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 two main factors: pipetting technique and washing technique

Pipetting Technique

1.    Pipette reagents and samples into the center of each well but do not touch the bottom or the wall.

2.    Tap the plate gently after pipetting to ensure thorough 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  necessary.

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 fitted properly. A pipette tip that is not fitted properly may not draw or dispense the volume accurately.

Washing Technique

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 washing, 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 too slowly may result in poor precision. Do not allow the wells standing to dry. Proceed immediately to the next step  in the kit insert.

3.   Using less Wash Buffer might often lead to poor CVs. Be sure to wash the wells with the volume specified in the manual to ensure that each well has been  thoroughly washed.

4.   If the Wash Buffer has run out, please don’t use Wash Buffer from another kit because not all Wash Buffers are general. You can contact Technical Service  for assistance.

5.   Don’t skip or shorten the soak time in the washing procedure, otherwise it may lead to poor precision. Always follow the directions in the manual for optimal  performance.

6.  It is recommended that the number of washing specified in the kit manual be adhered to. Changing the number of washing can affect the precision of the assay.

Reservoir

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 manual. Take necessary precautions to protect the reagents.

The plate sealer

A reused plate sealer may contain residual from the previous step which can contaminate the current component in the well, thus leading to poor precision. Using a new plate sealer for each incubation is recommended.