An Explanation of Recovery and Linearity

In ELISA development, recovery and linearity experiments are used to assess the compatibility of a particular sample diluent to be used for assaying analytes from a particular sample type such as serum, plasma, saliva, urine, etc. Specifically, recovery tests are used to determine if the assay is affected by the difference between the diluent used to prepare the standard curve and the sample matrix. Linearity tests determine the extent to which the dose-response of the analyte is linear in a particular diluent. Calculations for both rely on dividing the Observed values by the Expected values.

It is preferable to do recovery and linearity testing with samples that have known high concentrations of each analyte. However, if such natural samples are unavailable, testing may also be done by adding (spiking) known amounts of the analyte into samples.

Example Spike Recovery Protocol

1. Thaw samples to be tested.
2. Follow the assay protocol to reconstitute the calibrator and make the standard curve.
3. Prepare a 7-point 1:2 dilution series of the calibrator plus one blank – make 110 uL per sample and diluent.
4. Prepare eight 100 uL aliquots of the kit diluent and each endogenous sample to be tested.
5. Dilute the sample and diluent aliquots 1:2 (50%) with 100 uL of the 1:2 calibrator series.
Calculate the %Recovery for each spiked aliquot:

  • %Recovery = ((Observed Concentration – Endogenous Concentration)/ Spiked Diluent Concentration)*100.
  • The mean percent recovery for any sample type should meet design specifications, which are typically 80-120%

Example Linearity Protocol

1. Thaw samples to be tested.
2. Follow the assay protocol to reconstitute the calibrator and make the standard curve.
3. If samples are expected to have high levels of the analyte, spiking is not necessary. Make a 1:2 serial dilution curve of the samples to be tested in sample diluent.
4. If samples are expected to have very low levels of the analyte being tested, spike a 1:8 dilution of the calibrator into samples. Then perform a 1:2 serial dilution curve of the spiked samples in sample diluent.
5. Calculate the %Linearity for each dilution of the samples:


  • %Linearity = (Observed Concentration / (Previous observed value in the dilution series / Dilution Factor))*100.
  • The mean percent linearity for each should be 80-120%, preferably 90-110%.

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