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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 how much variation in signal there is between runs. It is a ratio of the standard deviation to the mean, expressed as a percentage. You should aim for a CV of < 20 %. 

Inconsistent results and high coefficient of variation (CV)

The coefficient of variation (CV) indicates the variation in signal between runs. It is a ratio of the standard deviation to the mean, expressed as a percentage. You should aim for a CV of < 20 %. 

Possible causes

Solution

Bubbles in wells.

The proper pipetting technique should prevent bubbles from forming in the well, but it's not always possible to follow this.

  • Ensure no bubbles are present prior to reading the plate.

  • You can try to remove bubbles by gently pipetting up and down.

Wells not washed equally/thoroughly.

Always wash wells as recommended. Automatic wash systems can be more effective and efficient in washing wells. 

  • Check that all ports of the plate washer are unobstructed. Wash wells as recommended.

Inconsistent pipetting

  • Use calibrated pipettes and proper pipetting technique.

  • Ensure all reagents are mixed thoroughly by gently pipetting up and down.

Inconsistent sample preparation or storage.

  • Ensure all samples are stored and prepared consistently, according to recommendations.

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 drying out, cover all wells with sealing film or tape.

  • You should also 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.