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Zieba, A., Ponten, F., Uhlen, M. & Landegren, U. (2018). In situ protein detection with enhanced specificity using DNA-conjugated antibodies and proximity ligation. Modern Pathology, 31(2), 253-263
Open this publication in new window or tab >>In situ protein detection with enhanced specificity using DNA-conjugated antibodies and proximity ligation
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
National Category
Immunology in the medical area Other Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-347648 (URN)10.1038/modpathol.2017.102 (DOI)000424761400003 ()28937142 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, 2008:0143EU, FP7, Seventh Framework Programme, 222635 241481Swedish Research CouncilVINNOVAEU, European Research Council, 294409
Available from: 2018-04-06 Created: 2018-04-06 Last updated: 2018-04-06Bibliographically approved
Shen, Q., Björkesten, J., Galli, J., Ekman, D., Broberg, J., Nordberg, N., . . . Landegren, U. (2018). Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays. Clinical Chemistry and Laboratory Medicine, 56(4), 582-594
Open this publication in new window or tab >>Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays
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2018 (English)In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 56, no 4, p. 582-594Article in journal (Refereed) Published
Abstract [en]

Background: A number of factors regarding blood collection, handling and storage may affect sample quality. The purpose of this study was to assess the impact on plasma protein profiles by delayed centrifugation and plasma separation and multiple freeze-thaw cycles.

Methods: Blood samples drawn from 16 healthy individuals were collected into ethylenediaminetetraacetic acid tubes and kept either at 4 degrees C or 22 degrees C for 1-36 h prior to centrifugation. Plasma samples prepared 1 h after venipuncture were also subjected to two to eight cycles of freezing at -80 degrees C and thawing at 22 degrees C. Multiplex proximity extension assay, an antibody-based protein assay, was used to investigate the influence on plasma proteins.

Results: Up to 36 h delay before blood centrifugation resulted in significant increases of 16 and 40 out of 139 detectable proteins in samples kept at 4 degrees C or 22 degrees C, respectively. Some increases became noticeable after 8 h delay at 4 degrees C but already after 1 h at 22 degrees C. For samples stored at 4 degrees C, epidermal growth factor (EGF), NF-kappa-B essential modulator, SRC, interleukin 16 and CD6 increased the most, whereas the five most significantly increased proteins after storage at 22 degrees C were CD40 antigen ligand (CD40-L), EGF, platelet-derived growth factor subunit B, C-X-C motif chemokine ligand 5 and matrix metallopeptidase 1 (MMP1). Only matrix metallopeptidase 7 (MMP7) decreased significantly over time and only after storage at 22 degrees C. No protein levels were found to be significantly affected by up to eight freeze-thaw cycles.

Conclusions: Plasma should be prepared from blood after a limited precentrifugation delay at a refrigerated temperature. By contrast, the influence by several freeze-thaw cycles on detectable protein levels in plasma was negligible.

Place, publisher, year, edition, pages
WALTER DE GRUYTER GMBH, 2018
Keyword
biobank, protein detection, proteome, proximity extension assay (PEA), sample collection and handling
National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-350276 (URN)10.1515/cclm-2017-0648 (DOI)000426657400016 ()29040064 (PubMedID)
Funder
Swedish Research Council, 829-2009-6285EU, European Research Council, 313010, 294409
Available from: 2018-05-14 Created: 2018-05-14 Last updated: 2018-05-14Bibliographically approved
Lönn, P. & Landegren, U. (2017). Close Encounters: Probing Proximal Proteins in Live or Fixed Cells. TIBS -Trends in Biochemical Sciences. Regular ed., 42(7), 504-515
Open this publication in new window or tab >>Close Encounters: Probing Proximal Proteins in Live or Fixed Cells
2017 (English)In: TIBS -Trends in Biochemical Sciences. Regular ed., ISSN 0968-0004, E-ISSN 1362-4326, Vol. 42, no 7, p. 504-515Article, review/survey (Refereed) Published
Abstract [en]

The well-oiled machinery of the cellular proteome operates via variable expression, modifications, and interactions of proteins, relaying genomic and transcriptomic information to coordinate cellular functions. In recent years, a number of techniques have emerged that serve to identify sets of proteins acting in close proximity in the course of orchestrating cellular activities. These proximi dependent assays, including BiFC, BioID, APEX, FRET, and isPLA, have opened up new avenues to examine protein interactions in live or fixed cells. We review herein the current status of proximity-dependentin situ techniques. We compare the advantages and limitations of the methods, underlining recent progress and the growing importance of these techniques in basic research, and we discuss their potential as tools for drug development and diagnostics.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-333831 (URN)10.1016/j.tibs.2017.05.003 (DOI)000403569300004 ()28566215 (PubMedID)
Available from: 2017-11-24 Created: 2017-11-24 Last updated: 2017-11-24Bibliographically approved
Löf, L., Arngården, L., Olsson-Strömberg, U., Siart, B., Jansson, M., Dahlin, J. S., . . . Kamali-Moghaddam, M. (2017). Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia. Scientific Reports, 7, 1-9, Article ID 623.
Open this publication in new window or tab >>Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, p. 1-9, article id 623Article in journal (Refereed) Published
Abstract [en]

Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.

National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-319726 (URN)10.1038/s41598-017-00755-y (DOI)000398162400034 ()28377570 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 294409Swedish Cancer SocietySwedish Research Council
Available from: 2017-04-07 Created: 2017-04-07 Last updated: 2017-05-15Bibliographically approved
Padhan, N., Yan, J., Boge, A., Scrivener, E., Birgisson, H., Zieba, A., . . . Landegren, U. (2017). Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation. Scientific Reports, 7, Article ID 1490.
Open this publication in new window or tab >>Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 1490Article in journal (Refereed) Published
Abstract [en]

Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-323033 (URN)10.1038/s41598-017-01516-7 (DOI)000400554200002 ()28473697 (PubMedID)
Funder
EU, European Research Council, 294409EU, FP7, Seventh Framework Programme, 241481Swedish Research CouncilSwedish Cancer Society
Available from: 2017-06-02 Created: 2017-06-02 Last updated: 2017-06-02Bibliographically approved
Schuette, M., Risch, T., Abdavi-Azar, N., Boehnke, K., Schumacher, D., Keil, M., . . . Yaspo, M.-L. (2017). Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors. Nature Communications, 8, Article ID 14262.
Open this publication in new window or tab >>Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors
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2017 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 14262Article in journal (Refereed) Published
Abstract [en]

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling 44,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.

National Category
Cancer and Oncology Genetics
Identifiers
urn:nbn:se:uu:diva-316929 (URN)10.1038/ncomms14262 (DOI)000393656400001 ()28186126 (PubMedID)
Funder
Swedish Research CouncilVINNOVAEU, European Research Council
Available from: 2017-03-09 Created: 2017-03-09 Last updated: 2017-11-29Bibliographically approved
Björkesten, J., Enroth, S., Shen, Q., Wik, L., Hougaard, D., Cohen, A., . . . Landegren, U. (2017). Stability of Proteins in Dried Blood Spot Biobanks.. Molecular & Cellular Proteomics, 16(7), 1286-1296
Open this publication in new window or tab >>Stability of Proteins in Dried Blood Spot Biobanks.
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2017 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, no 7, p. 1286-1296Article in journal (Refereed) Published
Abstract [en]

An important motivation for the construction of biobanks is to discover biomarkers that identify diseases at early, potentially curable stages. This will require biobanks from large numbers of individuals, preferably sampled repeatedly, where the samples are collected and stored under conditions that preserve potential biomarkers. Dried blood samples are attractive for biobanking because of the ease and low cost of collection and storage. Here we have investigated their suitability for protein measurements. 92 proteins with relevance for oncology were analyzed using multiplex proximity extension assays (PEA) in dried blood spots collected on paper and stored for up to 30 years at either +4&deg;C or -24&deg;C.</p> <p>Our main findings were that 1) the act of drying only slightly influenced detection of blood proteins (average correlation of 0.970), and in a reproducible manner (correlation of 0.999), 2) detection of some proteins was not significantly affected by storage over the full range of three decades (34% and 76% of the analyzed proteins at +4&deg;C and -24&deg;C, respectively), while levels of others decreased slowly during storage with half-lives in the range of 10 to 50 years, and 3) detectability of proteins was less affected in dried samples stored at -24&deg;C compared to at +4&deg;C, as the median protein abundance had decreased to 80% and 93% of starting levels after 10 years of storage at +4&deg;C or -24&deg;C, respectively. The results of our study are encouraging as they suggest an inexpensive means to collect large numbers of blood samples, even by the donors themselves, and to transport, and store biobanked samples as spots of whole blood dried on paper. Combined with emerging means to measure hundreds or thousands of protein, such biobanks could prove of great medical value by greatly enhancing discovery as well as routine analysis of blood biomarkers.

Keyword
Absolute quantification, Affinity proteomics, Biobanking, Bioinformatics splicing, Biomarkers, Blood*, DBS, Diagnostic, Dried Blood Spot, Multiplex protein detection, PCR, Plasma or serum analysis, Predictive markers*, Protein Stability, Proximity Extension Assay
National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-322568 (URN)10.1074/mcp.RA117.000015 (DOI)000404597500009 ()28501802 (PubMedID)
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme, 294409Novo Nordisk
Available from: 2017-05-25 Created: 2017-05-25 Last updated: 2017-11-29Bibliographically approved
Larssen, P., Wik, L., Czarnewski, P., Eldh, M., Löf, L., Ronquist, G., . . . Kamali-Moghaddam, M. (2017). Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assay. Molecular & cellular proteomics (online), 16(3), 502-511
Open this publication in new window or tab >>Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assay
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2017 (English)In: Molecular & cellular proteomics (online), ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, no 3, p. 502-511Article in journal (Refereed) Published
Abstract [en]

Extracellular vesicles (EVs) are membrane-coated objects such as exosomes and microvesicles, released by many cell-types. Their presence in body fluids and the variable surface composition and content render them attractive potential biomarkers. The ability to determine their cellular origin could greatly move the field forward. We used multiplex proximity extension assays (PEA) to identify with high specificity and sensitivity the protein profiles of exosomes of different origins, including seven cell lines and two different body fluids. By comparing cells and exosomes, we successfully identified the cells originating the exosomes. Furthermore, by principal component analysis of protein patterns human milk EVs and prostasomes released from prostate acinar cells clustered with cell lines from breast and prostate tissues, respectively. Milk exosomes uniquely expressed CXCL5, MIA and KLK6, while prostasomes carried NKX31, GSTP1 and SRC, highlighting that EVs originating from different origins express distinct proteins. In conclusion, PEA provides a powerful protein screening tool in exosome research, for purposes of identifying the cell source of exosomes, or new biomarkers in diseases such as cancer and inflammation.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-314142 (URN)10.1074/mcp.M116.064725 (DOI)000395670900013 ()28111361 (PubMedID)
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme, 259796 294409Swedish Cancer Society, 2013/867Stockholm County Council, 20140405Swedish Heart Lung Foundation, 20140497The Karolinska Institutet's Research FoundationCancer and Allergy Foundation
Available from: 2017-01-28 Created: 2017-01-28 Last updated: 2017-04-20Bibliographically approved
Lind, A.-L., Yu, D., Freyhult, E., Bodolea, C., Ekegren, T., Larsson, A., . . . Kamali-Moghaddam, M. (2016). A Multiplex Protein Panel Applied to Cerebrospinal Fluid Reveals Three New Biomarker Candidates in ALS but None in Neuropathic Pain Patients. PLoS ONE, 11(2), Article ID e0149821.
Open this publication in new window or tab >>A Multiplex Protein Panel Applied to Cerebrospinal Fluid Reveals Three New Biomarker Candidates in ALS but None in Neuropathic Pain Patients
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 2, article id e0149821Article in journal (Refereed) Published
Abstract [en]

The objective of this study was to develop and apply a novel multiplex panel of solid-phase proximity ligation assays (SP-PLA) requiring only 20 μL of samples, as a tool for discovering protein biomarkers for neurological disease and treatment thereof in cerebrospinal fluid (CSF). We applied the SP-PLA to samples from two sets of patients with poorly understood nervous system pathologies amyotrophic lateral sclerosis (ALS) and neuropathic pain, where patients were treated with spinal cord stimulation (SCS). Forty-seven inflammatory and neurotrophic proteins were measured in samples from 20 ALS patients and 15 neuropathic pain patients, and compared to normal concentrations in CSF from control individuals. Nineteen of the 47 proteins were detectable in more than 95% of the 72 controls. None of the 21 proteins detectable in CSF from neuropathic pain patients were significantly altered by SCS. The levels of the three proteins, follistatin, interleukin-1 alpha, and kallikrein-5 were all significantly reduced in the ALS group compared to age-matched controls. These results demonstrate the utility of purpose designed multiplex SP-PLA panels in CSF biomarker research for understanding neuropathological and neurotherapeutic mechanisms. The protein changes found in the CSF of ALS patients may be of diagnostic interest.

National Category
Neurology Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-281233 (URN)10.1371/journal.pone.0149821 (DOI)000371175700030 ()26914813 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationEU, European Research Council, 316929EU, European Research Council, 294409
Available from: 2016-03-21 Created: 2016-03-21 Last updated: 2017-11-30Bibliographically approved
Landegren, U. (2016). AFFINOMICS and the prospects for large-scale protein analyses. New Biotechnology, 33(5), 491-493
Open this publication in new window or tab >>AFFINOMICS and the prospects for large-scale protein analyses
2016 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 33, no 5, p. 491-493Article in journal, Editorial material (Other academic) Published
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-300128 (URN)10.1016/j.nbt.2015.09.006 (DOI)000378026000002 ()26455639 (PubMedID)
Available from: 2016-08-03 Created: 2016-08-03 Last updated: 2017-11-28Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-7820-1000

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