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In the wake of the reproducibility crisis and numerous discussions on how commercially available antibodies as research tool contribute to it, The Antibody Society developed a series of 10 webinars to address the issues involved. The webinars were delivered by speakers with both academic and commercial backgrounds. This report highlights the problems, and offers solutions to help the scientific community appropriately identify the right antibodies and to validate them for their research and development projects. Despite the various solutions proposed here, they must be applied on a case-by-case basis. Each antibody must be verified based on the content of the product sheet, and subsequently through experimentation to confirm integrity, specificity and selectivity. Verification needs to focus on the precise application and tissue/cell type for which the antibody will be used, and all verification data must be reported openly. The various approaches discussed here all have caveats, so a combination of solutions must be considered.

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.

There is a particular need in protein analysis and purification for specific, functional, and generic methods of protein immobilization on solid supports. Here we describe a double-hexahistidine ([His.sub.6]) tag sequence, comprising two hexahistidines separated by an 11-amino acid spacer, which shows at least 1 order of magnitude stronger binding to Ni-NTA-modified surfaces than a conventional single-[His.sub.6] tag or two single-[His.sub.6] tags at N- and C-termini. Using, as a model, tagged versions of green fluorescent protein (GFP), stable and tight binding of the double-[His.sub.6] tag/Ni-NTA interaction was demonstrated by competitive elution from Ni-NTA agarose beads, surface plasmon resonance on a Ni-NTA chip, and ELISA in Ni-NTA microwell plates. Protein purification by Ni-NTA chromatography was improved by a 6-8-fold increase in imidazole concentration required for elution, while the dissociation rate of double-[His.sub.6] GFP from Ni NTA chips in SPR (BIAcore) was 10 times slower than for single-[His.sub.6]-tagged proteins. ELISA assays and protein microarrays constructed with double-[His.sub.6] GFP demonstrated greater detection sensitivity with anti-His antibodies and Ni-NTA conjugates. Moreover, the double-[His.sub.6] tag could serve simultaneously both for protein immobilization and for detection on surfaces. The double-[His.sub.6] peptide has the potential to be a universal tag for protein immobilization and detection on arrays and single-step purification of proteins from crude mixtures.

Specific antibodies are investigated to determine their structural antigens which bind them to high affinity antibodies and the restrictions on antibody combining sites. X-ray structures of five conformationally different steroids are compared with Fab fragments of anti-progesterone antibody DB3 at 2.7 angstroms. The conserved interactions of DB3 with steroid D-ring explains the specificity of the antigen. Other types of crossreactivity are realized from the orientations of the A rings into the pockets.