We have an in house ELISA, how do you calculate unknown concentrations from optical density values?
We produced a standard curve but don't know whether to use sigma plot or excel.
Also, Which line fitting algorithm should we be using e.g. Linear, 4 parametric, polynomial etc.
Linear doesn't seem to fit my curve.
Thanks in advance
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Use whatever function provides a good fit to the curve. If none of them seems to work well, use linear interpolation. Do not include standards for which the absorbance exceeds the dynamic range of the plate reader, which is probably about 3 absorbance units.
Hi Terun,
To calculate the concentration of a unknown sample I used to prepare a standard curve (linear plot) of known concentration. Linear fit of samples absorption give you the best result of unknown sample, though you can use any function you want but that should fit the data curve.
Please try to prepare a standard curve using a large range of sample concentration as the unknown sample absorption should present at the curve. Unknown samples absorption value shouldn't be beyond the curve range.
Please have a look at this article's supplementary section- https://pubs.acs.org/doi/suppl/10.1021/acsami.6b11798/suppl_file/am6b11798_si_001.pdf
Dear Terun Desai, for a 2nd degree poly calibration you can use the attached excel workbook. This simple version only allows you to perform interpolation and does not assess the model goodness of fit nor estimates any statistical parameter. Therefore, it will not allows you to compare different models. Hope it helps. Regards Luis
See a good analytical chemistry book or lab manual and look up Beer-Lambert Law. Best wishes, David Booth
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