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ELISA Data Analysis - Guide to make ELISA standard curve and calculate concentration of target proteins ELISA assay is used for determining the concentration of target protein in biological samples. Three data output can be yielded. Quantitative: A standard curve can be generated by ODs of serial diluted standard proteins and corresponding known concentrations.

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ELISA data of samples can be interpolated from standard curve to calculate the concentrations of target proteins in samples. Qualitative:we simply get a negative or positive result from the assay to determine whether there is any target protein existing in a certain sample by comparing to the negative control. Semi-Quantitative: With this type of, we are able to get a negative or positive result as well as to compare the target protein levels in assay samples, since the level of samples'absorbance will directly correspond to the level of target protein concentration.

However, we can't calculate the exact concentration since there isn't any standard protein in the kit. Generally speaking, with quantitative ELISA kit, you can quantify target protein in samples since you test a serial of standard proteins of known concentration.

When you analyze ELISA data with quantitative ELISA kit, you have to plot the mean absorbance against the protein concentration and draw a curve fitting your standard result best, and then interpolate absorbance of samples to the curve to calculate the concentration. This ELISA standard curve protocol will give you a comprehensive instruction on how to calculate ELISA results with quantitative ELISA kit step by step. Firstly, you need to run the ELISA kit according to our guide of plate layout and get raw data from ELISA reader, and then let's go to the next step—statistical analysis of ELISA results, which is the last and the key step to get the concentration of target protein in your samples. We recommend you an ELISA data analysis software curve expert 1.3 or curve expert 1.4 that is simple to use and you can download at the end of this page to process data calculation. There are many other curve fitting softwares available in the market to conduct ELISA calculation as well such as or you can also use normal MS Excel for analysis. There are three steps to process ELISA data analysis. Input ELISA data of standard into the software.

Choose the best fitting curve. Calculate the concentration of target protein by interpolation Now I will show you how to make an ELISA standard curve step by step. Input ELISA data of standard into software After running up the ELISA assay, you can separate your raw data into three parts.

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It is recommended to run standards and samples in duplicate or triplicate. As a best practice, you’d better control the CV of replicates less than 8%. Part 1: absorbance of standards with known concentration. Part 2: absorbance of blank well/background. Part 3: absorbance of samples with unknown concentration Standards are serially diluted with Sample Diluent. Samples may also need to be diluted.

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Since sample diluent has absorbance even when there is no protein present at the detection wavelength, we run blank well with sample diluent in the assay to get the background OD. It is common practice to subtract the absorbance of the blank well from standards and all sample absorbance. Choose the best fitting curve 2.1 Click Run button and let software examine your data to choose the best possible curve fit. Select the model families to include in calculation.

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Usually, we click All On to include all model families. If the polynomial family is included for consideration, you must specify the maximum degree of the polynomial that software will consider in the 'Polynomial Constraint' area. We recommend setting the maximum degree of polynomial as “4”. Of course, if the polynomial family is not included, the polynomial constraint will simply be ignored.

Then press OK. This is an example to show you how different standard curves affect result. If a test sample produces an absorbance of 1.4, the corresponding two concentrations calculated by linear curve (r= 0.92255796) and nonlinear curve (r= 0.99993479) have a huge difference (199.955 VS 114.898). In this example we finally select “Rational Function” since its “r” value is closest to “1” compared with other standard curves. By pressing the right mouse button or by pressing Ctrl-M in any graphing window, you can pick from the graphing menu. By clicking Copy button you can paste standard graph to excel sheet or word document.

Calculate the concentration of target protein by interpolation How to calculate concentration? You have two ways to calculate the concentration of target protein. Analyze ELISA data in the software. The software allows easy location of x/y points, differentiation, and integration of a curve fit that has been performed by choosing Analyze from the graphing menu or pressing Ctrl-L.

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In this example we would like to find the concentration of target protein (Y value) by evaluating the curve fit at a given OD (X value). Just type in the point at which you want to evaluate the curve fit (the x value) in the 'At X=' field and press Calculate or Enter on your keyboard.

The calculator will then show the corresponding y value. Coefficients The Coefficients section gives the values of the relevant parameters for the current model - these coefficients are always expressed as a, b, c, d, and so on. These coefficients match the model shown in the Model section. If the list of coefficients exceeds the extent of the window that they are shown in, a scrollbar will appear to allow you to scroll through the parameters. If we click Copy , the list of coefficients is copied to the clipboard. Then paste them in any Excel sheet.