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 (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.
Nilsson
Peter
P
Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
Paavilainen
Linda
L
Larsson
Karin
K
Odling
Jenny
J
Sundberg
Mårten
M
Andersson
Ann-Catrin
AC
Kampf
Caroline
C
Persson
Anja
A
Al-Khalili Szigyarto
Cristina
C
Ottosson
Jenny
J
Björling
Erik
E
Hober
Sophia
S
Wernérus
Henrik
H
Wester
Kenneth
K
Pontén
Fredrik
F
Uhlen
Mathias
M
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000302:isolation & purification; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D040081:Protein Array Analysis; D020543:Proteome / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D020543:Proteome; D011994:Recombinant Proteins