High Variability Between Replicates (CV >15%)

Symptom

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.

1. 1 Solutions insufficiently mixed before addition, or uneven plate coating due to pipetting error
2. 2 Evaporation during coating step (no plate sealer used) or coating antibody concentration too low to saturate wells
3. 3 Inadequate washing leaving residual solution in wells; automatic washer ports obstructed
4. 4 Bubbles in wells affecting optical reading
5. 5 Contaminated buffers, pipette tips, or carry-over between samples

1. 1 Thoroughly mix each solution before adding to plate; verify equal volume addition per well
2. 2 Use plate sealer during coating; extend coating time to overnight at 4°C for low antibody concentrations
3. 3 Ensure complete solution removal between washes; check washer ports; add 30-second soak and rotate plate mid-wash
4. 4 Centrifuge plate before reading to remove bubbles
5. 5 Prepare fresh buffers; use fresh tips for each sample; avoid tip reuse

Related Video (3)

Bilibili (China-Accessible Mirrors) ★ 85

How to Run an R&D Systems Quantikine ELISA

"This is the exact kit brand (R&D Quantikine) and includes troubleshooting guidance directly applicable to replicate variability."

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How to Run an ELISA Assay – Invitrogen Kit Step-by-Step Tutorial

"Tutorial emphasizes accurate and reproducible protein detection, and properly mixing solutions is critical for replicate consistency."

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DuoSet ELISA — Sandwich ELISA Hands-on Protocol (Bio-Techne)

"Shows plate coating and benchwork steps where uneven coating can cause high CV."