Non-specific background signal is too high Test to see if the detector conjugate is binding non-specifically to the plate.
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 prior to the bottom of the standard curve Make sure that the standard curve was pipetted and diluted properly.
Standard improperly constituted Standard curve concentration is too low for the dynamic range of the ELISA.
No signal for the standard curve; signal for zero standard is normal Concentration of the standard is too high; decrease the amount of standard to within the dynamic range of the assay.
OD without standard present is too low Increase antibody concentration.
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 in replicates. If the variance is too high, consider increasing the concentration of standard by one.