ELISA Troubleshooting: Poor Reproducibility

When it comes to the optical density (OD) readings of ELISA, a common problem is poor reproducibility-poor reproducibility of replicates within a 96-well plate, or poor reproducibility from plate to plate. This part is intended to give possible causes for ELISA poor reproducibility problem about these two cases as well as relative troubleshooting.

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Replicates Within a Plate Show Poor Reproducibility

Possible causes Recommended troubleshooting
Excessive time was taken to add samples controls or reagents to the assay plate. Be sure to have all materials set up and ready to use quickly. Use a multichannel pipette to add reagents to multiple wells simultaneously. Rack controls with samples and dispense them onto the plate at the same time as the samples.
Multichannel pipette was not functioning properly. Verify pipette calibration and check that tips are on tight. Be sure all channels of the pipette draw and dispense equal volumes.
There was inconsistent washing or washer system malfunctioning. Verify the performance of the washer system. Have the system repaired if any ports drip or dispense/aspirate poorly.
There was poor distribution of antibody in the sample. If the sample was thawed or refrigerated, make sure it was mixed prior to dilution. Diluted samples also need to be mixed prior to adding them to the plate.

Poor Reproducibility Plate to Plate

Possible causes Recommended troubleshooting
Inconsistent incubation times occurred from plate to plate. Time each plate separately to ensure that plates have consistent incubation periods.
Inconsistent washing occurred from plate to plate. Use the same number of washes for each plate. Verify the performance of the washer system. Have the system repaired if any ports drip or dispense or aspirate poorly.
Pipette was working improperly. Be sure to allow sufficient time for sample diluent, samples and kit controls to come to room temperature by removing them from the kit box. Larger volumes will require longer equilibration time. If using a water bath to hasten equilibration, make sure it is maintained at room temperature; do not use a warm water bath for controls, samples or diluent.
Kit controls and samples were at different temperatures. Ensure that the correct reagents were used, that working solutions were prepared correctly and that contamination has not occurred.
Reagents were being used from different kit lots. If running two different kit lots at the same time, make sure to label reagent trays, etc., so all reagents within a lot are used with the corresponding plates.