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Question about Plate surface variability

Question about Plate surface variability - ELISA Assay Forum

Question about Plate surface variability - 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 09-13-2011, 03:21 AM
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Default Question about Plate surface variability



Hi all:
I am new here and am looking forward to the discussions. I have a question for all of you ELISA experts out there: is surface variability a big issue for you? My impression is that surface variability would be especially undesirable for competitive assays? Is it more of a problem with 384 and 1536 plates than with 96-well plates? It has been a while since I did ELISAs myself so I appreciate your input.
Tina
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Old 09-14-2011, 02:07 AM
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Default Re: Question about Plate surface variability

Hi Tina,

Not sure about the larger plate formats, as I don't work with them. I have had issues with surface variability, especially with antigen down sandwich formats, and this includes antibody fragment down formats too. If variability is a concern, and it is not the result of a process issue (such as drift during loading) I perform uniformity assessments loading a mid curve spike in all wells and target response CVs of <7%. This is useful for detecting edge effects as well as random variability between wells. I have seen plate batch dependent variability in response ranging by as much as 50% or so from highest to lowest wells. A switch of plate type is often useful, or pre-selection of a plate batch. Manufacturer's QC process involves consistency of coating of a single labeled protein, or antibody and does not cater for the wide variety of charge/conformations of proteins that may be used in coat plating. Also, beware of pre-coated plates (streptavidin for example) which are manufacturer QCd with biotin-fluorocein and not with the relative skyscraper sized layers we can produce in assay design, so steric hindrance can exacerbate a uniformity issue, especially at high analyte concentration (for a sandwich format).

A trick I have found useful, is to pre-mix the coating reagent with an equal mass or slightly more of carrier protein (albumin or casein for example) and this can improve consistency between wells.

Hope this is useful
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Old 09-19-2011, 08:48 PM
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Default Re: Question about Plate surface variability

Thank you so much. Very useful!
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