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The program involves two major sites. These sites are hosted by the Albanova University Center at the Royal Institute of Technology (KTH, Stockholm) and the Rudbeck Laboratory (Uppsala University, Uppsala), respectively. The Stockholm site is responsible for generating high-quality monospecific antibodies, and the Uppsala site is responsible for large-scale protein profiling in tissues and cells.
The manufacturing process performed in Stockholm involves molecular methodology prior to immunization, such as high-throughput cloning and expression of the protein fragments (PrESTs) representing putative human genes. It also entails post-immunization handling of antibodies, such as affinity purification and quality assurance.
Protein profiling is performed in Uppsala using immunohistochemistry on tissue- and cellmicroarrays. The arrays comprise a large variety of normal human tissues, cancer tissues and a set of human cell lines. The Uppsala site is also responsible for generating digital images representing outcome of staining and annotation of the respective images.
We have recently described a standardized approach for generating recombinant proteins based on coding sequences identified in the human genome sequence (see reference). This approach has been further developed with regard to (i) bioinformatics algorithms that allow the identification of coding sequences that are amenable both to recombinant protein expression and the generation of specific antibodies, (ii) a robust E. coli recombinant protein expression system, (iii) systematic antibody production associated with antigen purification of polyclonal sera and, (iv) systematic approaches to utilize these reagents, in particular using tissue arrays. The figure shows the different steps within the HPA program. These methodologies are all developed to allow high throughput on a genome wide scale.
Reference: Uhlen M, Ponten F. Antibody-based Proteomics for Human Tissue Profiling. Mol Cell Proteomics. 2005 Apr;4(4):384-393.
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