High Variability Between Replicates (CV 15%) (ELISA (R&D Guide))

Question

High Variability Between Replicates (CV >15%) (ELISA (R&D Guide))

Accepted Answer

Replicate wells for the same sample or standard show high coefficient of variation (>15%), with inconsistent absorbance values that suggest uneven treatment or technical errors. Common causes: 1) Solutions insufficiently mixed before addition, or uneven plate coating due to pipetting error 2) Evaporation during coating step (no plate sealer used) or coating antibody concentration too low to saturate wells 3) Inadequate washing leaving residual solution in wells; automatic washer ports obstructed 4) Bubbles in wells affecting optical reading 5) Contaminated buffers, pipette tips, or carry-over between samples Solutions: 1) Thoroughly mix each solution before adding to plate; verify equal volume addition per well 2) Use plate sealer during coating; extend coating time to overnight at 4°C for low antibody concentrations 3) Ensure complete solution removal between washes; check washer ports; add 30-second soak and rotate plate mid-wash 4) Centrifuge plate before reading to remove bubbles 5) Prepare fresh buffers; use fresh tips for each sample; avoid tip reuse