The issue of antibody cross-reactivity is one that has often been often overlooked or sidelined in the past, with typical antibody QC involving evidence of binding to the intended target, while neglecting potential cross-reactivity. In recent years, however, many examples have come to light where poor validation of antibodies has led to misleading or erroneous conclusions for assays in which they are used as reagents, due to unrecognised cross-reactions and even a failure to react with their supposed target altogether. It is now clear that off-target reactions can severely compromise an antibody’s usefulness and the interpretation and reproducibility of results, damaging research credibility and with potentially dangerous consequences in the diagnostics field.
One obvious way to confirm specificity of antibodies against protein targets is to screen their binding against the widest possible number of proteins, and this is where protein arrays find one of their principal applications. Protein array data can address some of the essential questions around antibody specificity, including the basic one of whether the intended target is actually recognised, either in native conformation or denatured, and whether the antibody is monospecific or cross-reactive with other proteins. Where the latter is the case, the arrays reveal the number, identities and strengths of the cross-reactions, and indicate their relevance to the proposed uses of the antibody.
Screening of antibodies on HuProt v4 human proteome arrays enables identification of reactivities across 80% of the human proteome, the widest range of individual proteins available today on a single microarray. As well as validation of antibody reagents, this is of particularly high value for the characterisation of candidate therapeutic or diagnostic antibodies, where the investment in downstream development and clinical approval is considerable and where eliminating candidates showng cross-reactivities early in the development process can lead to major savings in cost and time. Note that such a screen is also applicable to antibody fragments and single domains, and alternative binder types, such as engineered scaffolds.
The accompanying video illustrates the application of HuProt human proteome arrays in antibody specificity screening. See here for references of HuProt applications in binder profiling
For a wider discussion of the issue of antibody validation, please see our article from New BIOTECHNOLOGY.
Array detail images of equivalent areas: specific or x-reactive antibodies
Illustrating a protein array result for three antibodies nominally targeting the same protein (circled in red).
Left: specific; Middle: cross-reactive; Right: highly nonspecific
Assay workflow
Typically, the antibody of interest is incubated on the HuProt array, followed by a suitable fluorophore-labelled secondary reagent. Alternatively, if the antibody of interest is fluorophore-conjugated itself, incubation and detection can proceed in one step (not shown).
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