Antibody validation: a view from the mountains

Abstract

Validation of antibodies and other protein binders is a subject of pressing concern for the research community and one which is uppermost in the minds of all who use antibodies as research and diagnostic reagents. Assessing an antibody's fitness for purpose includes accurate ascertainment of its target specificity and suitability for the envisaged task. Moreover, standardised procedures are essential to guarantee sample quality in testing procedures. The problem of defining precise standards for antibody validation has engendered much debate in recent publications and meetings, but gradually a consensus is emerging. At the 8th Alpbach Affinity Proteomics workshop (March 2017), a panel of leaders in the antibody field discussed suggestions which could bring this complex but essential issue a step nearer to a resolution. 'Alpbach recommendations' for best practice include tailoring binder validation processes according to the intended applications and promoting greater transparency in publications and in the information available from commercial antibody developers/providers. A single approach will not fit all applications and end users must ensure that the reported validation holds for their specific requirements, highlighting the need for adequate training in the fundamentals of antibody characterisation and validation across the user community.

Keywords: Affinity proteomics; Alpbach workshop; Antibody characterization; Antibody validation; Antibody validation guidelines; Pre-analytics.

Figure 1.

Application-specific validation of target-specific mAbs. The Euler diagram depicts subsets of ELISA-positive mAbs that exhibit efficacy and specificity in applications on native brain tissue. WB: Western blot. IHC: conventional IHC on brain sections. AT: Immunofluorescence labelling of brain sections prepared for Array Tomography.