Problems with standard curve fit in ELISA
Accurate standard curves are needed to determine the concentration of target protein in your sample.
Before performing quantitative ELISA, you need a standard curve that performs well. This ensures you can reliably determine the concentration of your samples.
Note: If using one of our ELISA kits, the measurement values can vary considerably from the examples shown on the datasheet or protocol booklet. This is usually to be expected, so long as the curve has a good fit, as measured by the regression coefficient (R2). As long as the R² is >0.98, the standard curve can be used with confidence.
What are common issues with standards?
Possible issues
Solution
Standard may be degraded.
- Store and handle the standard as recommended.
- Avoid freeze/thaw cycles, if possible.
Pipetting error.
- Use calibrated pipettes and proper pipetting technique.
- Mix all solutions by pipetting up and down gently before adding them to the plate.
The standard solution has not been diluted correctly.
- Confirm dilutions and dilution calculations were made according to recommendations.
The standard has been improperly reconstituted.
- Briefly spin the vial before opening; inspect for undissolved material after reconstituting.
Why is the curve fitting model is not working with my data?
Possible causes
Solution
Curve-fitting model doesn't work
You may need a different curve-fitting model.
- You should always follow the manufacturer's instructions in the first instance. However, if the curve-fit doesn't seem to work, try plotting using different models.
- The most common curve fitting model for ELISA is the Four-Parameter Logistic (4PL). In some instances, a linear or 5PL model may be appropriate.
- Check that the curve not fitting isn’t due to replicate variation or incorrect pipetting.
- Run the standards in duplicates or triplicates.