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Analyzing Salivary Analyte Results

Data Analysis Resources

Calculating Inter- and Intra-Assay Coefficients of Variability

Description: In order to express the precision, or repeatability, of immunoassay test results, researchers in the social and behavioral sciences typically report two measures of the Coefficient of Variability (CV) in their publications.


Interpreting Differences in ELISA/EIA Results

Description: Occasionally, differences in EIA results are observed from what is expected. In most cases, these differences relate to one or more factors: kit storage conditions, faulty laboratory equipment, technician error, or other factors that are likely to be causing the poor performance.


Guidelines for Interpreting Cotinine Levels: United States

Description: We recommend the following guidelines when interpreting results from the Salimetrics Salivary Cotinine ELISA.


Data Analysis Faqs

Why are my results different than I expected?

Please contact us for technical support if this question is directed at the assay performance specifically or request a consultation if the question is related to data supporting your research hypothesis.

How do I interpret my data?

You can easily Ask an Expert to schedule a follow-up phone call with you or your team to discuss your project.

Why am I getting high CV’s?

Common causes of CV problems and tips to prevent CV problems:

  1. Pipetting:  The key to accuracy in pipetting is a consistent technique. Consistent speed and smoothness while pipetting is necessary.  Avoid any sudden motions when drawing or dispensing fluids.  Saliva samples can be viscous and you may want to set your pipettors to a slower speed.
  2. Pipette calibration:  Ensure that the pipettes being used are properly calibrated by checking the calibration.  Most manufacturers recommend pipetting 10 replicate samples of DI water at the minimum and maximum volume of the pipette.  Generally, the CV of the 10 replicates should be less than 2-3%.
  3. Always pre-wet the pipette tips – see; Pre-wetting the pipette tip(s) below. (Always change pipette tips between each standard, control and sample!)
  4. Plate washing:  If your plate washer probes are too close to the bottom of the well, it could strip off some of the antibody that is bound to the wells.    When the plate comes out of the plate washer is it dry?  There should be a small equal amount of coating liquid left in each of the wells after washing.  This is removed by blotting the plate onto some type of absorbent material.  These problems could product high CV’s and/or low OD’s since it would most likely be that this stripping or washing away of antibody would be unequal in the various wells.
  5. Insufficient mixing of reagents, standards and/or controls before use.
  6. Particulates in samples
  7. Samples not centrifuged long enough.
  8. Samples not re-centrifuged after a freeze or 24 hours since previous centrifugation.
  9. Plate Washer has a dispense and/or aspirate probe blocked – if necessary, see plate washer instructions for details on how to check for this problem.
  10. Mixing the plate too fast and splashing occurs.

NOTE: Be sure not to let your plate dry completely after washing.

Pre-wetting the pipette tip(s) (# 3 above) will result in more accurate and consistent pipetting of solutions which is very important for pipetting duplicates onto an immunoassay plate.

  1. Firmly place the correct size pipette tip onto the end of the pipetter.
  2. Depress the plunger button at the top of the pipetter with your thumb until you feel the first stop point.
  3. Immerse the pipette tip under the surface of the liquid to be dispensed to a depth of 2-5 cm, staying clear of the container walls and bottom.  Release the plunger button slowly to draw liquid up into the tip.
  4. Pause with the tip in the liquid for one or two seconds after aspirating.  The liquid in the tip “bounces” slightly when the plunger is released.  A slow, consistent pause helps minimize errors due to this effect.
  5. Withdraw the tip from the liquid & dispense back into the container by gently pressing the plunger all the way down.  This “pre-wets” the tip.

There are times when a high CV (>15%) may be acceptable (according to Salimetrics policy); you should set your own policies. When the assay values are near the lower limit of the assay, the CV is more likely to be skewed.

What statistical methods should I be using?

You can easily Ask an Expert to schedule a follow-up phone call with you or your team to discuss your project. The paper below also provides a very good overview;

Granger, D. A., Fortunato, C. K., Beltzer, E. B., & Virag, M.., Bright., M. & Out, D. (2012).  Salivary Bioscience and  research on adolescence: A integrated perspective.  Journal of Adolescence., 32, 1081-1095 PMID 22401843

Is my assay valid if my NSB OD value is > 0.1?

There are multiple reasons why the NSB wells can have OD values:
If the wells were not swapped out with clear plastic non-coated wells, then the OD values will be similar to that obtained in the Zero wells
If the wells were swapped out, then some background OD values may be occurring due to non-specific binding such as the following:

  •  Inadequate plate washing
  • Contamination of components and carryover of substances that are contributing to signal
  • Inaccurate conjugate dilution or prolonged incubation with conjugate
  • Incubation temperature too high or too low
  • Plate not read within the 10 minute window after adding Stop Solution or TMB incubation was longer than 30 minutes.

How do I process my data without software?

Software to complete the data reduction for the assays should be available from the plate reader manufacturer.  Salimetrics does not have 4-parameter excel worksheets.

If you do not have access to a data reduction software protocol with 4PL curve fitting capabilities, we recommend the free, online software available through www.myassays.com. This website has some pre-populated Salimetrics ELISA assays that simply require copying the OD450 and ODRef values into their site and the program is able to provide data that can be imported into an excel file. We recommend you create a login so that you have access to the expanded capabilities of the program. However, Salimetrics has no relationship with this organization and is unable to provide technical support.

Why don’t my values match those of other manufacturers’ kits?

Salimetrics assay kits are calibrated to National Institute for Standards and Technology (NIST) Standards and results should only be comparable to other assays that have also incorporated NIST Standards for their assays. Kit manufacturers may use different antibodies to detect desired analytes and each antibody has slightly different affinity and specificity profiles for the analyte being tested, which can lead to slightly differing concentrations for the same analyte. Assay design can also have an effect on sample values (direct competitive, sandwich indirect, kinetic enzyme reaction, extractions, etc.). Ideally, if done right, all manufacturers will match their results to NIST standardized controls and have results that are comparable.

Do I need to normalize my data?

We do not recommend “normalizing” data in enzyme immunoassays. B/Bo is a normalizing response variable used in Salimetrics assays; we feel that no further normalization is necessary. Controls are run against the standards on each plate to gauge the accuracy of the run.