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The response (the resulting concentration) is interpolated from the standard curve and OD that resulted due to the addition of a known quantity of recombinant protein standard. This is accomplished by adding a known amount of the recombinant protein standard (spike) into the natural test sample matrix and observing its response (recovery) relative to adding the same known amount to the assay diluent. The sample matrix can be a neat (undiluted) biological sample or biological sample diluted in assay diluent. In spike-and-recovery experiments, the primary objective is to determine whether analyte detection is affected by a difference between the assay diluent/buffer used to prepare an end-user’s standard curve and the sample matrix. To ensure accurate detection and quantification in a sample and mitigate sample matrix effects, an end-user may need to consider sample validation via spike-and-recovery and linearity-of-dilution experiments. On the other hand, when analyte levels are elevated significantly above the limit of quantification (LOQ) of the assay, optimizing the dilution of the sample becomes a strategy to overcome this interference. Exchanging the buffer may be necessary to resolve the interference in order to detect true analyte concentrations in your sample. Interfering components can also be pH-dependent, detergent, organic solvent, and buffering salt composition and concentration which may dictate buffer compatibility. Aside from the endogenous substances inherent to the sample and exogenous contaminants produced through human error, an end-user may need to assess the compatibility of the assay diluent buffering solution in contributing to potential analytical interference. Some examples of protein-rich samples or samples with potential matrix interference are urine, cell lysates, and blood components such as serum and plasma. In this analytical interference, interfering endogenous substances or exogenous contaminants in your sample matrices can result in false increases or decreases to true analyte concentration levels reflected in optical density (OD).
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These interfering substances are compounds with chemical differences but structural similarities that cross-react with the antibody targeting the analyte of interest. This is otherwise known as sample matrix interference. Samples need to demonstrate robust detection and quantification by a kit’s antibodies, which can initially be challenging due to differing antibody-analyte binding characteristics and differences in the endogenous substances present in the background amongst the analyte of interest that are inherent to sample matrices. In commercially available ELISA kits, a key component needed for accurate measurement of an analyte is performing sample validation for your unique samples and/or sample types not validated by the kit manufacturer. The technique depends on two elements: (1) the highly specific antibody-antigen interactions measured via development of a colorimetric product generated by the interaction of an enzyme conjugated to a secondary detection antibody and a complementary colorimetric substrate and (2) accurate comparison of this same interaction relative to a recombinant protein that is used as a reference standard to generate a standard curve and interpolate the concentration of analyte present in a biological sample.
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Enzyme-linked immunosorbent assay (ELISA) is a microplate-based technique frequently used for detecting and quantifying low-abundance analytes of interest in a variety of biological samples.