High CV from Plate Edge Effects (ELISA (Inconsistent Results / High CV))

Question

Accepted Answer

Outer (edge) wells show systematically different absorbance values compared to inner wells, with CV >15%. Edge wells may show higher or lower signal depending on evaporation or temperature variation. Visual inspection from the side reveals lower buffer levels in edge wells. Common causes: 1) Edge wells exposed to greater temperature variations during incubation 2) Outer wells experience higher evaporation rates due to humidity differences 3) Plates used directly from refrigerator; inner wells take longer to equilibrate to room temperature 4) Wells not sealed properly during incubation, allowing differential evaporation 5) Edge wells dry out faster due to increased surface area exposure to air Solutions: 1) Cover all wells with sealing film or tape during all incubation steps to prevent evaporation 2) Allow plate and all reagents to equilibrate to room temperature for 30 minutes before use 3) Do not use plates straight from the refrigerator; inner wells need more time to reach room temperature 4) Fill outer wells with buffer only (not sample) and use only inner wells for assay if edge effects persist 5) Incubate plates in a humidified chamber or sealed container to minimize evaporation