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Calculation of ELISA results


1. ELISA samples are always tested in duplicate or in triplicate, then average the absorbance values for each set of standards and samples. Duplicates should be within 20% of the mean.

2. Subtract the average value of zero standard OD (optical density).

3. Standard curve.

Create a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. We suggest the suitable software should be used for this, such as CurveExpert 1.3. The software is recommended that various methods (e.g. linear, semi-log, log/log, 4 parameter logistics) be tried to see which curve best fits the data. One way to determine if the curve is to backfit the standard curve O.D. values. Treat standards as unknowns and interpolate the O.D. values from the standard curve. They should be close to the expected value (+/- 10%). Use the curve that gives the best correlation (r) value and backfit.

Step by step as shown:

Step 1: Input information of the OD value and concentration of the standards

Step 2: Create and select a standard curve.

We recommend generating a standard curve for each set of samples assayed.

Step 3: Calculate the concentration according to the curve


Input the OD value of the sample and click , then user can get the actual concentration of target protein in the sample.

Or get the relevant equation

4. Samples that have an absorbance value falling out of the range of the standard curve

If the OD of the sample surpasses the upper limit of the standard curve, these samples should be diluted before proceeding with the ELISA to obtain an accurate result. As for these samples, the concentration obtained from the standard curve must be multiplied by the dilution factor when analyzing the results.

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