I am using the ELISA kits from R&D Systems and am supposed to let the plates incubate with color buffers for 20 minutes before reading. I accidentally pulled the plates out at 15 or 17 minutes, added stop buffer, and read on the spec. The standards are low (highest, 1000pg/ml, averaged 1.7 OD, about where my second-highest standard usually reads). The standard curve looks good and my samples, which are high in concentration, are all in range and fall in the middle of the curve. Everything is just downshifted a bit in the readings. Does that seem like a problem for accuracy?
I think no problem because you make the same circumstances for all samples and standards when you work the procedure.
That's fine when appropriate negative and more so positive controls are put alongside. You will get relative absorbance values. That's fine!
I think no problem because you make the same circumstances for all samples and standards when you work the procedure.
Dear Beatitude Steffen ,
There is no problem in what happened, as all the values are based on the same temporal reaction and it occurs simultaneously with your calibration curve, in which you will base your results. The only thing is that, whether you repeat the assay, you should stop the reaction after the same time in this procedure, as not all chemical reactions have linear kinetics. Normally R&D Elisa kits use HCl 2 N to stop the reaction and a peroxidase based color substrate. Nevertheless it may use TMB, or other reagents. That's why I would advise the time aspect for your future assays.
Eduardo.
One must follow the same procedure for reproducibility of results and comparison with new samples or treatments.
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