Poor Reproducibility Between Duplicate Wells (ELISA (Sigma Guide))

Question

Accepted Answer

Coefficient of variation (CV) between duplicate or triplicate wells exceeds 10-15%. Individual replicates show inconsistent absorbance values that cannot be attributed to biological variation. Common causes: 1) Uneven plate coating with inconsistent antibody or antigen coverage across wells 2) Insufficient or non-uniform washing with obstructions in washing ports 3) Variation in incubation temperature across plate positions 4) Inconsistent coating and blocking volumes between wells Solutions: 1) Use proper high-binding ELISA plates; verify coating and blocking volumes are consistent (typically 100 µL per well) 2) Follow uniform washing procedures; inspect and clear any obstructions in washer aspiration or dispensing ports; validate wash cycles 3) Use temperature-controlled incubator; avoid placement near heat sources or air vents; allow plates to equilibrate to room temperature before reading 4) Use calibrated multichannel pipettes; dispense from reagent reservoirs; verify pipette accuracy and precision