How to read ELISA Results?

[joey20]

Hi, how do you ELISA experts read elisa assay results?

i am confused from the data as its my first time. thank you all

[danfive]

You read absorbance on a spectrophotometer.
Calculate average absorbance for each std and sample. Use the avg absorbance for calculations.
Plot Absorbance values of standards only (x-axis) vs Protein conc (y-axis).
Extrapolate the linear or quadratic (best fit) line(and equation) for Abs values of the standards.
Use the formula for std curve to calculate the protein conc of samples/unknowns.
You do this by substituting Avg Absorbance (of sample) as x into the linear/quadratic formula; y =protein conc, x = avg absorbance.
I do this easily on excel, some spectrophotometers have built-in programs.

[Lawlence Zobofo]

Protein concentration should be on the x-axis and absorbance on the y-axis because the amount of absorbance depends upon protein concentration.

[Archonster]

For most of results, protein conc. should be on x-axis and ab. on y-axis.
As the cell based ELISA for compds screening, x should be the conc. of compds and y inhibition ratio calculated from ab.
So you have to know what kind of ELISA you are doing.

[danfive]

Originally Posted by danfive

You read absorbance on a spectrophotometer.
Calculate average absorbance for each std and sample. Use the avg absorbance for calculations.
Plot Absorbance values of standards only (x-axis) vs Protein conc (y-axis).
Extrapolate the linear or quadratic (best fit) line(and equation) for Abs values of the standards.
Use the formula for std curve to calculate the protein conc of samples/unknowns.
You do this by substituting Avg Absorbance (of sample) as x into the linear/quadratic formula; y =protein conc, x = avg absorbance.
I do this easily on excel, some spectrophotometers have built-in programs.

Like I mention in the above post, I do this on Excel--the spreadsheet program. I do this because the thermo spectrophotometer I use has horrible software on it.

Anyways, although, it is common sense to put the independent variable--protein conc on the x-axis, and the dependent variable (Abs) on the y-axis. I do it my way, because it makes no difference, and I get excel to solve for x (protein conc) by flipping the axes. That is by switching the x and y values, excel, which only solves for y, solves the equation for protein conc.

I can prove that it makes no difference, because I can just as easily switch the axes just as the last two posters mentioned. Resulting in an equation that requires me to manually solve for x. Solving for x, again gives me the same equation as flipping the axes.

In summation, following the advice of the last two posts, I get this:
Abs=Protein conc*constant + y-intercept
*this requires I manipulate and solve for Protein conc. I have Abs, I need Protein conc.

My way, w/ axes switched, gives this:
Protein conc=Abs*constant + y-intercept

**Excel program solves it for me!

[bubble]

Hi, could anyone help me with Elisa readings, please? I need to calculate a protein concentration using absorbance values.

I have a quadratic equation: y= -3E-06x^2+.0064x+.0483 (statdard curve equation), and absorbance values: 1.363, 1.592, 1.516, 2.426, 1.644, 2.715, 2.092, 0.824, for each of them I have to clacualte protein concentration using standard curve as stated above. I know that I have to calculate X1/X2 from quadratic equation, but I don't know what to do next??? enyone has an idea???? Thanks for your help!

P.s I plotted absorbance as a y-axis and concentration as a x-axis and then I generated the standard curve.