High CV from Inconsistent Well Washing (ELISA (Inconsistent Results / High CV))

Question

Accepted Answer

High coefficient of variation (>15%) between replicates with uneven background signal across the plate. Some wells show higher background than others after washing steps. Common causes: 1) Wells not washed equally or thoroughly during wash steps 2) Plate washer nozzles are clogged or misaligned, causing uneven aspiration 3) Manual washing with single-channel pipettor leads to inconsistent technique 4) Insufficient wash buffer volume or inadequate number of wash cycles 5) Plate washer vacuum pressure not properly calibrated Solutions: 1) Always wash wells exactly as recommended in the protocol (volume and number of cycles) 2) Check that the plate washer is functioning properly and has been recently cleaned 3) Use a multi-channel pipettor when washing plates manually to ensure equal treatment 4) Perform regular maintenance and cleaning of automated plate washer nozzles 5) Verify vacuum pressure settings meet manufacturer specifications