High CV from Inconsistent Pipetting Technique (ELISA (Inconsistent Results / High CV))

Question

Accepted Answer

High intra-assay coefficient of variation (>15%) between technical replicates. Standard curve shows poor reproducibility between duplicate points. Common causes: 1) Pipettes not properly calibrated, delivering inaccurate volumes 2) Multi-channel pipettor tips filling unequally due to clogged or damaged tips 3) Reagents not mixed thoroughly before dispensing, causing concentration gradients 4) Rushing through plate loading leads to inconsistent timing and technique 5) Pipetting technique varies between wells (depth, angle, speed) Solutions: 1) Use calibrated pipettes and verify calibration according to manufacturer schedule 2) Visualize that tips are filling equally when using a multi-channel pipettor; replace damaged tips 3) Ensure all reagents are mixed thoroughly by gently pipetting up and down 5-8 times before use 4) Do not rush loading the plate; ensure adequate time (typically 30-45 min) to properly perform the assay 5) Maintain consistent pipetting technique: same depth, angle, and aspiration/dispensing speed