Evaluating Precision and Reproducibility in your ELISA

You’ve established that your ELISA assay is compatible with your sample matrices and identified your sample MRD. Confirming the reproducibility of the assay in your lab is the next step toward qualifying your ELISA for its intended purpose.

Defining Precision and Reproducibility

Precision is defined as the closeness of agreement between measurements and is expressed as a percent coefficient of variation (%CV). For samples and controls this is calculated by dividing the standard deviation by the mean concentration of the replicates measured. Using this formula, both intra-assay and inter-assay precision can be determined. Intra-assay precision is a measure of the assay reproducibility between the different replicates in a single assay, while inter-assay precision demonstrates the reproducibility between multiple assays. Generally, tight intra-assay CV measurements indicate that the sample readings are not affected by the sample well positioning or procedure. Ensuring inter-assay CVs are within the acceptable range (< 15-20%, depending on your regulatory requirements) is another important metric that suggests the assay will continue to be reproducible over time.

Troubleshooting Assay Performance

If your CV measurements are above the expected values outlined in your kit documentation, it is likely that there is either a procedural or equipment issue causing the variability, as our kit lots are subjected to numerous QC and stability tests. We recommend the following steps to help you troubleshoot higher-than-expected CVs:

1. Evaluate your washing technique

Many automated plate washers and hand-held vacuum aspiration devices can adversely affect assay performance. Overly aggressive washing is a common cause of imprecision, as this can dissociate antibody-bound reactants in a highly variable manner. Automated vacuum aspiration devices or overly hard banging of the ELISA plate when using a manual washing method can negatively impact antibody reactivity, resulting in higher CVs. If your organization decides to use an automated plate washer, the setting should be as gentle as possible for aspiration and dispense of wash solution. Washing with a multi-channel pipette can also cause problems, ranging from low ODs to poor precision. For these reasons, we recommend implementing our manual (squirt bottle) washing procedure for optimal reproducibility. It is important that each well is subjected to washing for approximately the same amount of time; this is best accomplished when alternating the starting direction for washing methods that do not use a 96 well head. This involves rotating the plate 180 degrees between the washing cycles.

Review more details and watch the video in our “Guide to ELISA plate washing” article to help you master the recommended washing technique.

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2. Assess your plate reader

If your ELISA results are variable at the low end of your assay (<0.1 OD), you may have an issue with your plate reader. A failing light source, monochromator, or filter could result in intermittent variability/noise not readily detected by routine calibration, which will have a significant impact on the sensitivity, and ultimately, LOQ/LOD of your assay. This variability usually manifests as standard deviations of 0.020 OD units or less from well-to-well or read-to-read in empty wells or wells containing non-adsorbing liquid. To confirm whether these issues are caused by your instrument, you can check absorbance at dual wavelengths, as recommended for our HRP-TMB colorimetric ELISAs. Dual wavelength readings allow you to subtract any “off peak” OD values (e.g. 650 nm) from another reading measuring the accumulation of the substrate at the detection wavelength (e.g. 450 nm). This strategy should minimize any well-to-well variability resulting from smudges or plastic imperfections to improve the precision and accuracy of your assay. However, keep in mind that Cygnus ELISA microtiter strips have very minimal absorbance at off-peak wavelengths by design; the absorbance at 650nm is on the order of 0.040 ODs in our instruments, with a standard deviation of only about 0.002 OD units across 96 wells. While these readings can vary from instrument to instrument, if your readings are well above 0.040 with a standard deviation greater than 0.004 ODs, this may indicate problems with your plate reader and will have a significant impact on the assay precision and sensitivity.

3. Monitor reagents for contamination

Cygnus ELISA kits are designed to be highly sensitive; as such, they are capable of measuring analytes in the pg/mL to ng/mL range. Contamination of kit reagents by concentrated sources of the analyte that may be present in your facility will result in poor reproducibility. Many laboratories may have sources of high concentrations of the analyte in close proximity to the area where ELISA is performed: for example, culture media or upstream processing samples may contain numerous HCPs or growth media additives (e.g. BSA) in the mg/mL range. Such upstream samples will have on the order of a million-fold greater concentration than the sensitivity of the assay for that analyte. In such cases, it is easy to contaminate some of the kit reagents such as random microtiter plate wells, a standards vial, or the conjugate bottle. Whenever possible, be sure to set up your ELISA in an area away from where these upstream samples are handled. Check our “Avoidance of contamination of kit reagents” FAQ for more details on how to prevent contamination.

4. Evaluate operator-specific technique & instrumentation

Operator technique, poorly calibrated pipets, or poor-quality pipet tips can all adversely affect assay precision. Inviting a second analyst to perform the assay in another lab using different equipment may help you identify whether the source of the imprecision is due to operator inexperience, or perhaps, problems with routine laboratory tools, such as poorly calibrated pipets or pipet tips.

Have more questions for our technical support team? Reach out to us with your questions and our technical experts will help you get your assay performance back on track.