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  • 1.
    Alderborn, Anders
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Kamali, Masood
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Söderberg, Ola
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Schlingemann, Heidi
    Nilsson, Mats
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    The hunt for cancer biomarkers: Proximity ligation – a new technology for ultra-sensitive protein analysis2006Other (Other (popular scientific, debate etc.))
  • 2.
    Antson, D.O.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Isaksson, A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    PCR-generated padlock probes detect single nucleotide variation in genomic DNA2000In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 28, no 12, p. E58-Article in journal (Refereed)
    Abstract [en]

    Circularizing oligonucleotide probes, so-called padlock probes, have properties that should prove valuable in a wide range of genetic investigations, including in situ analyses, genotyping and measurement of gene expression. However, padlock probes can be difficult to obtain by standard oligonucleotide synthesis because they are relatively long and require intact 5'- and 3'-end sequences to function. We describe a PCR-based protocol for flexible small-scale enzymatic synthesis of such probes. The protocol also offers the advantage over chemical synthesis that longer probes can be made that are densely labeled with detectable functions, resulting in an increased detection signal. The utility of probes synthesized according to this protocol is demonstrated for the analysis of single nucleotide variations in human genomic DNA both in situ and in solution.

  • 3.
    Arvidsson, Per I.
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Karolinska institutet.
    Domeij, Bengt
    Uppsala University, Disciplinary Domain of Humanities and Social Sciences, Faculty of Law, Department of Law.
    Hansson, Mats G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Lind, Anna-Sara
    Uppsala University, Disciplinary Domain of Humanities and Social Sciences, Faculty of Law, Department of Law. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics.
    Ullerås, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Öppenheten förstör chansen till patent2015In: Svenska dagbladet, ISSN 2001-3868Article in journal (Other (popular science, discussion, etc.))
  • 4. Banér, Johan
    et al.
    Gyarmati, Péter
    Yacoub, Alia
    Hakhverdyan, Mikhayil
    Stenberg, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ericsson, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Belák, Sándor
    Microarray-based molecular detection of foot-and-mouth disease, vesicular stomatitis and swine vesicular disease viruses, using padlock probes2007In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 143, no 2, p. 200-206Article in journal (Refereed)
    Abstract [en]

    The World Organization for Animal Health (Office International des Epizooties, OIE) includes the diseases caused by foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV), as "Diseases Notifiable to the OIE". Foot-and-mouth disease (FMD) outbreaks have severe economical as well as social effects and cannot be differentiated from the diseases caused by the other two viruses on the basis of clinical symptoms. Efficient laboratory techniques are therefore required for detection and identification of the viruses causing similar vesicular symptoms in swine. A rapid method is described using padlock probes and microarrays to detect simultaneously and differentiate the three viruses in a single reaction, as well as providing serotype information in cases of VSV infection. The padlock probe/microarray assay detected successfully and identified 39 cDNA samples of different origin representing the three viruses. The results were in complete agreement with identities and serotypes determined previously. This novel virus detection method is discussed in terms of usefulness and further development.

  • 5.
    Banér, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Isaksson, Anders
    Waldenström, Erik
    Jarvius, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Parallel gene analysis with allele-specific padlock probes and tag microarrays2003In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, no 17, p. e103-Article in journal (Refereed)
    Abstract [en]

    Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes.

  • 6.
    Banér, Johan
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Marits, Per
    Nilsson, Mats
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Winqvist, Ola
    Landegren, Ulf
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Analysis of T-Cell Receptor V{beta} Gene Repertoires after Immune Stimulation and in Malignancy by Use of Padlock Probes and Microarrays.2005In: Clin Chem, ISSN 0009-9147, Vol. 51, no 4, p. 768-75Article in journal (Other scientific)
  • 7.
    Beghini, A.
    et al.
    Univ Milan, Dept Hlth Sci, Milan, Italy..
    Lazzaroni, F.
    Univ Milan, Dept Hlth Sci, Milan, Italy..
    Del Giacco, L.
    Univ Milan, Dept Biosci, Milan, Italy..
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Biasci, D.
    Univ Cambridge, Cambridge Inst Med Res, Cambridge, England..
    Turrini, M.
    Valduce Hosp, Dept Internal Med, Como, Italy..
    Prosperi, L.
    Univ Milan, Dept Biosci, Milan, Italy..
    Brusamolino, R.
    Osped Niguarda Ca Granda, Dept Pathol, Milan, Italy..
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Cairoli, R.
    Osped Niguarda Ca Granda, Dept Oncol, Hematol Unit, Milan, Italy..
    Clinical Relevance Of Recurrent Allele-Specific Recombination Expressing The Wnt10Bivs1 Allele Variant In Acute Myeloid Leukemia2016In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 101, p. 668-669Article in journal (Other academic)
  • 8.
    Birgegård, Gunnar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Dahl, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Glimelius, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. Enheten för onkologi.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Evaluation of beta globin mRNA as an early marker of haemoglobin response to epoetin treatment2007In: Medical Oncology, ISSN 1357-0560, E-ISSN 1559-131X, Vol. 24, no 3, p. 318-322Article in journal (Refereed)
    Abstract [en]

    Approximately 60% of anaemic cancer patients respond to epoetin treatment. An early marker of response would be valuable in order to avoid ineffective treatment. We have previously shown that beta globin mRNA increases rapidly after epoetin beta treatment of healthy controls. In the present study we have evaluated whether a change of this marker during the first 2 weeks of epoetin treatment could predict later Hb response in anaemic cancer patients. Twenty cancer patients with Hb <11 g/dl received epoetin beta (NeoRecormon®) 10,000 IU three times weekly during 6 weeks. Hb, reticulocytes and β-globin mRNA were followed. The latter was measured quantitatively using PCR via the 5′ nuclease assay. Eleven patients responded with a Hb increase of >1 g/dl, nine were nonresponders. All responders increased in β-globin mRNA within 2 weeks, mean 7.7× base-line. With a cut-off of an increase of 3× base-line value, we obtained a specificity of 45% and a sensitivity of 91% for the prediction of a later increase of Hb >1 g/dl. With a cut-off of 4× base-line, the specificity increased to 66%, but the sensitivity decreased to 82%. Beta globin mRNA increases before Hb in all responding patients. However, some non-responding patients also show an increase, and there is a trade-off between specificity and sensitivity as the cut-off level is set at different levels. Compared to reticulocyte count, β-globin mRNA is more reliable in the individual patient, but the clinical usefulness of the assay needs to be evaluated in further studies.

     

  • 9.
    Björkesten, Johan
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Enroth, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Shen, Qiujin
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Wik, Lotta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hougaard, David
    Statens Serum Inst, Danish Ctr Neonatal Screening, Copenhagen, Denmark.
    Cohen, Arieh
    Statens Serum Inst, Danish Ctr Neonatal Screening, Copenhagen, Denmark.
    Sörensen, Lene
    Karolinska Univ Hosp, Ctr Inherited Metab Dis, Stockholm, Sweden.
    Giedraitis, Vilmantas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Stability of Proteins in Dried Blood Spot Biobanks.2017In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, no 7, p. 1286-1296Article in journal (Refereed)
    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.

  • 10.
    Blokzijl, Andries
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Chen, Lei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gustafsdottir, Sigrun M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Vuu, Jimmy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Ullenhag, Gustav
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Kämpe, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Autoimmunity.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Hedstrand, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
    Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, article id e0154214Article in journal (Refereed)
    Abstract [en]

    Background

    The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder.

    Objectives

    To investigate SOX10 as a potential biomarker for melanoma and vitiligo.

    Methods

    In this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes.

    Results

    The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically.

    Conclusions

    We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.

  • 11.
    Blokzijl, Andries
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Nong, Rachel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Darmanis, S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Hertz, E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Protein biomarker validation via proximity ligation assays2014In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1844, no 5, p. 933-939Article, review/survey (Refereed)
    Abstract [en]

    The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PIA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. (C) 2013 Elsevier B.V. All rights reserved.

  • 12.
    Blokzijl, Andries
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. YUMAB GmbH, Rebenring 33, D-38106 Braunschweig, Germany..
    Zieba, Agata
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Hust, Michael
    Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany..
    Schirrmann, Thomas
    Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany.;YUMAB GmbH, Rebenring 33, D-38106 Braunschweig, Germany..
    Helmsing, Saskia
    Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany..
    Grannas, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Hertz, Ellen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Morén, Anita
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Chen, Lei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Dubel, Stefan
    Tech Univ Carolo Wilhelmina Braunschweig, Inst Biochem Biotechnol & Bioinformat, Dept Biotechnol, Spielmannstr 7, D-38106 Braunschweig, Germany..
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens2016In: Molecular & cellular proteomics (online), ISSN 1535-9476, E-ISSN 1535-9484, Vol. 15, no 6, p. 1848-1856Article in journal (Refereed)
    Abstract [en]

    The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF- signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF- signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF- signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA. Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.

  • 13.
    Clausson, Carl-Magnus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Allalou, Amin
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Centre for Image Analysis. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Weibrecht, Irene
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mahmoudi, Salah
    Farnebo, Marianne
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Increasing the dynamic range of in situ PLA2011In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 8, no 11, p. 892-893Article in journal (Refereed)
  • 14.
    Conze, Tim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Carvalho, Ana Sofia
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Almeida, Raquel
    Reis, Celso A.
    David, Leonor
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas2010In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 2, p. 199-206Article in journal (Refereed)
    Abstract [en]

    Changes in mucin protein expression and in glycosylation are common features in pre-neoplastic lesions and cancer and are therefore used as cancer-associated markers. De novo expression of intestinal mucin MUC2 and cancer-associated sialyl-Tn antigen are frequently observed in intestinal metaplasia (IM) and gastric cancer. However, despite that these antigens often co-localize, MUC2 has not been demonstrated to be a carrier of sialyl-Tn. By using the in situ proximity ligation assay (in situ PLA), we herein could show that MUC2 is a major carrier of the sialyl-Tn antigen in all IM cases and in most gastric carcinoma cases. The requirement by in situ PLA for the presence of both antigens in close proximity increases the selectivity compared to measurement of co-localization, as determined by immunohistochemistry. Identification of the mucin which is the carrier of a carbohydrate structure offers unique advantages for future development of more accurate diagnostic and prognostic markers.

  • 15.
    Conze, Tim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Göransson, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Razzaghian, Hamidreza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ericsson, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Öberg, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Single molecule analysis of combinatorial splicing2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 16, p. e163-Article in journal (Refereed)
    Abstract [en]

    Alternative splicing forms diverse mRNA isoform populations from a single ancestral pre-mRNA and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns into a library of binary strings of nucleic acid tags, encoding the exon composition of transcripts. The transcript abundance is registered by counts of individual molecules and individual exon inclusion patterns are represented as strings of binary data.

    The technique is illustrated by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits different genes to be analyzed in parallel and will be valuable for elucidating the complex effects of combinatorial splicing.

  • 16.
    Conze, Tim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Shetye, Alysha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Tanaka, Yuki
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gu, Jijuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Larsson, Chatarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Göransson, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Tavoosidana, Gholamreza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Analysis of Genes, Transcripts, and Proteins via DNA Ligation2009In: Annual review of analytical chemistry, ISSN 1936-1327, Vol. 2, p. 215-239Article, review/survey (Refereed)
    Abstract [en]

    Analytical reactions in which short DNA strands are used in combination with DNA ligases have proven useful for measuring, decoding, and locating most classes of macromolecules. Given the need to accumulate large amounts of precise molecular information from biological systems in research and in diagnostics, ligation reactions will continue to offer valuable strategies for advanced analytical reactions. Here, we provide a basis for further development of methods by reviewing the history of analytical ligation reactions, discussing the properties of ligation reactions that render them suitable for engineering novel assays, describing a wide range of successful ligase-based assays, and briefly considering future directions.

  • 17.
    Dahl, Fredrik
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Banér, Johan
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Gullberg, Mats
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Mendel-Hartvig, Maritha
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Circle-to-circle amplification for precise and sensitive DNA analysis.2004In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 101, no 13, p. 4548-53Article in journal (Other scientific)
  • 18.
    Darmanis, Spyros
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Gallant, Caroline Julie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Marinescu, Voichita Dana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Niklasson, Mia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Segerman, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Flamourakis, Georgios
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Fredriksson, Simon
    Olink Biosci, S-75237 Uppsala, Sweden..
    Assarsson, Erika
    Olink Biosci, S-75237 Uppsala, Sweden..
    Lundberg, Martin
    Olink Biosci, S-75237 Uppsala, Sweden..
    Nelander, Sven
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Westermark, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells2016In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 14, no 2, p. 380-389Article in journal (Refereed)
    Abstract [en]

    Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell's phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

  • 19.
    Darmanis, Spyros
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Gallant, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    PCR-Based Multiparametric Assays in Single Cells.2012In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 58, no 12, p. 1618-1619Article in journal (Refereed)
  • 20.
    Darmanis, Spyros
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kähler, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Spångberg, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Schallmeiner, Edith
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Self-assembly of proximity probes for flexible and modular proximity ligation assays2007In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 43, no 4, p. 443-450Article in journal (Refereed)
    Abstract [en]

    Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.

  • 21.
    Darmanis, Spyros
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nong, Rachel Yuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hammond, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gu, Jijuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Alderborn, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Vänelid, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Siegbahn, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Gustafsdottir, Sigrun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ericsson, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sensitive plasma protein analysis by microparticle-based proximity ligation assays2010In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 2, p. 327-335Article in journal (Refereed)
    Abstract [en]

    Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.

  • 22.
    Darmanis, Spyros
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Nong, Rachel Yuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Vänelid, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Siegbahn, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Ericsson, Olle
    Halo Genomics AB, Dag Hammarskjölds väg 36B, SE-752 37 Uppsala Sweden.
    Fredriksson, Simon
    Olink Biosciences, Dag Hammarskjölds väg 52B, SE-752 37 Uppsala, Sweden.
    Bäcklin, Christofer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Gut, Marta
    Centro Nacional de Análisis Genómico, C/Baldiri Reixac 4, 08028 Barcelona, Spain.
    Heath, Simon
    Centro Nacional de Análisis Genómico, C/Baldiri Reixac 4, 08028 Barcelona, Spain.
    Gut, Ivo Glynne
    Centro Nacional de Análisis Genómico, C/Baldiri Reixac 4, 08028 Barcelona, Spain.
    Wallentin, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, UCR-Uppsala Clinical Research Center.
    Gustafsson, Mats G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    ProteinSeq: high-performance proteomic analyses by proximity ligation and next generation sequencing2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, p. e25583-Article in journal (Refereed)
    Abstract [en]

    Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 μl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use. 

  • 23.
    Ebai, Tonge
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    de Oliveira, Felipe Marques Souza
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Löf, Liza
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Wik, Lotta
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Schweiger, Caroline
    Charité Comprehensive Cancer Center, University of Berlin, Berlin, Germany; Institute of Pathology, Medical University of Graz, Graz, Austria; .
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Keilholtz, Ulrich
    Institute of Pathology, Medical University of Graz, Graz, Austria; .
    Haybaeck, Johannes
    Charité Comprehensive Cancer Center, University of Berlin, Berlin, Germany; Department of Pathology, Otto von Guericke University Magdeburg, Magdeburg, Germany..
    Landegren, Ulf
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification2017In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 63, no 9, p. 1497-1505Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals.

    METHODS: Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition, these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader.

    RESULTS: We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor-15 by PLARCA and conventional sandwich ELISA or immuno RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA.

    CONCLUSIONS: PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories.

  • 24.
    Ebai, Tonge
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Parallel protein detection by solid-phase proximity ligation assay with real-time PCR or sequencing2015In: Current Protocol in Molecular Biology, ISSN 1934-3647, Vol. 109, no 20Article in journal (Refereed)
    Abstract [en]

    Proximity ligation assays are a group of protein detection techniques in which reagents with affinity for target proteins, typically antibodies, are coupled to short strands of DNA. DNA-modified affinity reagents are combined in assays constructed such that the coordinated binding of individual target molecules or complexes of interacting proteins by two or more of the reagents, followed by DNA ligation and/or polymerization reactions, gives rise to amplifiable DNA reporter strands. Proximity ligation assays have been shown to exhibit excellent sensitivity in single and multiplexed protein assays for individual or interacting proteins, both in solution and in situ. This unit describes procedures for developing solid-phase proximity ligation assays for soluble proteins using either real-time PCR or DNA sequencing as the readout. In addition, critical steps for assay optimization are discussed.

  • 25.
    Eriksson, Ronnie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Jobs, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Ekstrand, Charlotta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Ullberg, Måns
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout2009In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 78, no 2, p. 195-202Article in journal (Refereed)
    Abstract [en]

    A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.

  • 26. Flanigon, James
    et al.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Burbulis, Ian
    Annink, Carla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Steffen, Martin
    Oeth, Paul
    Brent, Roger
    van den Boom, Dirk
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Cantor, Charles
    Multiplex protein detection with DNA readout via mass spectrometry2013In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 30, no 2, p. 153-158Article in journal (Refereed)
    Abstract [en]

    Multiplex protein quantification has been constrained by issues of assay specificity, sensitivity and throughput. This research presents a novel approach that overcomes these limitations using antibody-oligonucleotide conjugates for immuno-polymerase chain reaction (immuno-PCR) or proximity ligation, coupled with competitive PCR and MALDI-TOF mass spectrometry. Employing these combinations of technologies, we demonstrate multiplex detection and quantification of up to eight proteins, spanning wide dynamic ranges from femtomolar concentrations, using only microliter sample volumes.

  • 27.
    Fredriksson, Simon
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gullberg, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jarvius, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Olsson, Charlotta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Pietras, Kristian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Gustafsdottir, Sigrún Margrét
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Östman, Arne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Protein detection using proximity-dependent DNA ligation assays2002In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 20, no 5, p. 473-477Article in journal (Refereed)
    Abstract [en]

    The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 x 10(-21) mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.

  • 28.
    Fryknäs, Mårten
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wickenberg Bolin, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Göransson, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gustafsson, Mats G
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Signal Processing.
    Foukakis, Theodoros
    Lee, Jia-Jing
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Höög, Anders
    Larsson, Catharina
    Grimelius, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wallin, Göran
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Isaksson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Molecular markers for discrimination of benign and malignant follicular thyroid tumors2006In: Tumor Biology, ISSN 1010-4283, Vol. 27, no 4, p. 211-220Article in journal (Refereed)
  • 29.
    Galli, Joakim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Oelrich, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Taussig, Michael J
    Andreasson, Ulrika
    Ortega-Paino, Eva
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    The Biobanking Analysis Resource Catalogue (BARCdb): a new research tool for the analysis of biobank samples.2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no D1, p. D1158-D1162Article in journal (Refereed)
    Abstract [en]

    We report the development of a new database of technology services and products for analysis of biobank samples in biomedical research. BARCdb, the Biobanking Analysis Resource Catalogue (http://www.barcdb.org), is a freely available web resource, listing expertise and molecular resource capabilities of research centres and biotechnology companies. The database is designed for researchers who require information on how to make best use of valuable biospecimens from biobanks and other sample collections, focusing on the choice of analytical techniques and the demands they make on the type of samples, pre-analytical sample preparation and amounts needed. BARCdb has been developed as part of the Swedish biobanking infrastructure (BBMRI.se), but now welcomes submissions from service providers throughout Europe. BARCdb can help match resource providers with potential users, stimulating transnational collaborations and ensuring compatibility of results from different labs. It can promote a more optimal use of European resources in general, both with respect to standard and more experimental technologies, as well as for valuable biobank samples. This article describes how information on service and reagent providers of relevant technologies is made available on BARCdb, and how this resource may contribute to strengthening biomedical research in academia and in the biotechnology and pharmaceutical industries.

  • 30.
    Ghanipour, Lana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Colorectal Surgery.
    Darmanis, Spyros
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Glimelius, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science.
    Påhlman, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Birgisson, Helgi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Detection of prognostic biomarkers with solid-phase proximity ligation assay in patients with colorectal cancer2016In: Translational Oncology, ISSN 1944-7124, E-ISSN 1936-5233, Vol. 9, no 3, p. 251-255Article in journal (Refereed)
    Abstract [en]

    Background: In the search for prognostic biomarkers a significant amount of precious biobanked blood samples is needed if conventional analyses are used. Solid-phase proximity ligation assay (SP-PLA) is an analytic method with the ability to analyse many proteins at the same time in small amounts of plasma. The aim of this study was to explore the potential use of  SP-PLA in patients with colorectal cancer (CRC).

    Material and methods: Plasma from patients with stage I-IV CRC, with (n=31) and without (n=29) disease dissemination at diagnosis or later, was analysed with SP-PLA using 35 antibodies targeting an equal number of proteins in 5 ml plasma. Carcinoembryonic antigen (CEA), analysed earlier on this cohort, was used as a reference.

    Results: A total of 21 of the 35 proteins were detectable with SP-PLA. Patients in stage II-III with disseminated disease had lower plasma concentrations of HCC-4 (p=0.025). Low plasma levels of TIMP-1 were seen in patients with disseminated disease stage II (p=0.003). The level of CEA was higher in patients with disease dissemination compared to those without (p=0.007).

    Conclusion: SP-PLA has the ability to analyse many tumour markers simultaneously in a small amount of blood. However, none of the markers selected for the present SP-PLA analyses gave better prognostic information compared with CEA. 

  • 31.
    Giandomenico, Valeria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Oncology.
    Modlin, Irvin M.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Khan, Mohid S.
    Millar, Robert P.
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
    Borlak, Jurgen
    Eriksson, Barbro
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Tumor Biology.
    Nielsen, Bengt
    Baltzer, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
    Waterton, John C.
    Ahlström, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Öberg, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Oncology.
    Improving the Diagnosis and Management of Neuroendocrine Tumors: Utilizing New Advances in Biomarker and Molecular Imaging Science2013In: Neuroendocrinology, ISSN 0028-3835, E-ISSN 1423-0194, Vol. 98, no 1, p. 16-30Article in journal (Refereed)
    Abstract [en]

    Neuroendocrine tumors (NET) are malignant solid tumors that arise in hormone-secreting tissue of the diffuse neuroendocrine system or endocrine glands. Although traditionally understood to be a rare disease, the incidence and prevalence of NET have increased greatly in the past 3 decades. However, during this time, progress in diagnosis and outcome of NET has generally been modest. In order to achieve improved outcome in NET, a better understanding of NET biology combined with more reliable serum markers and better techniques to identify tumor localization and small lesions are needed. Although some NET biomarkers exist, sensitive and specific markers that predict tumor growth and behavior are generally lacking. In addition, the integration of new molecular imaging technologies in patient diagnosis and follow-up has the potential to enhance care. To discuss developments and issues required to improve diagnostics and management of NET patients, with specific focus on the latest advances in molecular imaging and biomarker science, 17 global leaders in the fields of NET, molecular imaging and biomarker technology gathered to participate in a 2-day meeting hosted by Prof. Kjell Oberg at the University of Uppsala in Sweden. During this time, findings were presented regarding methods with potential prognostic and treatment applications in NET or other types of cancers. This paper describes the symposium presentations and resulting discussions.

  • 32.
    Gu, Gucci Jijuan
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Friedman, Mikaela
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Jost, Christian
    Johnsson, Kai
    Kamali-Moghaddam, Masood
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Plückthun, Andreas
    Landegren, Ulf
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Söderberg, Ola
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation2013In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 30, no 2, p. 144-152Article in journal (Refereed)
    Abstract [en]

    While antibodies currently play a dominant role as affinity reagents in biological research and for diagnostics, a broad range of recombinant proteins are emerging as promising alternative affinity reagents in detection assays and quantification. DNA-mediated affinity-based assays, such as immuno-PCR and proximity ligation assays (PLA), use oligonucleotides attached to affinity reagents as reporter molecules. Conjugation of oligonucleotides to affinity reagents generally employs chemistries that target primary amines or cysteines. Because of the random nature of these processes neither the number of oligonucleotides conjugated per molecule nor their sites of attachment can be accurately controlled for affinity reagents with several available amines and cysteines. Here, we present a straightforward and convenient approach to functionalize recombinant affinity reagents for PLA by expressing the reagents as fusion partners with SNAP protein tags. This allowed us to conjugate oligonucleotides in a site-specific fashion, yielding precisely one oligonucleotide per affinity reagent. We demonstrate this method using designed ankyrin repeat proteins (DARPins) recognizing the tumor antigen HER2 and we apply the conjugates in different assay formats. We also show that SNAP or CLIP tags expressed as fusion partners of transfected genes, allow oligonucleotide conjugations to be performed in fixed cells, with no need for specific affinity reagents. The approach is used to demonstrate induced interactions between the fusion proteins FKBP and FRB by allowing the in situ conjugated oligonucleotides to direct the production of templates for localized rolling circle amplification reactions.

  • 33.
    Gu, Gucci Jijuan
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Friedman, Mikaela
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Ren, Ping
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Torn, Carina
    Fex, Malin
    Hampe, Christiane S.
    Lernmark, Ake
    Landegren, Ulf
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Kamali-Moghaddam, Masood
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Elevated Serum GAD65 and GAD65-GADA Immune Complexes in Stiff Person Syndrome2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 11196Article in journal (Refereed)
    Abstract [en]

    Glutamic acid decarboxylase 65 (GAD65) and autoantibodies specific for GAD65 (GADA) are associated with autoimmune diseases including Stiff Person Syndrome (SPS) and Type 1 diabetes (T1D). GADA is recognized as a biomarker of value for clinical diagnosis and prognostication in these diseases. Nonetheless, it remains medically interesting to develop sensitive and specific assays to detect GAD65 preceding GADA emergence, and to monitor GADA-GAD65 immune complexes in blood samples. In the present study, we developed a highly sensitive proximity ligation assay to measure serum GAD65. This novel assay allowed detection of as little as 0.65 pg/ml GAD65. We were also able to detect immune complexes involving GAD65 and GADA. Both free GAD65 and GAD65-GADA levels were significantly higher in serum samples from SPS patients compared to healthy controls. The proximity ligation assays applied for detection of GAD65 and its immune complexes may thus enable improved diagnosis and better understanding of SPS.

  • 34.
    Gu, Gucci Jijuan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lund, Harald
    Wu, Di
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Blokzijl, Andries
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Classon, Christina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    von Euler, Gabriel
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sunnemark, Dan
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Role of Individual MARK Isoforms in Phosphorylation of Tau at Ser(262) in Alzheimer's Disease2013In: Neuromolecular medicine, ISSN 1535-1084, E-ISSN 1559-1174, Vol. 15, no 3, p. 458-469Article in journal (Refereed)
    Abstract [en]

    The microtubule-affinity regulating kinase (MARK) family consists of four highly conserved members that have been implicated in phosphorylation of tau protein, causing formation of neurofibrillary tangles in Alzheimer's disease (AD). Understanding of roles by individual MARK isoform in phosphorylating tau has been limited due to lack of antibodies selective for each MARK isoform. In this study, we first applied the proximity ligation assay on cells to select antibodies specific for each MARK isoform. In cells, a CagA peptide specifically and significantly inhibited tau phosphorylation at Ser(262) mediated by MARK4 but not other MARK isoforms. We then used these antibodies to study expression levels of MARK isoforms and interactions between tau and individual MARK isoforms in postmortem human brains. We found a strong and significant elevation of MARK4 expression and MARK4-tau interactions in AD brains, correlating with the Braak stages of the disease. These results suggest the MARK4-tau interactions are of functional importance in the progression of AD and the results also identify MARK4 as a promising target for AD therapy.

  • 35.
    Gu, Gucci Jijuan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wu, Di
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lund, Harald
    Dept. of Neuroscience, iMed, CNS and Pain Södertälje, AstraZeneca Research and Development.
    Sunnemark, Dan
    Dept. of Neuroscience, iMed, CNS and Pain Södertälje, AstraZeneca Research and Development.
    Kvist, Alexander
    Antibody Generation Group, Discovery Sciences, iMed, AstraZeneca Research and Development.
    Milner, Roy
    Antibody Generation Group, Discovery Sciences, iMed, AstraZeneca Research and Development.
    Eckersley, Sonia
    Antibody Generation Group, Discovery Sciences, iMed, AstraZeneca Research and Development.
    Nilsson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Agerman, Karin
    Dept. of Neuroscience, iMed, CNS and Pain Södertälje, AstraZeneca Research and Development.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamali‐Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elevated MARK2-Dependent Phosphorylation of Tau in Alzheimer's Disease2013In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 33, no 3, p. 699-713Article in journal (Refereed)
    Abstract [en]

    The appearance of neurofibrillary tangles (NFT), one of the major hallmarks of Alzheimer's disease (AD), is most likely caused by inappropriate phosphorylation and/or dephosphorylation of tau, eventually leading to the accumulation of NFTs. Enhanced phosphorylation of tau on Ser(262) is detected early in the course of the disease and may have a role in the formation of tangles. Several kinases such as microtubule-affinity regulating kinase (MARK), protein kinase A, calcium calmodulin kinase II, and checkpoint kinase 2 are known to phosphorylate tau on Ser(262) in vitro. In this study, we took advantage of the in situ proximity ligation assay to investigate the role of MARK2, one of the four MARK isoforms, in AD. We demonstrate that MARK2 interacts with tau and phosphorylates tau at Ser(262) in stably transfected NIH/3T3 cells expressing human recombinant tau. Staurosporine, a protein kinase inhibitor, significantly reduced the interaction between MARK2 and tau, and also phosphorylation of tau at Ser(262). Furthermore, we observed elevated interactions between MARK2 and tau in post-mortem human AD brains, compared to samples from non-demented elderly controls. Our results from transfected cells demonstrate a specific interaction between MARK2 and tau, as well as MARK2-dependent phosphorylation of tau at Ser(262). Furthermore, the elevated interactions between MARK2 and tau in AD brain sections suggests that MARK2 may play an important role in early phosphorylation of tau in AD, possibly qualifying as a therapeutic target for intervention to prevent disease progression.

  • 36.
    Gullberg, Mats
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gústafsdóttir, Sigrun M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Schallmeiner, Edith
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jarvius, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Bjarnegård, Mattias
    Betsholtz, Christer
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Fredriksson, Simon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Cytokine detection by antibody-based proximity ligation2004In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, no 22, p. 8420-4Article in journal (Other academic)
    Abstract [en]

    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.

  • 37.
    Gustafsdottir, Sigrun M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nordengrahn, Ann
    Fredriksson, Simon
    Wallgren, Per
    Rivera, Esteban
    Schallmeiner, Edith
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Merza, M.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Detection of individual microbial pathogens by proximity ligation2006In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 52, no 6, p. 1152-1160Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Nucleic acid amplification allows the detection of single infectious agents. Protein-based assays, although they provide information on ongoing infections, have substantially less detection sensitivity.

    METHODS: We used proximity ligation reactions to detect proteins on bacteria and virus particles via nucleic acid amplification. Antibodies recognizing viral or bacterial surface proteins were equipped with DNA strands that could be joined by ligation when several antibodies were bound in proximity to surface proteins of individual infectious agents.

    RESULTS: Detection sensitivities similar to those of nucleic acid-based detection reactions were achieved directly in infected samples for a parvovirus and an intracellular bacterium.

    CONCLUSIONS: This method enables detection of ligated DNA strands with good sensitivity by real-time PCR and could be of value for early diagnosis of infectious disease and in biodefense.

  • 38.
    Gustafsdottir, Sigrun M
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Schallmeiner, Edith
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Fredriksson, Simon
    Gullberg, Mats
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Söderberg, Ola
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Jarvius, Malin
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Jarvius, Jonas
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Howell, Mathias
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Proximity ligation assays for sensitive and specific protein analyses.2005In: Anal Biochem, ISSN 0003-2697, Vol. 345, no 1, p. 2-9Article, review/survey (Other (popular scientific, debate etc.))
  • 39.
    Gustafsdottir, Sigrun M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Schlingemann, Jörg
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Rada-Iglesias, Alvaro
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Schallmeiner, Edith
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    In vitro analysis of DNA-protein interactions by proximity ligation2007In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 104, no 9, p. 3067-3072Article in journal (Refereed)
    Abstract [en]

    Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro method for specific and sensitive solution-phase analysis of interactions between proteins and nucleic acids in nuclear extracts, based on the proximity ligation assay. The reagent consumption is very low, and the excellent sensitivity of the assay enables analysis of as few as 1-10 cells. We show that our results are highly reproducible, quantitative, and in good agreement with both EMSA and predictions obtained by using a motif finding software. This assay can be a valuable tool to characterize in-depth the sequence specificity of DNA-binding proteins and to evaluate effects of polymorphisms in known transcription factor binding sites.

  • 40.
    Gustafsdottir, Sigrun M
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wennström, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Fredriksson, Simon
    Schallmeiner, Edith
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hamilton, Andrew D
    Sebti, Said M
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Use of Proximity Ligation to Screen for Inhibitors of Interactions between Vascular Endothelial Growth Factor A and Its Receptors2008In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 54, no 7, p. 1218-1225Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Improved methods are required to screen drug candidates for their influences on protein interactions. There is also a compelling need for miniaturization of screening assays, with attendant reductions in reagent consumption and assay costs. METHODS: We used sensitive, miniaturized proximity ligation assays (PLAs) to monitor binding of vascular endothelial growth factor A (VEGF-A) to 2 of its receptors, VEGFR-1 and VEGFR-2. We measured the effects of proteins and low molecular weight compounds capable of disrupting these interactions and compared the results with those obtained by immunoblot analysis. We analyzed 6 different inhibitors: a DNA aptamer, a mixed DNA/RNA aptamer, a monoclonal VEGF-A neutralizing antibody, a monoclonal antibody directed against VEGFR-2, a recombinant competitive protein, and a low molecular weight synthetic molecule. RESULTS: The PLAs were successful for monitoring the formation and inhibition of VEGF-A-receptor complexes, and the results correlated well with those obtained by measuring receptor phosphorylation. The total PLA time is just 3 hours, with minimal manual work and reagent additions. The method allows evaluation of the apparent affinity [half-maximal inhibitory concentration (IC(50))] from a dose-response curve. CONCLUSIONS: The PLA may offer significant advantages over conventional methods for screening the interactions of ligands with their receptors. The assay may prove useful for parallel analyses of large numbers of samples in the screening of inhibitor libraries for promising agents. The technique provides dose-response curves, allowing IC(50) values to be calculated.

  • 41. Gyarmati, Péter
    et al.
    Conze, Tim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Zohari, Siamak
    LeBlanc, Neil
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Banér, Johan
    Belák, Sándor
    Simultaneous genotyping of all hemagglutinin and neuraminidase subtypes of avian influenza viruses by use of padlock probes2008In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 46, no 5, p. 1747-1751Article in journal (Refereed)
    Abstract [en]

    A subtyping assay for both the hemagglutinin (HA) and neuraminidase (NA) surface antigens of the avian influenza virus (AIV) has been developed. The method uses padlock probe chemistry combined with a microarray output for detection. The outstanding feature of this assay is its capability to designate both the HA and the NA of an AIV sample from a single reaction mixture. A panel of 77 influenza virus strains was tested representing the entire assortment of the two antigens. One hundred percent (77/77) of the samples tested were identified as AIV, and 97% (75/77) were subtyped correctly in accordance with previous examinations performed by classical diagnostic methods. Testing of heterologous pathogens verified the specificity of the assay. This assay is a convenient and practical tool for the study of AIVs, providing important HA and NA data more rapidly than conventional methods.

  • 42.
    Hammond, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wik, Lotta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Deslys, Jean-Philippe
    Comoy, Emmanuel
    Linné, Tommy
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sensitive detection of aggregated prion protein via proximity ligation2014In: Prion, ISSN 1933-6896, E-ISSN 1933-690X, Vol. 8, no 3, p. 261-265Article in journal (Refereed)
    Abstract [en]

    The DNA assisted solid-phase proximity ligation assay (SP-PLA) provides a unique opportunity to specifically detect prion protein (PrP) aggregates by investigating the collocation of three or more copies of the specific protein. We have developed a SP-PLA that can detect PrP aggregates in brain homogenates from infected hamsters even after a 10(7)-fold dilution. In contrast, brain homogenate from uninfected animals did not generate a detectable signal at hundred-fold higher concentration. Using either of the two monoclonal anti-PrP antibodies 3F4 and 6H4 we successfully detected low concentrations of aggregated PrP. The presented results provide a proof of concept that this method might be an interesting tool in the development of diagnostic approaches of prion diseases.

  • 43. Hardenbol, Paul
    et al.
    Banér, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jain, Maneesh
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Namsaraev, Eugeni A.
    Karlin-Neumann, George A.
    Fakhrai-Rad, Hossein
    Ronaghi, Mostafa
    Willis, Thomas D.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Davis, Ronald W.
    Multiplexed genotyping with sequence-tagged molecular inversion probes2003In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 21, no 6, p. 673-8Article in journal (Refereed)
    Abstract [en]

    We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.

  • 44.
    Howell, W. Mathias
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Grundberg, Ida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Faryna, Marta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 7, p. e99-Article in journal (Refereed)
    Abstract [en]

    The current arsenal of molecular tools for site-directed cleavage of single-stranded DNA (ssDNA) is limited. Here, we describe a method for targeted DNA cleavage that requires only the presence of an A nucleotide at the target position. The procedure involves hybridization of a complementary oligonucleotide probe to the target sequence. The probe is designed to create a deliberate G:A mismatch at the desired position of cleavage. The DNA repair enzyme MutY glycosylase recognizes the mismatch structure and selectively removes the mispaired A from the duplex to create an abasic site in the target strand. Addition of an AP-endonuclease, such as Endonuclease IV, subsequently cleaves the backbone dividing the DNA strand into two fragments. With an appropriate choice of an AP-cleaving enzyme, the 3'- and 5'-ends of the cleaved DNA are suitable to take part in subsequent enzymatic reactions such as priming for polymerization or joining by DNA ligation. We define suitable standard reaction conditions for glycosylase/AP-cleaving enzyme (G/AP) cleavage, and demonstrate the use of the method in an improved scheme for in situ detection using target-primed rolling-circle amplification of padlock probes.

  • 45.
    Howell, WM
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Stenberg, Johan
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Detection techniques for single nucleotide polymorphisms.2006In: Nucleic acid testing in human disease, CRC , 2006, p. 139-169Chapter in book (Other scientific)
  • 46.
    Jarvius, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    DNA Skyline: fonts to facilitate visual inspection of nucleic acid sequences2006In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 40, no 6, p. 740-Article in journal (Other academic)
  • 47.
    Jarvius, Jonas
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Teknisk-naturvetenskapliga vetenskapsområdet, Physics, Department of Physics and Materials Science, Materials Science.
    Melin, Jonas
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Technology, Department of Engineering Sciences. Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Teknisk-naturvetenskapliga vetenskapsområdet, Physics, Department of Physics and Materials Science, Materials Science. Materialvetenskap.
    Göransson, Jenny
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Teknisk-naturvetenskapliga vetenskapsområdet, Physics, Department of Physics and Materials Science, Materials Science.
    Göransson, Henrik
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Teknisk-naturvetenskapliga vetenskapsområdet, Physics, Department of Physics and Materials Science, Materials Science.
    Nikolajeff, Fredrik
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Technology, Department of Engineering Sciences. Teknisk-naturvetenskapliga vetenskapsområdet, Physics, Department of Physics and Materials Science, Materials Science. Materialvetenskap.
    Landegren, Ulf
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Teknisk-naturvetenskapliga vetenskapsområdet, Physics, Department of Physics and Materials Science, Materials Science.
    Nilsson, Mats
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Teknisk-naturvetenskapliga vetenskapsområdet, Physics, Department of Physics and Materials Science, Materials Science.
    Amplified Single Molecule Detection in a Thermoplastic Microfluidic System2005In: Poster at microTAS-2005 October 9-13, Boston, USA, 2005Conference paper (Other scientific)
  • 48.
    Jarvius, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Oligonucleotide ligation assay2003In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 212, p. 215-28Article in journal (Refereed)
  • 49.
    Jarvius, Malin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Paulsson, Janna
    Weibrecht, Irene
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Leuchowius, Karl-Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Andersson, Ann-Catrin
    Wählby, Carolina
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis.
    Gullberg, Mats
    Botling, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sjöblom, Tobias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Markova, Boyka
    Östman, Arne
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method2007In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 9, p. 1500-1509Article in journal (Refereed)
    Abstract [en]

    Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor β (PDGFRβ) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRβ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRβ in porcine aortic endothelial cells transfected with the β-receptor, but not in cells transfected with the α-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRβ. We furthermore visualized tyrosine phosphorylated PDGFRβ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

  • 50. Johansson Wensman, Jonas
    et al.
    Leuchowius, Karl-Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Walter and Eliza Hall Institute of Medical Research, Australia .
    Yan, Jiting
    Berg, Anna-Lena
    Bode, Liv
    Ludwig, Hanns
    Belák, Sándor
    Landegren, Ulf
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Berg, Mikael
    Visualization of Borna Disease Virus Protein Interactions with Host Proteins using in situ Proximity Ligation Assay2016In: British journal of virology, ISSN 2055-6128, Vol. 3, no 1, p. 11-23Article in journal (Refereed)
    Abstract [en]

    Borna disease virus type 1 (BDV) comprises highly conserved neurotropic non-segmented negative strand RNA-virus variants causing neurological and behavioral disorders in a wide range of mammalian animals, possibly including humans. Viral persistence in the brain has been frequently observed, however, the exact mechanisms behind BDV’s ability to establish persistence despite a prominent immune response are not known. Here we have used in situ proximity ligation assay (in situ PLA), a selective tool for studying virus-host protein-protein interactions. BDV P (phosphoprotein) and N (nucleoprotein) have previously been reported to interact with several host proteins, thereby interfering with various signaling pathways. In this study, we focused on some of these interactions (BDV P-HMGB1, BDV N/P-Cdc2). First, we used rat glioma cell cultures persistently infected with a laboratory strain of BDV (C6BV) to establish the assay. Next, in situ PLA was applied to detect BDV P in brain tissues of infected animals. Finally, protein-protein interactions were visualized in both C6BV and brain tissues of experimentally as well as naturally infected animals (rat and horse, respectively). BDV proteins and their interactions with host proteins could be shown in cell cultures (HMGB1, Cdc2) and in brain tissues of rat (HMGB1, Cdc2) and horse (Cdc2 only) infected with BDV. In this study, we have for the first time directly visualized protein-protein interactions between BDV and its host, and thereby confirmed previous data to demonstrate findings in cell cultures to be applicable also in experimentally and naturally infected animals.

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