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Antibody-based profiling of cerebrospinal fluid within multiple sclerosis
KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-0056-1313
KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-7843-2960
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
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2013 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, no 15, 2256-2267 p.Article in journal (Refereed) Published
Abstract [en]

Antibody suspension bead arrays have proven to enable multiplexed and high-throughput protein profiling in unfractionated plasma and serum samples through a direct labeling approach. We here describe the development and application of an assay for protein profiling of cerebrospinal fluid (CSF). While setting up the assay, systematic intensity differences between sample groups were observed that reflected inherent sample specific total protein amounts. Supplementing the labeling reaction with BSA and IgG diminished these differences without impairing the apparent sensitivity of the assay. We also assessed the effects of heat treatment on the analysis of CSF proteins and applied the assay to profile 43 selected proteins by 101 antibodies in 339 CSF samples from a multiple sclerosis (MS) cohort. Two proteins, GAP43 and SERPINA3 were found to have a discriminating potential with altered intensity levels between sample groups. GAP43 was detected at significantly lower levels in secondary progressive MS compared to early stages of MS and the control group of other neurological diseases. SERPINA3 instead was detected at higher levels in all MS patients compared to controls. The developed assay procedure now offers new possibilities for broad-scale protein profiling of CSF within neurological disorders.

Place, publisher, year, edition, pages
2013. Vol. 13, no 15, 2256-2267 p.
Keyword [en]
Antibody microarrays, Biomarker discovery, Cerebrospinal fluid, Multiplexed proteomics technology, Protein arrays, Proteome profiling
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-134062DOI: 10.1002/pmic.201200580ISI: 000327008300007Scopus ID: 2-s2.0-84881230169OAI: oai:DiVA.org:kth-134062DiVA: diva2:664555
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceVinnovaKnut and Alice Wallenberg Foundation
Note

QC 20131115

Available from: 2013-11-15 Created: 2013-11-15 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Neuroproteomic profiling of human body fluids
Open this publication in new window or tab >>Neuroproteomic profiling of human body fluids
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis provides results from affinity based studies where human body fluids were profiled to find markers for neurological diseases. Both proteins and autoantibodies were analysed using microarray technologies that can profile hundreds of analytes and hundreds of samples in parallel using small sample volumes. A central element in this work was to develop and apply new methods to study cerebrospinal fluid (CSF), which is the fluid in direct contact with the brain. CSF contains proteins reflecting the physiological state of the central nervous system and therefore offers a unique insight into proteins associated to neurological disorders. As a complement to CSF, bloodderived samples such as serum and plasma, were also investigated as these represent potential sources of disease related proteins. The work presented here summarises the development of assay protocols to study protein and autoantibodies in CSF and blood using planar and bead-based microarrays.

In Paper I, an antibody-based protocol was developed to enable multiplexed protein profiling in CSF. The protocol was then applied for a first analysis within multiple sclerosis (MS) patients. In Paper II, the results were further evaluated in additional CSF as well as plasma samples. Based on the CSF analysis we found two proteins associated to MS; GAP43, a protein related to disease progression and SERPINA3, a protein involved in inflammation. In addition, four other proteins; IRF8, METTL14, IL7 and SLC30A7, were found to have altered plasma levels between the patient groups. The expression of these proteins were further investigated by immunofluorescent staining of human brain tissue, revealing differential localisation of proteins in diseased and healthy brain. In Paper III, a study on extensive protein profiling of plasma in the context of another neurodegenerative disorder, amyotrophic lateral sclerosis (ALS), is described. The levels of three proteins, namely NEFM, RGS18 and SCL25A20, were found to be elevated in ALS patients compared to controls. Among these, NEFM also indicated association to disease subtype as the levels were elevated in patients with definite compared to suspected diagnosis.

In addition to antibodies, we also utilised antigens on microarrays to screen for the presence of autoantibodies in body fluids. In Paper IV, a strategy for this analysis was developed using protein fragments and two types of microarrays. This strategy was then applied for profiling of the autoantibody repertoire of MS patients, revealing 51 protein fragments with potential disease relevance. Interestingly, comparison of plasma and CSF samples obtained from the same patients indicated high concordance of antibodies between the two body fluids. In Paper V, a similar strategy was applied to narcolepsy, another neurological disorder. Our investigation of antibodies in serum revealed higher reactivity towards METTL22, NT5C1A and TMEM134 compared to controls in two independent sample materials.

In conclusion, the presented work constitutes a framework of proteomic assays for enhanced exploration of proteins and autoantibodies in neuroscience. Moreover, we have reported identification of several potential disease markers to be further investigated within neurological disorders.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. viii, 67 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:2
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-158944 (URN)978-91-7595-402-8 (ISBN)
Public defence
2015-02-06, Rockefellersalen, KI, Solna, 09:00 (English)
Opponent
Supervisors
Note

QC 20150116

Available from: 2015-01-16 Created: 2015-01-15 Last updated: 2015-01-16Bibliographically approved
2. Affinity assays for profiling disease-associated proteins in human plasma
Open this publication in new window or tab >>Affinity assays for profiling disease-associated proteins in human plasma
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affinity-based proteomics offers opportunities for the discovery and validation of disease-associated proteins in human body fluids. This thesis describes the use of antibody-based immunoassays for multiplexed analysis of proteins in human plasma, serum and cerebrospinal fluid (CSF). This high-throughput method was applied with the objective to identify proteins associated to clinical variables. The main work in this thesis was conducted within the diseases of multiple sclerosis and malignant melanoma, as well as mammographic density, a risk factor for breast cancer.

The suspension bead array (SBA) technology has been the main method for the work presented in this thesis (Paper I-IV). SBA assays and other affinity proteomic technologies were introduced for protein profiling of sample material obtained from clinical collaborators and biobanks. Perspectives on the validation of antibody selectivity by means of e.g. immuno-capture mass spectrometry are also provided.

Paper I describes the development and application of a protocol for multiplexed pro- tein profiling of CSF. The analysis of 340 CSF samples from patients with multiple sclerosis and other neurological disease revealed proteins with potential association to disease progression (GAP43) and inflammation (SERPINA3). Paper II continued on this work with an extended investigation of more than 1,000 clinical samples and included both plasma and CSF collected from the same patients. Comparison of disease subtypes and controls revealed five plasma proteins of potential diagnostic relevance, such as IRF8 and GAP43. The previously reported associations for GAP43 and SERPINA3 in CSF was confirmed. Subsequent immunohistochemical analysis of post-mortem brain tissue revealed differential protein expression in disease affected areas. In Paper III, 150 serum samples from patients with cutaneous malignant melanoma were analyzed. Protein profiles from antibody bead arrays suggested three proteins (RGN, MTHFD1L, STX7) of differential abundance between patients with no disease recurrence and low tumor thickness (T-stage 1 and 2) compared to patients with high tumor thickness (T-stage 3 and 4) and disease recurrence. We observed MTHFD1L expression in tissue of a majority of patients, while expression of STX7 in melanoma tissue had been reported previously. Paper IV describes the analysis of protein in plasma in relation to mammographic breast density (MD), one of the strongest risk factors for the development of breast cancers. More than 1,300 women without prior history of breast cancer were screened. Linear associations to MD in two independent sample sets were found for 11 proteins, which are expressed in the breast and involved in tissue homeostasis, DNA repair, cancer development and/or progression in MD.

In conclusion, this thesis describes the use of multiplexed antibody bead arrays for protein profiling of serum, plasma and CSF, and it shortlists disease associated proteins for further validation studies. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. 89 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:6
Keyword
proteomics, affinity proteomics, immunoassay, antibody microarray, suspension bead array, protein profiling, immuno-capture, plasma, serum, cerebrospinal fluid, biomarker discovery, multiple sclerosis, malignant melanoma, mammographic breast density
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-202616 (URN)978-91-7729-297-5 (ISBN)
Public defence
2017-03-31, Inghesalen, KI, Tomtebodavägen 18A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20170302

Available from: 2017-03-02 Created: 2017-03-02 Last updated: 2017-03-06Bibliographically approved

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Häggmark, AnnaAyoglu, BurcuUhlén, MathiasSchwenk, Jochen M.

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