In the ELISA experiment, it is essential to run each sample in duplicate or triplicate wells and calculate the average OD value for each standard concentration. The coefficient of variation (CV) between duplicate wells should be less than 20% to ensure reliability and consistency of results. After obtaining the average OD values, subtract the blank OD from each reading to eliminate background interference.
Next, construct a standard curve by plotting the adjusted OD values on the X-axis and the corresponding standard concentrations on the Y-axis. Use specialized software such as CurveExpert 1.4 to generate and fit the best curve. This software offers multiple fitting options, including linear, semi-logarithmic, logarithmic, and four-parameter logistic models. Choose the model that best fits your data. To validate the curve, input the known standard concentrations and check if the calculated values match the actual concentrations within a 10% margin of error. Select the curve with the highest correlation coefficient.
To use CurveExpert 1.4:
1. Copy and paste the OD values and standard concentrations into the data table.
2. Generate and select the most appropriate standard curve based on the data.
3. Input the sample OD values into the software and click "Calculate" to determine the exact sample concentration. Alternatively, export the equation and use Excel to compute the concentration manually.
If the sample’s OD value falls outside the range of the standard curve, it indicates that the concentration may be too high. In such cases, dilute the sample before the experiment and re-run the assay. Multiply the calculated concentration by the dilution factor to obtain the original sample concentration. Always ensure that the final result is within the linear range of the standard curve for accurate interpretation.
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