Protein affinity reagents are fundamental tools of both basic and applied biomedical research. They are used for a wide range of applications, including measurement of protein expression levels, detection of protein-protein and protein-nucleic acid interactions, and detection of disease biomarkers (1
). Currently, the most widely used protein affinity reagents are polyclonal and monoclonal antibodies. Monoclonal antibodies (mAbs)1
are indefinitely renewable and recognize a single protein epitope, making them the more desirable of the two reagents for most applications (2
). Indeed, with over 20 mAb-based drugs now in use and over 100 in clinical trials, they have also become a central pillar of the biopharmaceutical industry (4
). However, despite their widespread use, well characterized protein affinity reagents are not available for the great majority of human proteins. This lack of characterization has led to a major bottleneck in analyzing protein expression and function, often making interpretation of data obtained using any class of protein affinity reagent problematic (5
). Several recent studies have suggested that many commercially available mAbs may not even recognize their purported targets and cross-react extensively with other cellular antigens (7
). Antibody cross-reactivity is an even more pressing problem in diagnostic and therapeutic applications, as underlined by the recent withdrawal of several mAb-based pharmaceuticals from the market (8
Several large scale efforts are now underway to systematically identify high grade antibodies against much of the human proteome (4
). These approaches, which are primarily directed toward validation of polyclonal antibodies, rely heavily on immunocytochemistry and immunohistochemistry for validation, using both cell lines and tissue microarrays. Although these efforts provide a great deal of useful information, they neither cover all tissues nor confirm that the antibody in question is actually recognizing its target antigen in all of the tissues examined. To address this, it would be necessary to comprehensively measure cross-reactivity of any given antibody against the full proteome, something that is in principle possible using microarray-based analysis of antibody specificity (13
A protein microarray approach has been previously used to analyze the specificity of antibodies generated against viral (16
), microbial (17
), and mammalian (19
) proteins and is already used on a small scale as part of the Human Protein Atlas project (11
). However, existing human protein microarrays either are protein family-specific (22
) or are comprised of only a minority of the human proteome (20
). Although Goshima et al.
) described fabrication of a more comprehensive human protein microarray that contained a total of 13,364 human proteins, these proteins were not purified away from the in vitro
translation reaction mixtures used for protein synthesis, a fact that severely limits the potential usefulness of this reagent.
To remedy this situation, we have developed a microarray that includes nearly two-thirds of the annotated full-length human proteome. The proteins used to generate this microarray were purified under native conditions at low cost following galactose-induced expression from Saccharomyces cerevisiae
). Expressing recombinant eukaryotic proteins in yeast allows one to obtain higher success rate of purification and also improves the chances that proteins will retain biological activity relative to prokaryotic and in vitro
-based expression systems (24
). Furthermore, the use of an evolutionarily distant heterologous expression system like yeast minimizes the risk of contamination of recombinant human proteins with interacting cellular proteins, which is a potential complication that can result from the use of mammalian cells for protein expression.
A microarray with this level of coverage of the human proteome can potentially be used to identify antibodies that efficiently recognize proteins in their native conformation and that are thus useful for applications such as immunoprecipitation. We have used this tool as the backbone of an integrated platform that enables rapid and low cost identification of mAbs that both selectively bind a diverse assortment of human proteins and are useful in a wide range of experimental applications.