ELISA non-linear analysis. - (Mar/11/2011 )
I'm quite new to lab work so excuse me if this is a silly question .
I have carried out an competative ELISA and i'm not sure how to work out the concentration of my samples from the absobances using the standard curve data as my curve is non-linear. Normally my data is linear so I just use the equation from excel to work it out.
I'm trying to use graph pad prism, however i'm not sure how to do this??
Any help would be appreciated,
That's not a problem in GraphPad Prism.
I assume you have your data and you get vaguely sigmoidal shape of curve?...
(based on GraphPad v.4.x)
Analyze --> Curves & Regression --> Nonlinear regression --> Sigmoidal dose-response (variable slope)
tick on option: unknowns from standard curve
Now when you enter your unknowns in Y column under your standard curve data, you'll get new tab "Interpolated X values". Of course those results are in the same format and unit as your curve (eg. log of concentration). (You can do the re-calc in GraphPad: Analyze --> Data manipulation --> Transforms.)
Note one thing - this will calculate your results from anywhere on the curve so if you enter unknowns that are too far from linear range you'll get very strange results.
That's not a problem.
As I see from the picture - you have concentration on X-axis. Transform that to logarithm (with GraphPad function) to get evenly-spaced graph, then use sigmoidal curve.
That's right. If you don't have those software to plot the standard curve and give you the formula and values, Excel also works. Transform both concentration ( X axis ) and OD (Y axis ) into log10, the standard curve will be linear. Then refer to the instruction at http://www.novateinbio.com/index.php?main_page=tech_info.
One of the easiest software to use for ELISA analysis is MasterPlex ReaderFit (http://www.miraibio.com/masterplex-readerfit/curve-fitting-for-plate-readers.html). With an intuitive user interface and full video tutorials, ReaderFit helps all levels of ELISA researchers get their results quicker. It also works with 4 and 5 parameter logistics, competitive binding (downward curve) and EC50 / IC50.
I realize this reply may be a bit late, but I hope it will help others looking to work on ELISA analysis.