Competitive ELISA troubleshooting tips
Solutions to common problems with competitive ELISA.
What are the common problems with competitive ELISA?
Possible issues
Solution
Non-specific background signal is too high
- Test to see if the detector conjugate is binding non-specifically to the plate by running wells with the primary antibody and sample omitted.
- Decrease the concentration of the detector conjugate.
- Optimize blocking with a different blocking buffer or by increasing the time/temperature of the blocking step.
- Increase the number and/or length of the wash cycles.
B/B0 ratio for the end of the standard curve point is >95% or <5%
- Increase/decrease the amount of standard by one dilution factor to shift the standard curve.
Standard curve reaches a plateau at the top of the standard curve (high concentration; low OD)
- Make sure that the standard curve was pipetted and diluted properly.
- Verify non-specific binding isn’t too high.
- Increase the incubation time.
No signal for the standard curve; signal for zero standard is normal
- Verify the standard curve was pipetted and diluted properly.
- The concentration of the standard is too high; decrease the amount of standard to within the dynamic range of the assay.
OD value of the zero standard is too low
- Increase antibody concentration.
- Incubate longer with the substrate.
High CVs at the bottom of the standard curve
- Higher ODs (ie, lower concentrations) are more likely to demonstrate variance in replicates. If the variance is too high, consider increasing the concentration range of the standard curve or diluting the samples less so that they fall in the middle of the standard curve.