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Affinity Arrays for Profiling Proteins and Autoantibody Repertoires
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-7843-2960
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , xi, 159 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:12
National Category
Biomedical Laboratory Science/Technology
Identifiers
URN: urn:nbn:se:kth:diva-150398ISBN: 978-91-7595-209-3 (print)OAI: oai:DiVA.org:kth-150398DiVA: diva2:742805
Public defence
2014-09-05, Inghesalen, Karolinska Institute, Tomtebodavägen 18 A, Solna, 10:00 (English)
Opponent
Supervisors
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140902

Available from: 2014-09-02 Created: 2014-09-02 Last updated: 2014-09-02Bibliographically approved
List of papers
1. Autoantibody profiling in multiple sclerosis using arrays of human protein fragments
Open this publication in new window or tab >>Autoantibody profiling in multiple sclerosis using arrays of human protein fragments
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2013 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 9, 2657-2672 p.Article in journal (Refereed) Published
Abstract [en]

Profiling the autoantibody repertoire with large antigen collections is emerging as a powerful tool for the identification of biomarkers for autoimmune diseases. Here, a systematic and undirected approach was taken to screen for profiles of IgG in human plasma from 90 individuals with multiple sclerosis related diagnoses. Reactivity pattern of 11,520 protein fragments (representing ̃38% of all human protein encoding genes) were generated on planar protein microarrays built within the Human Protein Atlas. For more than 2,000 antigens IgG reactivity was observed, among which 64% were found only in single individuals. We used reactivity distributions among multiple sclerosis subgroups to select 384 antigens, which were then reevaluated on planar microarrays, corroborated with suspension bead arrays in a larger cohort (n = 376) and confirmed for specificity in inhibition assays. Among the heterogeneous pattern within and across multiple sclerosis subtypes, differences in recognition frequencies were found for 51 antigens, which were enriched for proteins of transcriptional regulation. In conclusion, using protein fragments and complementary high-throughput protein array platforms facilitated an alternative route to discovery and verification of potentially disease-associated autoimmunity signatures, that are now proposed as additional antigens for large-scale validation studies across multiple sclerosis biobanks.

Keyword
autoantibody, immunoglobulin G, adult, aged, article, controlled study, female, human, major clinical study, male, multiple sclerosis, priority journal, protein microarray, transcription regulation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-133813 (URN)10.1074/mcp.M112.026757 (DOI)000330536400021 ()2-s2.0-84884389479 (Scopus ID)
Funder
VinnovaKnut and Alice Wallenberg FoundationAFA InsuranceSwedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20131204

Available from: 2013-12-04 Created: 2013-11-11 Last updated: 2017-12-06Bibliographically approved
2. The calcium-activated chloride channel anoctamine 2 as an autoimmune component of multiple sclerosis
Open this publication in new window or tab >>The calcium-activated chloride channel anoctamine 2 as an autoimmune component of multiple sclerosis
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-150381 (URN)
Note

QS 2014

Available from: 2014-09-02 Created: 2014-09-02 Last updated: 2014-09-02Bibliographically approved
3. Bead Arrays for Antibody and Complement Profiling Reveal Joint Contribution of Antibody Isotypes to C3 Deposition
Open this publication in new window or tab >>Bead Arrays for Antibody and Complement Profiling Reveal Joint Contribution of Antibody Isotypes to C3 Deposition
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 5, e96403- p.Article in journal (Refereed) Published
Abstract [en]

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen beta and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen b peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism.

National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-147429 (URN)10.1371/journal.pone.0096403 (DOI)000336656000073 ()2-s2.0-84900387101 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140630

Available from: 2014-06-30 Created: 2014-06-27 Last updated: 2017-12-05Bibliographically approved
4. Affinity Proteomic Profiling of Plasma, Cerebrospinal Fluid, and Brain Tissue within Multiple Sclerosis
Open this publication in new window or tab >>Affinity Proteomic Profiling of Plasma, Cerebrospinal Fluid, and Brain Tissue within Multiple Sclerosis
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2014 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 11, 4607-4619 p.Article in journal (Refereed) Published
Abstract [en]

The brain is a vital organ and because it is well shielded from the outside environment, possibilities for noninvasive analysis are often limited. Instead, fluids taken from the spinal cord or circulatory system are preferred sources for the discovery of candidate markers within neurological diseases. In the context of multiple sclerosis (MS), we applied an affinity proteomic strategy and screened 22 plasma samples with 4595 antibodies (3450 genes) on bead arrays, then defined 375 antibodies (334 genes) for targeted analysis in a set of 172 samples and finally used 101 antibodies (43 genes) on 443 plasma as well as 573 cerebrospinal spinal fluid (CSF) samples. This revealed alteration of protein profiles in relation to MS subtypes for IRF8, IL7, METTL14, SLC30A7, and GAP43. Respective antibodies were subsequently used for immunofluorescence on human post-mortem brain tissue with MS pathology for expression and association analysis. There, antibodies for IRF8, IL7, and METTL14 stained neurons in proximity of lesions, which highlighted these candidate protein targets for further studies within MS and brain tissue. The affinity proteomic translation of profiles discovered by profiling human body fluids and tissue provides a powerful strategy to suggest additional candidates to studies of neurological disorders.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2014
Keyword
antibodies, suspension bead arrays, plasma, CSF, brain tissue, immunofluorescence, multiple sclerosis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-150383 (URN)10.1021/pr500609e (DOI)000344636500012 ()25231264 (PubMedID)2-s2.0-84908890596 (Scopus ID)
Funder
Swedish Research CouncilAFA InsuranceScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation
Note

QC 20141212

Updated from manuscript to article in journal.

Available from: 2014-09-02 Created: 2014-09-02 Last updated: 2017-12-05Bibliographically approved
5. Affinity proteomics within rare diseases: a BIO-NMD study for blood biomarkers of muscular dystrophies
Open this publication in new window or tab >>Affinity proteomics within rare diseases: a BIO-NMD study for blood biomarkers of muscular dystrophies
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2014 (English)In: EMBO Molecular Medicine, ISSN 1757-4676, E-ISSN 1757-4684, Vol. 6, no 7, 918-936 p.Article in journal (Refereed) Published
Abstract [en]

Despite the recent progress in the broad-scaled analysis of proteins in body fluids, there is still a lack in protein profiling approaches for biomarkers of rare diseases. Scarcity of samples is the main obstacle hindering attempts to apply discovery driven protein profiling in rare diseases. We addressed this challenge by combining samples collected within the BIO-NMD consortium from four geographically dispersed clinical sites to identify protein markers associated with muscular dystrophy using an antibody bead array platform with 384 antibodies. Based on concordance in statistical significance and confirmatory results obtained from analysis of both serum and plasma, we identified eleven proteins associated with muscular dystrophy, among which four proteins were elevated in blood from muscular dystrophy patients: carbonic anhydrase III (CA3) and myosin light chain 3 (MYL3), both specifically expressed in slow-twitch muscle fibers and mitochondrial malate dehydrogenase 2 (MDH2) and electron transfer flavo-protein A (ETFA). Using age-matched sub-cohorts, 9 protein profiles correlating with disease progression and severity were identified, which hold promise for the development of new clinical tools for management of dystrophinopathies.

Keyword
antibody-based proteomics, disease severity biomarkers, Duchenne muscular dystrophy, plasma profilling, protein profiling
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-149211 (URN)10.15252/emmm.201303724 (DOI)000339094500005 ()2-s2.0-84903740009 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme, 241665VinnovaKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140819

Available from: 2014-08-19 Created: 2014-08-18 Last updated: 2017-12-05Bibliographically approved

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