This whitepaper was published by CDI Laboratories in partnership with Arrayjet Ltd.
by Pedro Ramos, Andrew Leahy, Ignacio Pino, and Scott Paschke (April, 2016)
Summary
The use of protein microarrays to evaluate antibodies is likely to set new quality standards to evaluate antibody cross-reactivity and will also address the need for new methods to identify antibodies that can be used in robust scientific investigations, grant proposals and for commercialisation.
Introduction
A number of recent articles and commentaries published in high impact journals detail the problems with antibody cross-reactivity, its impact on data relevancy and the amount of time and money wasted on the use of poor antibodies. In addition, there is growing demand from the NIH regarding the need for antibody standardization that would ensure that reagents used in publications are actually detecting the proteins of interest.
Antibodies are among the most commonly employed biological research reagents—tools used primarily to identify and/or isolate molecules of interest. It is becoming clear that they are also the cause of many problems regarding data interpretation and may hinder researchers’ abilities to reach unambiguous conclusions. (Baker M. (2015) Reproducibility crisis: Blame it on the Antibodies. Nature 521: 274–276.)
Scientists—along with non-scientists—contend that the complex system for ensuring the reproducibility of biomedical research is failing at certain levels and is in need of restructuring. (Bradbury A and Plückthun A (2015) Reproducibility: Standardize antibodies used in research. Nature 518: 27–29.)
Glenn Begley, chief scientific officer at TetraLogic Pharmaceuticals in Malvern, Pennsylvania, and coauthor of the controversial article “Drug development: Raise standards for preclinical cancer research” (Begley and Ellis, Nature 483: 531–533, 2012) suggested that “poorly characterized antibodies probably contribute more to the problem than any other laboratory tool.” While antibodies are the undisputed workhorses of biological experimentation, they are also littering the field with false findings. A few crusaders are pushing for change. This recent and growing concern over commercial antibody quality indicates that the day is coming when all commercially available antibodies will need to undergo standardized testing to meet quality criteria. Only then will the data generated using these antibodies be accepted for journal publication or for inclusion in NIH grants.
As an example, some companies have begun using knockout cell lines to address antibody specificity issues. This is a good move forward but is still not a complete solution. A knockout cell line does give a snapshot of how a particular antibody behaves in a particular cell line under the given assay conditions. The knockout does not, however, guarantee that antibody will behave similarly in a different cell line under different conditions, or in a different tissue type.
This is because radically different expression levels of proteins can occur across cell lines, tissue types and treatments. In contrast, using high content protein microarrays produces a comprehensive picture of antibody cross-reactivity against a large part of the entire human proteome. Since the proteins spotted on the arrays are robustly expressed, array analysis can give a clear profile of cross-reactivity across proteins that are selectively expressed in a wide range of cell lines.
In addition, with tools such as the CDI Labs HuProt™ array, it is possible to measure antibody cross-reactivity to proteins in their native, folded conformation (e.g., in an immunoprecipitation or immunofluorescence assay), or to denatured proteins (as found on western blots, or in IHC) in which antibodies do not recognize native proteins and thus the tissues need to be processed by antigen retrieval methods. The HuProt™ arrays are ideal for both types of screening, as the proteins are initially spotted in native conformation, but the arrays can also be denatured with 9M urea and 5mM DTT. See Hu, et al. “Proteins on HuProt™ Array Are Well Folded”:
