How antibodies can cause false ELISA results

The Enzyme-Linked ImmunoSorbent Assay (ELISA) is a popular analytical biochemistry assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a sample.

The ELISA is widely used as a research tool, for example to look at cytokine levels in serum, and is also often used as a diagnostic tool in medicine. ELISAs also play an important role as a quality-control check in scientific production and manufacturing.
In one ELISA format, the sample is immobilised on the surface, and antigens detected by antibodies presented in solution. These antibodies may be directly labelled with a light- or colour-generating enzyme to allow their detection, or the ELISA may be developed by incubation with a secondary, labelled antibody. Measurement of light or colour generated by the label allows measurement of the antigen. However, antibodies that cross-react with an irrelevant antigen in the sample may cause false results in this format – and the problem can be due to either the primary or the secondary antibody to irrelevant proteins in the sample.
Sandwich ELISA

A sandwich ELISA being used with antibodiesIn a different format, the so-called sandwich ELISA, an antibody that is able to recognise the target antigen is immobilised, and the sample presented as a solution. After binding and washing away unbound proteins, a second antibody is added, this again being either directly labelled or later developed with a secondary, labelled, antibody. In theory, the label can only be bound to the plate if antigen has first been recruited by the immobilised antibody and then has recruited, to a different part of its surface, the second antibody. Antibodies can now cause cross-reactivity in multiple ways: an irrelevant protein can cross-link the immobilised and second antibodies, or even the immobilised antibody and the labelled secondary antibody. One common cause of this is a protein called rheumatoid factor – which is usually a multivalent IgM antibody that has the ability to bind to the constant region of the IgG immunoglobulins that are used in almost every immunological assay in the life sciences. Other ways that a false positive may arise would be if the second antibody binds to the surface in the absence of antigen, or if the labelled secondary antibody does so.

Non specific protein interactions such as those described above are more likely to occur when proteins are denatured, exposing their often hydrophobic cores. Proteins, including antibodies, are known to unfold when brought to surfaces – indeed this is the very process that allows the first ELISA format we described to work, as the unfolded proteins in the sample unfold and adsorb to the (usually) plastic assay surface. When devising a new sandwich ELISA, it can take a very long time, and a great deal of effort, to find antibodies that will work when immobilised on a surface, and/or the right kind of coating polymer that will allow the antibody to immobilise without so much unfolding that the assay parameters (sensitivity and specificity) cannot be achieved.
Although antibodies have been the default affinity reagent in the ELISA process, Affimer technology is likely to be a superior alternative to antibodies due to a number of key factors.
Firstly, as Affimer technology is not found in the blood, biological or clinical samples are unlikely to contain any molecules equivalent to rheumatoid factor that would drive cross-linking. Secondly, as Affimer reagents are selected on the basis of their specificity, they are unlikely to bind to irrelevant antigens in the sample, although as with any analytical assay this must always be confirmed and controlled for. Thirdly, since the Affimer scaffold has been engineered to have no natural binding partners in human samples, the incidence of false positive results is greatly reduced. And finally, Affimer binders remain functional when immobilised on any one of a range of solid supports – plastic, glass, gold, including 2d (planar) and 3d (bead) surfaces – and can be functionalised with enzymes such as HRP allowing a standard ELISA format to be performed.
The generation of Affimer pairs to build an assay is much easier than with antibodies because the in vitro selection process used to generate yields with multiple hits giving reasonable likelihood of finding Affimer binders for distinct epitopes on your target in the pool.