I have used excel and Prism to analyse the results.
I have some sample values which are below blank (OD).
If I substract the OD value of these samples from blank I do get VERY LOW or negative values which latter when I calculate the cytokine concentration based on standard curve , I get negative values.
What is the best to do inthis case?
What is the best program to analys the result from ELISA?
Should I subtract the OD of samples from OD of blank before past it in any program?
Any answer appriciated.
It is more usual to subtract the OD of the blank from the OD of standards and samples - often called "blank corrected data". It should not matter whether you use the raw OD or the blank corrected OD to fit you data.
The fit that you use for the standard curve to calculate the results from your sample ODs depends on the data in the standard curve, but for ELISA is usually a 4-parameter fit. This is readily calculated in PRISM. If you are unsure, PRISM have excellent help files, and as long as you have your license number you could contact PRISM directly for help.
IF the sample OD are lower than the blank OD (or the lowest standard), it is not possible to calculate an accurate concentration for those samples. You could report these data as lower than the limits of detection of the assay. For each assay it is important to know the limits of detection of the assay - often this information is provided by the manufacturer.
Thank you so much Lain . One more question: standard values should be also blank corrected .
Regards,
Thank you Dr. Lain Martin Sheldon,
I have another question;
I am working on a in vitro inflammatory model(a co-cultured model) which stimulated with IL-6. After stimulation , the control and control+IL-6 show no significatnt differences in expersion of proinflammatory cytokines TNF-a or IL-8.
I would like to know your opinion on this result. Iam thinking the model is not working! what do you think?
I suggest that you check that your model is functioning, as your data suggest there may be a problem if you expect an increase in IL-6 from your model. You might be able to use a positive control challenge and a known inflammatory mediator readout to confirm your model.
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