Cytokine detection by antibody-based proximity ligation

Gullberg, MatsUppsala universitet,Institutionen för genetik och patologi (author)

Efficient and precise detection techniques, along with extensive repertoires of specific binding reagents, will be needed to meet the challenges of proteome analyses. The recently established proximity ligation mechanism enables sensitive high-capacity protein measurements by converting the detection of specific proteins to the analysis of DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins and to form amplifiable tag sequences by ligation when brought in proximity. In our previous report, both the ligatable arms and the protein binders were DNA molecules. We now generalize the method by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences. Sufficient reagent for >100,000 proximity ligation assays can be prepared from 1 microg of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-microl samples, and we exemplify its utility in situations when only minute sample amounts are available.

SAMHÄLLSVETENSKAP Annan samhällsvetenskap Tvärvetenskapliga studier inom samhällsvetenskap hsv//swe
SOCIAL SCIENCES Other Social Sciences Social Sciences Interdisciplinary hsv//eng
Antibodies/*metabolism
Cell Line
Comparative Study
Cytokines/*analysis
Humans
Insulin/analysis
Molecular Probe Techniques
Oligonucleotides/metabolism
Research Support; Non-U.S. Gov't
Thrombin/analysis
MEDICINE
MEDICIN
protein-detection
immuno-pcr
affinity
antigen
selection
thrombin
gene

Gústafsdóttir, Sigrun M.Uppsala universitet,Institutionen för genetik och patologi (author)
Schallmeiner, EdithUppsala universitet,Institutionen för genetik och patologi(Swepub:uu)edsch087 (author)
Jarvius, JonasUppsala universitet,Institutionen för genetik och patologi(Swepub:uu)jonajarv (author)
Bjarnegård, Mattias,1970Gothenburg University,Göteborgs universitet,Institutionen för medicinsk och fysiologisk kemi,Institute of Medical Biochemistry(Swepub:gu)xbjama (author)
Betsholtz, ChristerGothenburg University,Göteborgs universitet,Institutionen för medicinsk och fysiologisk kemi,Institute of Medical Biochemistry (author)
Landegren, UlfUppsala universitet,Institutionen för genetik och patologi(Swepub:uu)ulfland (author)
Fredriksson, SimonUppsala universitet,Institutionen för genetik och patologi (author)
Uppsala universitetInstitutionen för genetik och patologi (creator_code:org_t)

In:Proceedings of the National Academy of Sciences of the United States of America: Proceedings of the National Academy of Sciences101:22, s. 8420-40027-84241091-6490

By the author/editor: Gullberg, Mats; Gústafsdóttir, S ...; Schallmeiner, Ed ...; Jarvius, Jonas; Bjarnegård, Matt ...; Betsholtz, Chris ...; show more...; Landegren, Ulf; Fredriksson, Sim ...; show less...

About the subject

SOCIAL SCIENCES: SOCIAL SCIENCES; and Other Social Sci ...; and Social Sciences ...

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