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Would you work with a gene, vector or oligonucleotide of unknown sequence? Today, the majority of scientists would answer with a resounding ‘No’. Biology is becoming more quantitative, digital and defined. The availability of genomic sequences in public databases allows the same genes to be effectively and reproducibly synthesized and studied in different laboratories everywhere: where a sequence is available there is no uncertainty. The genes, vectors, RNAs or oligonucleotides I order will be indistinguishable from the ones you describe in your experiments. This allows me to attempt to reproduce your experiments, and while there may be differences in the protocols we use, at least we can be sure that the underlying biological materials are essentially identical. However, the situation is very different when it comes to the specific detection of molecules in biological samples using ‘specific’ probes. The most widely used specific binding reagents are synthetic nucleic acid probes based on sequence knowledge. These are able to recognize RNA or DNA by hybridization with enormous specificity and affinity, and rely on well-understood base complementarity for their high specificity. For that reason, they are reproducible between laboratories, and even partial cross-reactivities are predictable to some degree. Nowadays, the sequences of oligonucleotides used in an experimental study are typically listed in a Supplementary Table in each publication, allowing relatively straightforward experimental duplication. For historical, as well as practical, reasons, antibodies are by far the most widely used class of specific detection reagents for essentially all other target classes, particularly proteins. Polyclonal antisera have been used in research for nearly a century, and monoclonal antibodies for four decades (Kohler and Milstein, 1975). However, although these reagents have been instrumental in addressing numerous biomedical research questions, they are never defined at the molecular level (we do not consider here the use of antibodies as biological pharmaceuticals, as these are all highly quality-controlled, recombinant reagents that have been exquisitely characterized). Animal-derived reagent antibodies are the main subject of this editorial. Without access to alternatives, researchers have become accustomed to—and usually do not question—the inadequate definition and characterization of these traditional reagents, even in an era when working with oligonucleotides, genes, vectors or even genomes of unknown sequence is inconceivable. Modern biomedical and clinical research relies on specific, high-affinity detection reagents that are functional in complex environments. They provide information on whether a particular component is present in a biological sample, how much of it there is, where it is found, and with which other macromolecules it interacts. The nature of the specific detection reagent in molecular terms (i.e. whether it is an antibody, another scaffold or an aptamer) is less important than its quality, assessed in terms of specificity, epitope recognized, affinity and functionality in different assays, and the ability to describe it sufficiently well that other scientists can reproducibly use the same reagent. Progress in research relies on reproducibility—the generation of reliable results upon which future studies can be dependably based. However, many experts, including NIH Director, Francis Collins, believe our ‘system for ensuring reproducibility of biomedical research is failing’ (Collins and Tabak, 2014). Clearly, the ability to repeat experiments with reagents identical to those used in previous publications is an essential part of creating a successful and reproducible biomedical research environment. For all those reagents defined at the molecular or sequence level described above, this is an achievable goal, and can be eliminated as a source of irreproducibility. However, when it comes to antibodies, the situation is very different for a number of different reasons. Obviously, the molecular definition of polyclonal antibodies will remain impossible at a practical level, andwhile the genes of monoclonal antibodies can be sequenced and therefore completely defined, this is rarely carried out. In addition to the problem of inadequate definition, the situation is further clouded by the fact that it is projected nomore than 35–50%of commercial antibodies actually recognize their targets with the claimed specificity (Berglund et al., 2008; Slaastad et al., 2011); although this projection is unevenly distributed, with some manufacturers producing consistently high, and others consistently low, quality antibodies (Berglund et al., 2008; Bordeaux et al., 2010). This is further exacerbated by intrinsic lot-to-lot variability, particularly Protein Engineering, Design & Selection, 2015, vol. 28 no. 10, pp. 303–305 doi: 10.1093/protein/gzv051 Commentary

Getting to reproducible antibodies: the rationale for sequenced recombinant characterized reagents