What causes high coefficient variation for my ELISA results?

The coefficient of variation (CV) is a key metric used to assess the consistency of ELISA results. It represents the ratio of the standard deviation to the mean, expressed as a percentage. The CV for duplicate samples in ELISA should ideally be ≤ 20%. A high CV indicates significant inconsistency and error in your results.

These are some of the possible causes of high coefficient variation for your ELISA results: 

• Bubbles in wells: Bubble formation in the well can be prevented by using the proper pipetting technique but this is not always possible. Ensuring no bubbles are present before reading the plate and removing bubbles by gently pipetting up and down can help prevent this problem. 
• Wells not thoroughly clean: Uneven or insufficient washing of wells can cause high coefficient variation. To avoid this problem, use an automatic wash system for better efficiency and check that all ports of the plate washer are unobstructed.
• Inconsistent pipetting: Using calibrated pipettes, ensuring proper pipetting technique, and thoroughly mixing reagents by gently pipetting up and down can help prevent high coefficient variation in ELISA results.
• Inconsistent sample preparation or storage: Storing and preparing all samples consistently according to recommendations will prevent this issue. 
• Edge effects due to incorrect plate handling: Exposing different wells to temperature and humidity variations can cause edge effects. Covering wells with sealing tape or film and letting plates and reagents equilibrate at room temperature before use can prevent them from drying out. Avoid using plates directly from the fridge.