Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (Pr...

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Bibliographic Details
Published in:Proteomics (Weinheim) 2005-11, Vol.5 (17), p.4327-4337
Main Authors: Nilsson, Peter, Paavilainen, Linda, Larsson, Karin, Ödling, Jenny, Sundberg, Mårten, Andersson, Ann-Catrin, Kampf, Caroline, Persson, Anja, Szigyarto, Cristina Al-Khalili, Ottosson, Jenny, Björling, Erik, Hober, Sophia, Wernérus, Henrik, Wester, Kenneth, Pontén, Fredrik, Uhlen, Mathias
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Animals
Antibodies - chemistry
Antibodies - isolation & purification
Antibody generation
Antibody Specificity
Bioengineering
Biological and medical sciences
Bioteknik
Chromatography, Affinity
Fundamental and applied biological sciences. Psychology
Humans
Immunohistochemistry - methods
Miscellaneous
Protein Array Analysis
Protein microarray
Proteins
Proteome - chemistry
Proteome - immunology
Proteome - isolation & purification
Proteome atlas
Rabbits
Recombinant Proteins - chemistry
Recombinant Proteins - immunology
TECHNOLOGY
TEKNIKVETENSKAP
Tissue microarray
Tissue profiling
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Summary:A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.
ISSN:1615-9853
1615-9861
1615-9861
DOI:10.1002/pmic.200500072