title: Cytokine detection by antibody-based proximity ligation.
abstract: 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.
authors.affiliation: The Beijer Laboratory, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Se-75185 Uppsala, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Gullberg
authors.forename: Sigrún M
authors.initials: SM
authors.lastname: Gústafsdóttir
authors.forename: Edith
authors.initials: E
authors.lastname: Schallmeiner
authors.forename: Jonas
authors.initials: J
authors.lastname: Jarvius
authors.forename: Mattias
authors.initials: M
authors.lastname: Bjarnegård
authors.forename: Christer
authors.initials: C
authors.lastname: Betsholtz
authors.forename: Ulf
authors.initials: U
authors.lastname: Landegren
authors.forename: Simon
authors.initials: S
authors.lastname: Fredriksson
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D002460:Cell Line; D016207:Cytokines / Q000032:analysis*; D006801:Humans; D007328:Insulin / Q000032:analysis; D015336:Molecular Probe Techniques; D009841:Oligonucleotides / Q000378:metabolism; D013917:Thrombin / Q000032:analysis
chemical_list: D000906:Antibodies; D016207:Cytokines; D007328:Insulin; D009841:Oligonucleotides; D013917:Thrombin