Problem with ELISA horizontal data drift
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I am seeking any input/advice regarding a serious problem we are having with data drift on our ELISA plates. We are coating Costar high-binding 96-well plates with a PDGF-beta receptor/Fc chimera in carbonate buffer overnight at 4 degrees C. The concentration of chimera is ~0.4 ug/ml as recommended by the manufacturer and we are loading equal volumes (100 ul) into each well of the plates using an 8-channel multichannel pipet. We have checked the pipet to be sure equal volumes are being dispensed from each tip. Solutions and wash buffers are dispensed in a left to right fashion across the plate, column by column. For detection we are using a biotinylated goat anti-PDGF antibody followed by streptavidin-HRP and TMB substrate.<br /><br />Following development of the assay, we are consistently finding that the response for a sample containing the same amount of PDGF at the top of the plate (ie. Row A) is ~30-40% higher than for the same sample at the bottom of the plate (ie. Row H). Thus, we see horizontal drifting of our data from top to bottom despite loading the plates in a left-to-right fashion. We are perplexed and any helpful comments or ideas from the community would be greatly appreciated.
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