Goodmorning! I need to se up a plasma ELISA assay. Once I put the plate in the plate reader and I obtain the results, what is the next step? I will first do the standard curve and a few samples with different dilutions, but then I do not know if I need to convert the numbers I obtained from the plate reader.
Thanks in advance!
Dear Federica Perrone
Once you put the plate in the plate reader and obtain the results (absorbance), you do the following.
You will obtain the absorbance readings (OD) from the plate reader. So, initially you need to subtract the absorbance value of the blank from the standard, the assay background control, and the sample values prior to creating the standard curve. Then you plot the standard curve with the absorbance values of the standards on the Y-axis and the concentration of the standards on the X-axis. Then with the help of the absorbance value of your diluted samples obtained from the plate reader you may obtain the concentration of the diluted samples from the standard curve. The absorbance value of the sample on the Y-axis corresponds to a concentration value on the X-axis.
The concentration of your diluted samples should fall towards the center of the curve as this area is the linear portion of the standard curve in which analyte concentration can be determined accurately. Concentration should not be extrapolated from the standard curve beyond the recommended standard range as outside this range the standard curve is non-linear. Samples for which the absorbance value exceeds the highest point of the standard range should be re-analyzed at a higher dilution.
Since you have diluted your samples, you need to multiple the concentration of the diluted sample with their respective dilution factor in order to obtain the final concentration of the analyte in the original undiluted sample.
Best Wishes.
Hi! Thank you for your quick and clear answer! Regarding the standard curve, I will do the following dilutions: 1/2, 1/4, 1/8, 1/16, 1/32, 1/64. When I plot the absorbance values of the standards for the curve, the concentration of the standards on the X-axis will be 1/2, 1/4, 1/8, 1/16, 1/32, 1/64?
Do you have perharps a ready-to-use excel file with all the calculations? It would be great, otherwise thank you so much anyway! :o)
There is an Excel program for this. It is called ADAMSEL and you can find it here http://www.malariaresearch.eu/content/software. It works great but you have to put a lot of time into understanding it.
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