Introduction

Well-characterized antibodies are essential tools for protein studies, global proteomics analysis, as well as for clinical diagnostics. However, although production of antibodies is a well-established process and a large number of antibodies are commercially available through many vendors, specific antibodies still do not exist for the majority of human proteins. An underlying factor limiting the available antibody repertoire is that commercial production tends to focus on popular targets. The current needs of the proteomics community demand a far more global approach (Blow, Nature, Vol. 447, 2007, 741-742; Editorial, Nature Methods, Vol. 1, 2007, 1-2). A second significant issue is the lack of a universally defined standard for antibody quality. This makes it difficult to compare antibodies of various sources without committing resources to using the antibody in its final application. Initial standardized testing for specificity and sensitivity followed by a thorough characterization would be of great interest to the end user, but this is a costly endeavor and most efficiently accomplished on a larger scale.

At present, there are a handful of large-scale high-throughput antibody production efforts initiated around the world (Persson et al, Curr. Opin. Mol. Ther., Vol. 8, 2006, 185-190). One such initiative is the Swedish Human Protein Atlas (HPA) program (Uhlén et al, Mol. Cell Proteomics, Vol. 4, 2005, 1920-1932). The aim of this program is to explore the entire human proteome using an antibody-based proteomics approach (Kampf et al, Clin. Proteomics, Vol.1, 2004, 285-300; Uhlén and Pontén, Mol. Cell Proteomics, Vol. 4, 2005, 384-393). Specifically, the HPA program generates protein expression profiles of the non-redundant set of human proteins, presented as immunohistological images from the majority of human tissues. All images are annotated and made publicly available via an open access database, the Human Protein Atlas (www.proteinatlas.org). An ambitious and thorough quality-control process has been developed by which all antibodies have to pass a set of criteria prior to being tested by immunohistochemistry (IHC) and other methods (Hober and Uhlén, Curr. Opin. Biotech., Vol. 19, 2007, 1-6).

The Human Protein Atlas Program

The Swedish Human Protein Atlas (HPA) program is an academic initiative, headed by Professor Mathias Uhlén at the Royal Institute of Technology in Stockholm, Sweden and the Rudbeck Laboratory in Uppsala, Sweden. The vision of the HPA program is to systematically generate quality-assured antibodies to all non-redundant human proteins, and to use these reagents to functionally explore human proteins, protein variants, and protein interactions. At present, 50 new antibodies are generated per week along with 50,000 new IHC images. In order to manage this large amount of material and data generated, methodologies have been developed to support high-throughput systems including data collection, image handling, and storage.

Antibody Development and Quality Control

The HPA proteomics approach is based on affinity-purified polyclonal antibodies (mono-specific antibodies, msAbs) raised toward bioinformatically-designed Protein Epitope Signature Tag (PrEST) antigens. The PrEST antigens, the monospecific antibodies and the resultant images for the Human Protein Atlas are generated in a high-throughput manner as outlined in Figure 1.

Figure 1. (Click to enlarge)

The initial step of the process is the antigen design. Tailormade bioinformatics software based on the Basic Local Alignment Search Tool (BLAST) function is used to design the PrEST antigens. The scanning procedure permits selection of fragments of a specified size, between 50-150 amino acids, with a minimal sequence similarity to other human proteins. Further, this program allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides.

The computer-selected PrEST regions are RT-PCR amplified from pools of human total RNA and cloned into an expression vector as a fusion to a histidine tag and an albumin binding protein (ABP) (Agaton et al, Mol. Cell Proteomics, Vol. 2, 2003, 405-414). All recombinant PrEST clones are fully sequenced to verify the correct insert and that no polymeraseintroduced mutations are present. The PrEST sequence analysis is the first of several quality control steps that must be passed before further processing (Figure 1).

The sequence-verified PrEST clones are expressed in E. coli and the produced PrEST antigens are affinity-purified by immobilized metal ion chromatography under denaturing conditions. The purified PrEST antigens are quality controlled by mass spectrometry (MS) for sequence accuracy, by SDS-PAGE analysis for protein purity analysis, and by bicinchoninic acid assay (BCA) for protein concentration determination.