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In situ protein detection with enhanced specificity using DNA-conjugated antibodies and proximity ligation
KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-8993-048X
2018 (English)In: Modern Pathology, ISSN 0893-3952, E-ISSN 1530-0285, Vol. 31, no 2, p. 253-263Article in journal (Refereed) Published
Abstract [en]

Antibodies are important tools in anatomical pathology and research, but the quality of in situ protein detection by immunohistochemistry greatly depends on the choice of antibodies and the abundance of the targeted proteins. Many antibodies used in scientific research do not meet requirements for specificity and sensitivity. Accordingly, methods that improve antibody performance and produce quantitative data can greatly advance both scientific investigations and clinical diagnostics based on protein expression and in situ localization. We demonstrate here protocols for antibody labeling that allow specific protein detection in tissues via bright-field in situ proximity ligation assays, where each protein molecule must be recognized by two antibodies. We further demonstrate that single polyclonal antibodies or purified serum preparations can be used for these dual recognition assays. The requirement for protein recognition by pairs of antibody conjugates can significantly improve specificity of protein detection over single-binder assays.

Place, publisher, year, edition, pages
Nature Publishing Group , 2018. Vol. 31, no 2, p. 253-263
Keywords [en]
antibody conjugate, APEX 1 protein, calvasculin, DNA conjugated antibody, immunoglobulin G antibody, polyclonal antibody, protein, protein A, protein G, rabbit antiserum, reagent, trefoil factor 1, unclassified drug, animal tissue, antibody affinity, antibody labeling, antibody specificity, Article, assay, click chemistry, controlled study, DNA strand, genetic transcription, immunohistochemistry, in situ proximity ligation assay, limit of detection, mRNA expression level, nonhuman, priority journal, protein analysis, protein expression, protein expression level, protein purification, tissue microarray, tissue section
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-227448DOI: 10.1038/modpathol.2017.102ISI: 000424761400003Scopus ID: 2-s2.0-85041805855OAI: oai:DiVA.org:kth-227448DiVA, id: diva2:1210262
Note

Export Date: 9 May 2018; Article; CODEN: MODPE; Correspondence Address: Landegren, U.; Department of Immunology, Genetics and Pathology, Science for Life Laboratory, BMC, Uppsala University, Husargatan 3, Sweden; email: Ulf.Landegren@igp.uu.se; Funding details: VINNOVA; Funding details: 222635; Funding details: 241481; Funding details: NCI, National Cancer Institute; Funding details: #2008:0143, Knut och Alice Wallenbergs Stiftelse; Funding details: FP5, Fifth Framework Programme; Funding details: FP/2007– 2013, FP7, Seventh Framework Programme; Funding details: ERC, European Research Council; Funding details: TRC, The Research Council; Funding details: 294409, ERC, European Research Council; Funding details: IngaBritt och Arne Lundbergs Forskningsstiftelse; Funding details: Uppsala Universitet; Funding text: This work was supported by the Knut and Alice Wallenberg Foundation (#2008:0143), the European Community's 7th Framework Program (FP7/2007–2013) under grant agreement n° 222635 (AffinityProteome) 241481 (Affinomics), The Swedish Research Council, Swedish Governmental Agency for Innovation Systems, IngaBritt and Arne Lundberg Foundation, the European Research Council under the European Union's Seventh Framework Programme (FP/2007– 2013) / ERC Grant Agreement n. 294409 (ProteinSeq), and Uppsala University. UL holds stock in Olink, having rights to the in situ proximity ligation assay technology. We would also like to thank Tara Hiltke at the National Cancer Institute for providing mAbs for in situ proximity ligation assay experiments. QC 20180528

Available from: 2018-05-28 Created: 2018-05-28 Last updated: 2018-05-28Bibliographically approved

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