| 1. |
- Bremer, Hanna, et al.
(author)
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ILF2 and ILF3 are autoantigens in canine systemic autoimmune disease
- 2018
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In: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 8
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Journal article (peer-reviewed)abstract
- Dogs can spontaneously develop complex systemic autoimmune disorders, with similarities to human autoimmune disease. Autoantibodies directed at self-antigens are a key feature of these autoimmune diseases. Here we report the identification of interleukin enhancer-binding factors 2 and 3 (ILF2 and ILF3) as autoantigens in canine immune-mediated rheumatic disease. The ILF2 autoantibodies were discovered in a small, selected canine cohort through the use of human protein arrays; a method not previously described in dogs. Subsequently, ILF3 autoantibodies were also identified in the same cohort. The results were validated with an independent method in a larger cohort of dogs. ILF2 and ILF3 autoantibodies were found exclusively, and at a high frequency, in dogs that showed a speckled pattern of antinuclear antibodies on immunofluorescence. ILF2 and ILF3 autoantibodies were also found at low frequency in human patients with SLE and Sjogren's syndrome. These autoantibodies have the potential to be used as diagnostic biomarkers for canine, and possibly also human, autoimmune disease.
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| 2. |
- Hellström, Cecilia, et al.
(author)
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High-density serum/plasma reverse phase protein arrays
- 2017
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In: Serum/Plasma Proteomics. - New York, NY : Humana Press. ; , s. 229-238
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Book chapter (peer-reviewed)abstract
- In-depth exploration and characterization of human serum and plasma proteomes is an attractive strategy for the identification of potential prognostic or diagnostic biomarkers. The possibility of analyzing larger numbers of samples in a high-throughput fashion has markedly increased with affinity-based microarrays, thus providing higher statistical power to these biomarker studies. Here, we describe a protocol for high-density serum and plasma reverse phase protein arrays (RPPAs). We demonstrate how a biobank of 12,392 samples was immobilized and analyzed on a single microarray slide, allowing high-quality profiling of abundant target proteins across all samples in one assay.
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| 3. |
- Pin, Elisa, et al.
(author)
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Array-based profiling of proteins and autoantibody repertoires in CSF
- 2019
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In: Cerebrospinal Fluid (CSF) Proteomics. - New York, NY : Humana Press Inc.. ; , s. 303-318
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Book chapter (peer-reviewed)abstract
- Protein profiling enabled through affinity proteomics represents a powerful strategy for analysis of complex samples such as human body fluids. Cerebrospinal fluid (CSF) is the proximal fluid of the central nervous system and is commonly analyzed in the context of neurological diseases. Through the presence of brain-derived proteins, this fluid can offer insight into the physiological state of the brain. Here, we describe multiplex and flexible protein and autoantibody profiling approaches using suspension bead arrays. Through minimal sample processing, these methods enable high-throughput analysis of hundreds of samples and proteins in one single assay and thereby provide powerful approaches for discovery of disease-associated proteins and autoantigens.
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| 4. |
- Qundos, Ulrika, et al.
(author)
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Affinity proteomics discovers decreased levels of AMFR in plasma from Osteoporosis patients
- 2016
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In: PROTEOMICS - Clinical Applications. - : Wiley-Blackwell. - 1862-8346 .- 1862-8354. ; 10:6, s. 681-690
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Journal article (peer-reviewed)abstract
- Purpose: Affinity proteomic approaches by antibody bead arrays enable multiplexed analysis of proteins in body fluids. In the presented study, we investigated blood plasma within osteoporosis to discovery differential protein profiles and to propose novel biomarkers candidates for subsequent studies. Experimental design: Starting with 4608 antibodies and plasma samples from 22 women for an untargeted screening, a set of 72 proteins were suggested for further analysis. Complementing these with targets from literature and other studies, a targeted bead array of 180 antibodies was built to profile for 92 proteins in plasma samples of 180 women from two independent population-based studies. Results: Differential profiles between osteoporosis patients and matched controls were discovered for 12 proteins in at least one of the two study sets. Among these targets, the levels of autocrine motility factor receptor (AMFR) were concordantly lower in plasma of female osteoporosis patients. Subsequently, verification of anti-AMFR antibody selectivity was conducted using high-density peptide and protein arrays, and Western blotting. Conclusions and clinical relevance: Further validation in additional study sets will be needed to determine the clinical value of the observed decrease in AMFR plasma levels in osteoporosis patients, but AMFR may aid our understanding of disease mechanisms and could support existing tools for diagnosis and monitoring of patient mobility within osteoporosis.
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| 5. |
- Sierra-Sanchez, Alvaro, et al.
(author)
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Screening and Validation of Novel Biomarkers in Osteoarticular Pathologies by Comprehensive Combination of Protein Array Technologies
- 2017
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In: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 16:5, s. 1890-1899
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Journal article (peer-reviewed)abstract
- Osteoarthritis (OA) is one of the most prevalent articular diseases. The identification of proteins closely associated with the diagnosis, progression, prognosis, and treatment response is dramatically required for this pathology. In this work, differential serum protein profiles have been identified in OA and rheumatoid arthritis (RA) by antibody arrays containing 151 antibodies against 121 antigens in a cohort of 36 samples. Then the identified differential serum protein profiles have been validated in a larger cohort of 282 samples. The overall immunoreactivity is higher in the pathological situations in comparison with the controls. Several proteins have been identified as biomarker candidates for OA and RA. Most of these biomarker candidates are proteins related to inflammatory response, lipid metabolism, or bone and extracellular matrix formation, degradation, or remodeling.
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| 6. |
- Sjöberg, Ronald, et al.
(author)
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Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling
- 2016
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In: New Biotechnology. - : Elsevier. - 1871-6784 .- 1876-4347. ; 33:5, s. 582-592
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Journal article (peer-reviewed)abstract
- High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt (TM) Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt (TM) Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions.
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| 7. |
- Sjöberg, Ronald, et al.
(author)
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High-density antigen microarrays for the assessment of antibody selectivity and off-target binding
- 2018
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In: Epitope Mapping Protocols. - New York, NY : Humana Press Inc.. - 9781493978397 ; , s. 231-238
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Book chapter (peer-reviewed)abstract
- With the increasing availability of collections of antibodies, their evaluation in terms of binding selectivity becomes an important but challenging task. Planar antigen microarrays are very suitable tools to address this task and provide a powerful proteomics platform for the characterization of the binding selectivity of antibodies toward thousands of antigens in parallel. In this chapter, we describe our in-house developed procedures for the generation of high-density planar antigen microarrays with over 21,000 features. We also provide the details of the assay protocol, which we routinely use for the assessment of binding selectivity of the polyclonal antibodies generated within the Human Protein Atlas.
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| 8. |
- Sjöberg, Ronald, 1974-
(author)
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On Generation and Applications of High-Density Protein Microarrays
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Affinity proteomics has experienced rapid development over the last two decades and one of the most promising platforms to emerge are the protein microarrays. The combination of affinity reagents and miniaturisation enables assays for simultaneous high throughput and sensitive protein analysis. Due to the combination of these desrable properties, a multitude of protein array platforms for rapid and efficient study of proteomes and protein interactions are in use today. Although the protein microarray field has more than two decades of history to look back on the development of new protein microarray platforms continues to this day and beyond.In the paper I in this thesis, a microarray of eluates from dried blood spot samples collected from neonates were designed and utilised for detection of complement factor 3 (C3) deficiency. The data acquired from the microarrays platform were compared to C3 levels obtained through enzyme-linked immunosorbant assay (ELISA), and the microarray assay were found to separate the C3 deficient samples from the controls. The conclusion of this investigation was that the microarray platform would be suitable for high-throughput screening of C3 deficiency in neonates. Paper II outlines the work in developing a multiplex platform for validation of affinity reagents. A set of 398 affinity binders, originating from five research groups, were profiled against 432 antigens and representing both polyclonal rabbit antibodies, monoclonal mouse antibodies, and recombinant single-chain variable fragments. Approximately 50% of the binders were found to preferably recognise their intended target while 10% of the binders did not generate any, or low, signals with their respective targets. For paper III, a reverse phase array (RPPA) platform using fluorescence-based detection of IgA deficiency in over 2.000 samples where validated on a label-free detection system and ELISA. The data from the label-free platform and the RPPA were found agree well with each other while data from ELISA did with neither of them. It was found that the label-free platform proved to be well-suited for detection of IgA in serum. Paper IV describes one of the world’s largest protein microarrays containing 21.120 recombinant protein fragments. We describe some of the possible applications of these large-scale arrays, such as binding profiles for the validation of antibodies with 11.520 and 21.120 recombinant proteins, as well as screening for autoimmunity in human serum samples.
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| 9. |
- Ssengendo, Ronald, et al.
(author)
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Computation of the Gravimetric Quasigeoid Model over Uganda Using the KTH Method
- 2015
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Conference paper (other academic/artistic)abstract
- The gravimetric quasigeoid can be determined either directly by Stokes formula or indirectly by computing the geoid first and then determining the quasigeoid-to-geoid separation which is then used to determine the quasigeoid. This paper presents the computational results of the gravimetric quasigeoid model over Uganda (UGQ2014) based on the later technique. UGQ2014 was derived from the Uganda Gravimetric Geoid Model (UGG2014) which was computed by the technique of Least Squares Modification of Stokes formula with additive corrections commonly called the KTH Method. UGG2014 was derived from sparse terrestrial gravity data from the International gravimetric Bureau, the 3 arc second SRTM ver4.1 Digital Elevation Model and the GOCE-only geopotential model GO_CONS_GCF_2_TIM_R5. The quasigeoid-to geoid separation was then computed from the Earth Gravitational Model 2008 (EGM08) complete to degree 2160 of spherical harmonics together with the global topographic model DTM2006.0 also complete to degree 2160.Another aim of this paper is to compare the approximate and strict formulas of computing the quasigeoid-to-geoid separation and evaluate their effects on the final quasigeoid model. Using 10 GNSS/levelling data points distributed over Uganda, the RMS fit of the quasigeoid model based on the approximate formula are 27 cm and 10 cm before and after a 4-parameter fit, respectively. Similarly, the RMS fit of the model based on the strict formula are 15 cm and 6 cm, respectively. The results show the improvement to the final quasigeoid model brought about by using the strict formula to model more effectively the terrain in the vicinity of the computation point. With an accuracy of 6 cm, UGQ2014 represents significant progress towards the computation of a final gravimetric geoid over Uganda which can be used with GNSS/levelling. However, with more data especially terrestrial gravity data and GNSS/levelling we anticipate that the accuracy of gravimetric quasigeoid modelling will improve in future.
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| 10. |
- Ssengendo, Ronald, et al.
(author)
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The Uganda Gravimetric Geoid Model computed by the KTH Method
- 2015
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In: Journal of Geodetic Science. - : De Gruyter Open. - 2081-9919 .- 2081-9943. ; 5:2, s. 35-46
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Journal article (peer-reviewed)abstract
- For many developing countries such as Uganda, precise gravimetric geoid determination is hindered by the low quantity and quality of the terrestrial gravity data. With only one gravity data point per 65 km2, gravimetric geoid determination in Uganda appears an impossible task. However, recent advances in geoid modelling techniques coupled with the gravity-field anomalies from the Gravity Field and Steady-State Ocean Circulation Explorer (GOCE) satellite mission have opened new avenues for geoid determination especially for areas with sparse terrestrial gravity. The present study therefore investigates the computation of a gravimetric geoid model over Uganda (UGG2014) using the Least Squares Modification of Stokes formula with additive corrections. UGG2014 was derived from sparse terrestrial gravity data from the International Gravimetric Bureau, the 3 arc second SRTM ver4.1 Digital Elevation Model from CGIAR-CSI and the GOCE-only global geopotential model GO_CONS_GCF_2_TIM_R5. To compensate for the missing gravity data in the target area, we used the surface gravity anomalies extracted from the World Gravity Map 2012. Using 10 Global Navigation Satellite System (GNSS)/levelling data points distributed over Uganda, the RMS fit of the gravimetric geoid model before and after a 4-parameter fit is 11 cm and 7 cm respectively. These results show that UGG2014 agrees considerably better with GNSS/levelling than any other recent regional/global gravimetric geoid model. The results also emphasize the significant contribution of the GOCE satellite mission to the gravity field recovery, especially for areas with very limited terrestrial gravity data. With an RMS of 7 cm, UGG2014 is a significant step forward in the modelling of a “1-cm geoid” over Uganda despite the poor quality and quantity of the terrestrial gravity data used for its computation.
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| 11. |
- Zhou, Xiamo, 1990-, et al.
(author)
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Thiol–ene–epoxy thermoset for low-temperature bonding to biofunctionalized microarray surfaces
- 2017
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In: Lab on a Chip. - : Royal Society of Chemistry. - 1473-0197 .- 1473-0189. ; 17:21, s. 3672-3681
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Journal article (peer-reviewed)abstract
- One way to improve the sensitivity and throughput of miniaturized biomolecular assays is to integrate microfluidics to enhance the transport efficiency of biomolecules to the reaction sites. Such microfluidic integration requires bonding of a prefabricated microfluidic gasket to an assay surface without destroying its biological activity. In this paper we address the largely unmet challenge to accomplish a proper seal between a microfluidic gasket and a protein surface, with maintained biological activity and without contaminating the surface or blocking the microfluidic channels. We introduce a novel dual cure polymer resin for the formation of microfluidic gaskets that can be room-temperature bonded to a range of substrates using only UVA light. This polymer is the first polymer that features over a month of shelf life between the structure formation and the bonding, moreover the fully cured polymer gaskets feature the following set of properties suitable for microfluidics: high stiffness, which prevents microfluidic channel collapse during handling; very limited absorption of biomolecules; and no significant leaching of uncured monomers. We describe the novel polymer resin and its characteristics, study through FT-IR, and demonstrate its use as microfluidic well-arrays bonded onto protein array slides at room temperature followed by multiplexed immunoassays. The results confirm maintained biological activity and show high repeatability between protein arrays. This new approach for integrating microfluidic gaskets to biofunctionalised surfaces has the potential to improve sample throughput and decrease manufacturing costs for miniaturized biomolecular systems.
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