Quality of histone modification antibodies undermines chromatin biology research

The lack of reproducibility is widely recognized as a serious issue in contemporary research (see (Buck, 2015; Freedman & Inglese, 2014; Freedman et al., 2015; McNutt, 2014a; McNutt, 2014b) and the Nature special “Challenges in irreproducible research” April 2, 2013). In molecular biology, the quality of antibodies has been identified and highlighted as one of the most recurring stumbling blocks that undermine the quality and validity of experimental results (Baker, 2015; Bordeaux et al., 2010; Bradbury & Pluckthun, 2015). This issue is even more pervasive in the field of molecular epigenetics and chromatin biology, where antibodies for various types of histone post translational modifications (PTMs) have been single-handedly used to translate the language of histone modifications into experimentally observable properties. Hence most of what we know about the distribution, role and function of histone modifications so far has been passed through an antibody as essential mediator.

Raising a specific histone modification antibody is not a trivial task; this is mostly due to the hypermodified state of the histone tail, coupled with the minute size and the chemical relatedness of many histone modifications and similarities in the amino acid sequence surrounding the modified residues. The antibody has to be able to discriminate between the unmodified and the modified state of the targeted amino acid residue, as well as between different forms of modifications (e.g. acetylations of different lysine residues, mono-, di- and trimethylation of lysine residues, or symmetric and asymmetric methylation of arginine residues). Moreover, the presence of an adjacent modification might prevent binding of an antibody to the target modification, causing false negative results. Another difficulty is that some histone modifications such as methylation or acetylation of H3K9 and H3K27 lie within an identical amino acid context (ARKS motif), which makes the readout of the target peptide sequence outside of this central motif very important as well.

In spite of the intricate task of producing histone modification antibodies and their crucial role in chromatin biology, surprisingly, they remain to be insufficiently characterized. In line with this, numerous scientific groups have alarmingly raised concerns about the promiscuous behavior of some histone modification antibodies and the undocumented effects of secondary modifications (Bock et al., 2011; Egelhofer et al., 2011; Hattori et al., 2013; Kungulovski et al., 2014; Nishikori et al., 2012; Rothbart et al., 2015). As mentioned above, the situation in chromatin biology is exceptional, because of the outstanding role of histone PTM antibodies as the sole research tool in this field. As a consequence, elaborate quality control criteria for histone PTM antibodies were put forward to ensure the integrity of research (Egelhofer et al., 2011; Kungulovski et al., 2015; Landt et al., 2012). To increase transparency, at least two databases for deposition of antibody quality data by researchers were put in place (http://compbio.med.harvard.edu/antibodies/; http://www.histoneantibodies.com/) (Egelhofer et al., 2011; Rothbart et al., 2015). However, in spite of being heroic attempts, these and similar databases have only a limited value, because most of the antibodies used in chromatin biology are polyclonal, and lab experience over the last years has demonstrated that the specificity data obtained for one batch of antibody do not necessarily reflect the properties of another one (Kungulovski et al., 2014), a caveat which is still often ignored by naïve end-users.

The necessary quality control steps for histone modification antibodies (Egelhofer et al., 2011; Kungulovski et al., 2015; Landt et al., 2012) currently burden the individual antibody user with high costs and workload. Given that antibodies are expensive reagents, which are of no use without appropriate quality documentation, these efforts must be redirected from the end-customer to the manufacturers of antibodies. Herein, we urgently ask the vendors and manufacturers of antibodies to provide the necessary product sheets for all types of antibodies on a regular basis, including quality control documentation for each batch of polyclonal and each catalog number of recombinant or monoclonal antibodies. The following information must be provided:

As proposed by others (Bradbury & Pluckthun, 2015) end-users should consider boycotting companies not complying with this demand, or at least stay away from products lacking a proper lot-specific documentation.

The batch-to-batch variability of critical properties like cross reactivity or inhibition by secondary marks makes the application of polyclonal antibodies intrinsically unsustainable, because experiments cannot be reproduced after the corresponding batch of an antibody is sold out. As a consequence of this, rigorously speaking, large data sets in chromatin biology exist in a “grey” area outside of natural science, since it is impossible to repeat the underlying experiments. In a long-term perspective, a shift away from polyclonal antibodies towards alternative reagents, which can be produced at constant quality, would help to reduce the necessary costs and workload associated with quality control of polyclonal antibodies and ensure sustainability. This applies to high quality monoclonal antibodies, recombinant antibodies (Hattori et al., 2013) or analogous recombinant reading domains (Kungulovski et al., 2014). This will not only help to reduce costs in chromatin research in the long run (once obtained, the documentation will be valid for all lots) but also help to standardize the affinity reagents used and ease the lab-to-lab comparison of data. Of note, in chromatin biology native reading domains designed by nature to specifically recognize relevant histone PTM marks are available as an alternative to antibodies (Kungulovski et al., 2014), which is an advantage over other fields, where recombinant production of antibodies is the only technical solution to the issue of reproducible performance and long term availability of these essential research reagents.