High Coefficient of Variation at Low Standard Concentrations (ELISA (Competitive))

Question

Accepted Answer

Replicate wells at the bottom of the standard curve (low analyte, high OD) show high CVs >15-20%, while mid-curve and high-concentration points demonstrate acceptable reproducibility. Common causes: 1) Higher OD values inherently more susceptible to pipetting and reader variability 2) Minimal competitive displacement at low concentrations amplifies measurement variance 3) Sample concentrations falling outside optimal middle range of standard curve 4) Edge effects on microplate causing differential evaporation or temperature gradients Solutions: 1) Shift standard curve concentration range upward to move samples into mid-curve range with lower CVs 2) Dilute samples less (e.g., 1:2 instead of 1:5) to increase measured concentration into optimal range 3) Use calibrated multichannel pipettes and increase number of replicates from duplicates to triplicates 4) Avoid using outer edge wells of plate or include blank rows/columns as thermal buffer zones