Inconsistent results and high coefficient of variation in ELISA
High variance indicates greater inconsistency and error in your results.
The coefficient of variation (CV) indicates the variation in signal between replicates (intra-assay variability) and runs (inter-assay variability). It is a ratio of the standard deviation to the mean, expressed as a percentage. You should aim for a CV of < 15 %.
What can cause inconsistent results and high coefficient of variation (CV)?
Bubbles in wells.
Solutions containing detergents can be especially susceptible to forming bubbles.
- Ensure no bubbles are present prior to reading the plate.
- Gently add solutions to the wells.
- Pop bubbles by touching them with a dry pipette tip.
Wells not washed equally/thoroughly.
Always wash wells as recommended. Automatic wash systems can be more effective and efficient in washing wells.
- Check that the plate washer is functioning properly and has been recently cleaned.
- Use a multi-channel pipettor when washing plates manually.
- Use calibrated pipettes and proper pipetting technique.
- Visualize that tips are filling equally when using a multi-channel pipettor.
- Ensure all reagents are mixed thoroughly by gently pipetting up and down.
- Don’t rush loading the plate; make sure that you have time to properly perform the assay before starting.
- Ensure all samples are stored and prepared consistently, according to recommendations.
- Keep the number of freeze/thaw cycles that samples are exposed to equal across all samples.
Edge effects due to incorrect handling of plates.
Edge effects occur in ELISA when different wells are exposed to slight variations in temperature and humidity. The outer (or ‘edge’) wells are usually the first to respond to any change in the environment, causing them to dry out or evaporate before the inner wells.
- In some cases, the edge effect is clearly visible. Try looking at the plate from the side – if the buffer level is lower on the outside wells, some of the buffer has dried out.
- To prevent wells from drying out, cover all wells with sealing film or tape.
- Ensure the plate and all reagents are at left to equilibrate to room temperature before incubation. Don’t use plates straight from the fridge; the inner wells need more time to reach room temperature.
- Verify that the instrument has been maintained and calibrated according to the manufacturer’s recommendations.