Questions from customers Posted April 24, 2017 Report Share Posted April 24, 2017 Dear Mabtech, Usually, we coat our plates ourselves but recently I had a chance to try the pre-coated sample kit and it made such a difference! But before moving forward I wanted to get your expert opinion on the data and whether I am correctly interpreting what I am seeing. In the past, when I ran the assay on our plates I got a lot of background, and I had trouble getting the correct focus on the reader. For my comparison, I ran the assays at the same time, using exactly the same samples oriented exactly the same way on the plate. After development, the pre-coated plates were much clearer. The spot resolution was much higher and there was much less background in the high responding wells, thereby increasing the contrast. I read the plates with the IFN count settings and did not adjust anything except the AOI. What I saw was much higher spot counts on the pre-coated plate. Visually, it is obvious that the level of specificity of the samples did not change, but the background is significantly decreased on the pre-coated plate. I’m guessing this then allows the camera to identify the spots more accurately. My questions: 1. Am I correct in assuming that the decrease in background is resulting in more accurate spot counts? 2. I did not have to adjust the reader setting much to get most of the spots counted on the pre-coated plate. Is this how the reader should perform, i.e. with minimal manipulation to settings? 3. Do you think the flexible underdrain on the manually coated plate could be contributing to poor spot resolution? 4. The protocol we currently follow uses antibodies from Mabtech, but ALP and color development reagents from other companies. In addition to variability from coating the plate ourselves, do you think there is added variability when mixing the different reagents from different manufacturers? Also, do you think the conjugated ALP might be helpful not only to save time with the assay but also to minimize non-specific color development? Thank you so much for your valued input! Best, R Link to comment Share on other sites More sharing options...
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