The ELISA Basics Guide has the right amount of detail to help you plan your experiment and achieve a successful ELISA.
Antigen - substance, protein, chemical compound, or virus that is able to elicit an immune response against which antibodies are raised.
AP (Alkaline Phosphatase) - phosphatase enzyme that removes a phosphate group from a substrate. In ELISAs the substrate is p-NitroPhenyl Phosphate (pNPP).
Assay diluent (see also buffer) - buffer solution in which the sample to be analyzed is diluted in.
Assay sensitivity - a measure of the ability of the ELISA to distinguish between small changes in concentration.
Background - the signal readout attributable to all reagents excluding the analyte. Should be low.
Blocking - application of reagents, generally buffers, to lower background by binding to the potential non-specific binding sites of antibodies and enzyme conjugates.
Buffer - solutions containing compounds, generally proteins, to reduce the non-specific binding of antibodies; used in blocking to reduce background.
Cross-reactivity - an antibody binding to a target that is very similar to but not the intended target analyte, i.e. a closely related molecule with structural similarities to the target antigen.
Detection limit - the smallest quantity of analyte that can be reliably measured by the ELISA assay; it is often set to 2 standard deviations (2 SD) above background level.
Dilution - addition of a buffer to a protein solution to make it less concentrated, used in optimizing antibody concentration but also applied to samples to obtain readings within the dynamic range of the assay.
Dynamic range - range in the ELISA over which the absorbance reading increases in a linear mode and the analyte can be reliably measured.
Edge effect - the result of inconsistencies in the production of ELISA multiwell plates or when assay conditions, such as stacking plates, cause the outer wells to behave differently. As a result, unexpected values can appear in the outer wells which may be out of line with neighboring well. This can best be controlled for by using duplicates or triplicates for all samples, and noting any large variations in the results for a given sample.
Heterophilic interference - arises from antibodies found in the sample analyzed by the ELISA; it is binding by these antibodies to the detection antibody used in the assay. The best known example of heterophilic interference is HAMA (human anti-mouse antibody) found in some patients where it interferes with the accurate analyte determination, showing false positive readings.
HRP (Horseradish Peroxidase) - an enzyme that breaks down hydrogen peroxide to water – a peroxidase. Chromogenic substrates such as TMB serve as indicators of that enzyme activity.
Hook effect - caused by very high levels of antigen in the sample. As a result specific binding of the antigen by the antibody is insufficient to match analyte levels and signal is lower than expected. The best way to avoid this issue is to test several dilutions of each sample.
Immunoassay - any type of assay that uses antibodies to measure the concentration of an analyte in a sample.
Interference - effects on the immunoassay that interfere with the accurate measurement of the analyte (e.g. matrix effect, heterophilic interference).
Matrix effect - the effect compounds in the sample have on the measurement of the analyte.
pNPP (para-Nitrophenylphosphate) - colorimetric substrate for alkaline phosphatase, it precipitates as a yellow substance.
Protein stabilizers - reagents that promote maintenance of the native structure of proteins during adsorbtion to the assay surface.
Substrate - compound such as pNPP and TMB that is used to measure the analyte in an immunoassay.
TMB - a colorimetric substrate for horseradish peroxidase, turning blue upon completion of the enzymatic reaction.