-2011, 04:48 PM
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Question Higher Titers on outside rows



I am using a indirect ELISA to detect antibody levels in serum from mice. For the past month, the titers on rows A and H are consistently higher than the duplicate of the sample (run on rows B and G, respectively). I incubate plates overnight for the coating step. Has anybody had this phenomena happen? If so, how do I combat this problem?
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Old 06-08-2011, 11:59 PM
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Default Re: Higher Titers on outside rows

assuming that you are sealing your plate during the incubations, edge effects are usually caused by a temperature gradient during one or more incubation steps.

so, for example, addition of cold substrate to a plate at room temp, will result in the edge wells warming more rapidly than those in the centre, so the reaction progresses more rapidly on the periphery.

The same effect will be seen for binding of a detection antibody (and I have seen antigen down overnight assay coating steps dramatically affected by the same thing)

Solution is to ensure that all the reagents are at the incubation temperature before they are added to the plate, and if you want to go the extra mile, ensure that the plate and wash buffers are also at the incubation temperature.

Perform a uniformity assessment, by plating the same thing in all 96 wells at mid curve concentration, and aim for response CVs in the 5 to 7% range and no evidence of edge effects for a robust assay.

Other things that can cause drift, rather than edge effects, include differential plate drying between plate washing and solution application. Lab humidity variations can dramatically influence this problem. I always ensure that the well is wet, by minimizing interval between washing and application of subsequent solution (30 seconds or less) and always use a 96 well polypropylene preload plate and a multichannel pipette to load samples to empty wells.
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Old 06-10-2011, 10:52 PM
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Default Re: Higher Titers on outside rows

Moderator,
I responded two days ago with information that j.ernst might find useful and the response has to be reviewed by a moderator
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Old 06-11-2011, 05:10 PM
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Default Re: Higher Titers on outside rows

in the absence of the full repsonse being approved by the moderator

Edge effects are usually caused by differential temperature gradients during the incubation by application of solutions, etc, to the plate at a temperature different from the incubation temperature (outside wells warm/cool quicker than inside wells)

Ensure wash buffers, antibody solutions, plates, etc, are prewarmed (or cooled) to that of the incubation temperature. Your problem will most likely be solved.
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Old 06-13-2011, 12:29 PM
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Default Re: Higher Titers on outside rows

Thank you Elisadeveloper for the suggestion. I will try this during my next ELISA
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