| | Re: Question about Plate surface variability
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