Hello,
I am currently doing a Human Transferrin Receptor ELISA https://www.abcam.com/human-transferrin-receptor-elisa-kitcd71-ab272469.html.
When doing the standard I didn't make the right dilutions, for example the first standard has a concentration of 3400 pg/ml according to the protocol, but my first standard according to the volume that I diluted, theoretically has a concentration of 6400 pg/ml.
Due to this problem some of my samples' O.D.s are outside of the range of my modified standards.
The question here is: would it be correct to make a standard curve with my modified concentration to get the concentrations of the samples that are within the range?
If you have samples outside the standard curve, their data is not valid. You can do some estimates (extrapolation), but you have no information about the detection limit and possibly the background and other effects of the actual assay.
If possible, repeat the test, at least for the affected samples.
If you have only material for the standards left, you could test with 3 or 4 samples in the lower range, how to extrapolate the standard curve, but that's really not a good practice. For research, it might be acceptable, depending on what you are deducting from the data, for diagnostic procedures, it's a no-go.
Hello Melissa Rivera
It would not be right to make a standard curve with modified concentration. The standards in your assay should be at a range of concentrations that yields data from essentially undetectable to maximum signal. The calibration standards should be spaced approximately evenly on a logarithmic scale within the anticipated range. A typical standard curve will have a sigmoidal shape in which the higher concentrations of standard dilutions will reach a plateau in absorbance. The concentration of your unknown samples should lie within the linear range of your ELISA standard curve. Make sure you don’t miss this range.
Best.
You need to modify the concentrations of curve if your data out of your curve linearity range.