ELISA is a complex, multistep assay involving sensitive enzymatic reporters, which means that there are many opportunities for assay failure, either through false-positive or false-negative results. Enzymatic reporter systems, such as used by ELISA, are sensitive to any native enzymatic activity present in the sample (peroxidases, phosphatases). The initial coating step in a highly alkaline buffer abolishes most of such background activity, as does the specific capture of virus particles in sandwich ELISA and the washes with PBS. However, the user should be aware of the possibility of residual enzymatic activity in the samples, particularly if the substrate incubation step is extended to allow more colour to develop (for instance, when trying to detect very low amounts of virus). Secondly, either the enzyme or the substrate may be faulty, preventing colour development even though there has been antibody recognition of the sample. Alternatively, the primary or secondary antibody may fail recognising the virus that is present in the sample, for a number of reasons. All ELISA assays should therefore have a number of controls to establish the correct functioning of the assay itself, thus validating the results from the samples.
- Quantification of background substrate absorbance.
- Absence of non-specific binding of antibodies/reporters to wells;blocking efficiency; washing efficiency.
- Absence of non-specific binding of reporters/secondary antibody.
- Quantification of background absorbance in system.
- Direct adsorbtion
in CB of primary antibody.
- Correct recognition of primary antibody by reporter conjugate.
- Direct adsorption
in CB of reporter conjugate.
- Activity of the reporter enzyme.
- Quantification of background enzyme activity in samples.
- Purified virus
- Correct recognition of virus by primary antibody.
- For quantification of virus titres, use as calibration standard.