In the ELISA experiment, it is essential to use each sample in duplicate or triplicate wells. The average OD value for each standard concentration should be calculated, and the coefficient of variation (CV) between duplicates should not exceed 20%. This ensures data reliability and consistency.
Next, subtract the blank OD value from the average OD values of all samples and standards. This step helps eliminate background interference and provides a more accurate measurement.
Afterward, create a standard curve by plotting the adjusted OD values on the X-axis and the corresponding standard concentrations on the Y-axis. To achieve the best fit, use software like CurveExpert 1.4, which offers multiple fitting methods such as linear, semi-logarithmic, logarithmic, and four-parameter logistic equations. Choose the model that best matches your data.
To validate the curve, input the known standard concentrations into the equation and calculate the expected OD values. If the results are within ±10% of the actual values, the curve is reliable. Select the curve with the highest correlation coefficient for accurate quantification.
Here’s how to use CurveExpert 1.4:
1. Copy and paste the OD values and corresponding standard concentrations into the software table.
2. Generate and select the most appropriate standard curve based on the data fit.
3. Use the selected curve to calculate the sample concentration by entering the OD value and clicking “Calculate.†The software will return the exact concentration.
Alternatively, you can export the equation and perform calculations in Excel for convenience.
If the OD value of a sample falls outside the range of the standard curve, it means the concentration is either too high or too low. In such cases, dilute the sample before testing. After obtaining the concentration from the curve, multiply it by the dilution factor to get the true value.
By following these steps, you can ensure accurate and reproducible results in your ELISA experiments. Always double-check your data and maintain proper documentation throughout the process.
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