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How to improve Precision/Reproducibility for ELISA?
Immunoassay precision is defined as the reproducibility between wells within an assay (intra-assay) and between assays (inter-assay). Poor precision, determined by high CVs, is often caused by 2 main factors: pipetting technique and washing technique
1. Pipette reagents and samples into the center of each well but not touching the bottom or the wall.
2. Tap the plate gently after pipetting to ensure mixing of reagents.
3. If your sample is viscous, please pre-rinse the pipette tip with the reagent to compensate for surface tension. Then wait a moment to allow the volume to reach equilibrium before removing the pipette tip from the reservoir.
4. Inadequate or uneven fill volumes in the wells is a good indication that the pipette needs to be calibrated. Check the pipette function and recalibrate if needed.
5. Always be sure to change pipette tips between reagents, standards, and samples to avoid carryover or cross-contamination.
6. Check to be sure that the pipette tips are on properly. A pipette tip that is not fitted properly may not draw or dispense the volume accurately.
7. As you remove the tip from the well, slide the tip on the edge of the well to remove any liquid remaining on the outside of the tip.
1. If using an automated washer, aspiration apparatus, or a multi-channel pipette, always be sure that each cannulae is dispensing and aspirating properly.
2. After the last wash, decant any remaining Wash Buffer by inverting the plate and blotting it dry by rapping it firmly against clean paper toweling on a hard surface. Washing the wells too rapidly or slowly may result in poor precision. Do not allow the wells to sit dry. Proceed immediately to the next step in the kit insert.
3. Using less Wash Buffer will often lead to poor CVs. Be sure to wash the wells with the volume specified in the kit manual to ensure that each well is thoroughly washed.
4. If you ran out of Wash Buffer, please don’t use Wash Buffer from another kit because not all Wash Buffers are identical. You can contact Technical Service for assistance.
5. Don’t skip or shorten the soak time in the wash step, which can lead to poor precision. Always follow the directions in the kit manual for optimal performance.
6. It is recommended that the number of washes specified in the kit manual be adhered to. Changing the number of washes can affect the precision of the assay.
Use separate reservoirs for each reagent to avoid contamination. Some analytes are highly susceptible to contamination (e.g., saliva, oxidizing reagents, etc.). Refer to the kit manual. Take necessary precautions to protect kit reagents.
The plate sealer
A reused plate sealer may contain residual from the previous step which can contaminate the current component in the well, leading to poor precision. Use a new plate sealer for each incubation is recommended.