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Interference is when some component of a sample prevents the appropriate binding events to take place. Percent recovery is the measurement used and is calculated as the amount of material measured (observed or Obs) divided by the concentration expected (Exp). This number is usually expressed as a percentage.
- Pipette an aliquot of a typical sample into a fresh tube.
- Spike the sample with a known concentrationof standard (provided in the kit). It is recommended to use a concentration that falls close to the top of the curve range to ensure even the 1:64 dilution will not fall below the lower end of the curve.
- Make serial dilutions of the spiked sample with Assay Buffer. Recommended dilutions are neat sample, 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64.
Run the spiked sample and all dilutions in the assay kit with standard curve diluted in the kit Assay Buffer. - Calculate the concentration of the + spiked volumes using a data reduction software program.
Set up a table where you record the dilution factors and the observed concentrations from the assay.
Calculate the expected concentrations for the sample dilutions by dividing the “neat” spiked concentration by the dilution factor. Generate the expected concentrations by running this calculation down the dilution series. - Calculate the percent recoveries using the following formula: % Recovery = (Obs / Exp)*100. Many researchers use a table containing the observed, Obs, expected, Exp, and % Recovery values for each dilution.
- Where the dilutions are adequate to eliminate any matrix interference, the recoveries will be about 100%. If at least two consecutive dilutions result in about 100% recoveries, they are considered to be dilutions at which the sample is interference-free.
- This pattern of calculating percent interference can be started from any dilution and worked downward or upward, however needed. If there is interference but none of the dilutions tried yields consecutive results, repeat the experiment using a wider dilution range.
- If no two dilutions yield about 100% recovery, one may need to take the samples through an extraction (either liquid/ liquid or C18 columns) to eliminate matrix-interference.