Antibodies are naturally occurring in humans, with the function to protect the body from pathogens. Occasionally, antibodies towards the body’s own proteins are produced. These so called autoantibodies are present in healthy individuals but are also highly associated with diseases with autoimmune involvement. Research on autoantibodies in healthy individuals as well as in patients is important to gain knowledge and facilitate prognostics, diagnostics and treatment. However, a method for purification of these antibodies has not previously been described. In the present project, an enrichment procedure of circulating autoantibodies found in human plasma is described. Twenty protein fragments previously known to be highly reactive were attached to magnetic microbeads, enabling autoantibodies from eight human plasma sample pools to be captured. The six antigens with highest shown reactivity were chosen for elution procedure. Using pH alterations and heat treatments, a successful elution and enrichment procedure was developed. With analysis of the eluted autoantibodies, it can be established that the enrichment was successful on multiple sample pools. In the scaled-up procedure, autoantibodies could be enriched in all positive antigen-sample combinations. Concentration measurements indicated amounts of up to 0.23 mg antibodies per ml eluate. This implies sufficient concentrations for further applications of the enriched autoantibodies. / Antikroppar förekommer naturligt i människor, med syftet att skydda kroppen från patogen. I vissa fall skapas av misstag antikroppar som angriper kroppens egna proteiner. Dessa autoantikroppar förekommer hos alla människor, såväl friska som sjuka, men de är också starkt förknippade med autoimmuna sjukdomar. Kunskapen om autoantikroppar hos friska personer och hos patienter är idag begränsad, men fortsatt forskning inom området förväntas i framtiden underlätta prognostik, diagnostik och behandling. Hittills har ingen metod för anrikning av autoantikroppar ur blodplasma beskrivits. I detta projekt beskrivs en anrikningsmetod för autoantikroppar ur blodplasma från människa. Tjugo tidigare kända högreaktiva proteinfragment fästes på magnetiska mikrokulor. Dessa antigen-täckta mikrokulor användes för att fånga in autoantikroppar från åtta plasmaprover. De sex proteinfragment som hade högst reaktivitet i dessa prover valdes ut för elueringsförsök. Eluering genomfördes under basiska följt av sura förhållanden, tillsammans med värmebehandling. Denna elueringsmetod fungerade för anrikning av några autoantikroppar från flera av plasmaproverna. I ett utökat experiment kunde autoantikroppar anrikas ur alla kombinationer av antigen och plasmaprov som förväntades ge signal. Koncentrationen av autoantikroppar i eluaten uppskattades till högst 0.23 mg/ml. Denna koncentration är tillräcklig för flera vanliga metoder där antikroppar används.

As the aging population is increasing worldwide, so is the prevalence of neurodegenerativediseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia(FTD) and amyotrophic lateral sclerosis (ALS). Reliable biomarkers able to aid the diagnosis anddifferentiation of these diseases are needed in order to start the right treatment as early as possible.Due to its representative state of the central nervous system, cerebrospinal fluid (CSF) is afavorable sample material for biomarker discovery within neurodegenerative diseases. Alteredprotein levels of this body fluid might serve as a biomarker, but further validation of earlierfindings is needed. The aim of this project was to validate earlier studies suggesting potentialprotein biomarkers in CSF. From a list of 80 potential biomarkers in the CSF of patient samples,eight were chosen to be included in this validation effort. By utilizing a suspension bead array ina sandwich assay setup, 21 antibodies were tested in an initial screening. Antibody pairs that couldmeasure the protein levels in a dilution dependent manner was further optimized before individualpatient samples were analyzed. Sandwich assays targeting the three proteins Amphiphysin(AMPH), Chitotriosidase-1 (CHIT1) and Beta-synuclein (SNCB) were successfully developed andcorrelated to earlier generated data using a suspension bead array with a single binder setup.Therefore, the earlier findings of elevated levels of AMPH and SNCB in AD patients and CHIT1in ALS patients were successfully validated. / Prevalensen av neurodegenerativa sjukdomar såsom Alzheimers sjukdom (AD), Parkinsonssjukdom (PD), frontallobsdemens (FTD) och amyotrofisk lateralskleros (ALS) ökar i takt med denåldrande populationen. Pålitliga biomarkörer som kan hjälpa till vid diagnostiseringen av dessasjukdomar behövs för att starta rätt behandling så tidigt som möjligt. Ryggmärgsvätska, enkroppsvätska tillhörande det centrala nervsystemet, kan ge en inblick i det centrala nervsystemetstillstånd. Förändrade proteinnivåer i denna kroppsvätska skulle därför kunna fungera sombiomarkörer. Målet i detta projekt var att validera tidigare föreslagna proteinbiomarkörer iryggmärgsvätska. Utifrån en lista av 80 tidigare analyserade proteiner i ryggmärgsvätska hospatienter, inkluderades åtta proteiner i detta valideringsförsök. En antikroppsbaserad så kalladsandwich assay användes i en suspension bead array för att testa 21 stycken antikroppar i ett initialtscreeningsförsök. Antikroppspar som kunde mäta proteinnivåer på ett spädningsberoende vis i detinitiala screeningsförsöket optimerades vidare innan den utvecklade sandwich assayn användes föratt analysera proteinnivåer i individuella prover. Sandwich assays gentemot Amphiphysin(AMPH), Chitotriosidase-1 (CHIT1) och Beta-synuclein (SNCB) kunde bli framtagna ochkorrelerade gentemot tidigare genererat data från en single binder assay på ett framgångsrikt sätt.Projektet kunde därmed validera tidigare fynd som indikerat förhöjda nivåer av AMPH och SNCBi AD patienter, samt förhöjda nivåer av CHIT1 i ALS patienter.

This thesis is about protein profiling in blood-derived samples using suspension bead ar- rays built with protein affinity reagents, and the evaluation of binding characteristics and potential disease relation of such profiles. A central aim of the presented work was to discover and verify disease associated protein profiles in blood-derived samples such as serum or plasma. This was based on immobiliz- ing antigens or antibodies on color-coded beads for a multiplexed analysis. This concept generally allow for a dual multiplexing because hundreds of samples can be screened for hundreds of proteins in a miniaturized and parallelized fashion. At first, protein antigens were used to study humoral immune responses in cattle suffering from a mycoplasma infec- tion (Paper I). Here, the most immunogenic of the applied antigens were identified based on reactivity profiles from the infected cattle, and were combined into an antigen cocktail to serve as a diagnostic assay in a standard ELISA set-up. Next, antibodies and their em- ployment in assays with directly labeled human samples was initiated. This procedure was applied in a study of kidney disorders where screening of plasma resulted in the discovery of a biomarker candidate, fibulin-1 (Paper II). In parallel to the disease related applica- tions, systematic evaluations of the protein profiles were conducted. Protein profiles from 2,300 antibodies were classified on the bases of binding properties in relation to sample heating and stringent washing (Paper III). With a particular focus on heat dependent de- tectability, a method was developed to visualize those proteins that were captured to the beads in an immunoassay by using Western blotting (Paper IV). In conclusion, this thesis presents examples of the possibilities of comparative plasma profiling enabled by protein bead arrays. / <p>QC 20130208</p>

Antibodies play an important role in the natural immune response to invading pathogens. The strong and specific binding to their antigens also make them indispensable tools for research, diagnostics and therapy. This thesis describes the development of methods for characterization of an- tibody specificity and the use of these methods to investigate the polyclonal antibody response after immunization. Paper I describes the development of an epitope-specific serum fractionation technique based on epitope map- ping using overlapping peptides followed by chromatographic separation of polyclonal serum. This technique together with another epitope mapping technique based on bacterial display of protein fragments were then used to generate antibody sandwich pairs (Paper I), investigate epitope variations of repeated immunizations (Paper II) and to determine the ratio of antibodies targeting linear and conformational epitopes of polyclonal antibodies (Paper III). Paper IV describes the optimization of in situ-synthesized high-density peptide arrays for epitope mapping and how different peptide lengths influ- ence epitope detection and resolution. In Paper V we show the development of planar peptide arrays covering the entire human proteome and how these arrays can be used for epitope mapping and off-target binding analysis. In Paper VI we show how polyclonal antibodies targeting linear epitopes can be used for peptide enrichment in a rapid, absolute protein quantification protocol based on mass spectrometry. Altogether these investigations demonstrate the usefulness of peptide arrays for fast and straightforward characterization of antibody specificity. The work also contributes to a deeper understanding of the polyclonal anti- body response obtained after immunization with recombinant protein frag- ments. / <p>QC 20141111</p>

Alzheimers sjukdom (AD, eng. Alzheimer’s disease) upptäcktes för 111 år sedan av Alois Alz-heimer. Idag är det den ledande orsaken till demens hos äldre, och incidencen förväntas öka med befolkningens ökande livslängd. År 2050 förutspås antalet patienter med AD nå 10 miljoner personer [1]. Det har gjorts många försök att angripa AD via dess främsta kännetäcken, såsom plack av beta-amyloid (Aβ), Aβ-oligomerer, och ansamlingar av tau-protein, kallat tau-trassel. Trots att forskning om AD bedrivits i flera årtionden är dess orsak alltjämt okänd.På sistone har det funnits ett fokus på de inflammatoriska komponenterna inom AD. Det finns en utbredd aktivering av immunförsvaret i det centrala nervsystemet hos patienter med AD, men varken dess orsak eller dess roll inom AD är känd. Däremot finns det tydliga tecken på att inflammationen är av autoimmun art. Med detta i åtanke är det tydligt att det finns ett stort behov att utröna auto-immunitetens roll inom AD. I denna forskningsstudie användes proteomik-metoder för att bestämma autoantikroppsprofilerna inom plasma och cerebrospinalvätska (CSF, eng. cerebrospinal fluid) hos AD-patienter och en frisk kontrollgrupp.I denna studie användes par av plasma- och CSF-prover från 23 friska individer och 49 patien-ter. Dessutom inkluderades 2 plasmaprover och 18 CSF-prover från patienter. En 380-faldig och en 314-faldig riktad analys gjordes med hjälp utav suspension bead array-teknologi (SBA). Varje SBA bestod av färgkodade, magnetiska mikrosfärer i suspension, med antigen immobiliserade på kulornas yta. Denna analysmetod användes för att undersöka autoantikropssprofilerna i alla prover. Resul-taten visade en ökad respons från autoantikroppar mot antigenen SLC17A6 (Solute Carrier Family 17 Member 6), MAP1A (Microtubule Associated Protein 1A), och MAP2 (Microtubule Associated Protein 2) i patiener gentemot friska individer. Dock har dessa antigen uppvisat en bred reaktivitet i tidigare, opublicerade studier. Därför behövs ytterligare forskning för att fastställa deras roll inom AD.Dessutom användes paren av plasma- och CSF-prover för att undersöka autoantikroppsprofilernas överrensstämmelse inom varje patient. Det visade sig att korrelationen följde en normalfördelning, med starkare korrelation inom antigen med starkare reaktivitet mot den motsvarande autoantikroppen. Denna studie utgör en av de första storskaliga forskningsstudierna av överrensstämmelsen mellan autoantikroppsprofilerna inom plasma och CSF. / Alzheimer’s disease (AD) was discovered 111 years ago by Alois Alzheimer. Today, it is the leading cause of dementia in elderly, and incidence is expected to increase with life expectancy. By 2050, the number of a˙ected individuals is predicted to reach 10 million [1]. There have been numerous attempts to describe AD by its primary hallmarks, including amyloid plaques, amyloid beta (Aβ) oligomers, and tau tangles. However, despite several decades of intense research, the cause of AD remains unknown.Recently, there has been a focus on the inflammatory components of AD. There is an extensive activation of the immune system within the CNS of AD patients, but neither its cause nor its role in AD is known. However, there are strong indications that the inflammation has an autoimmune character. Considering this, there is an imperative need to examine autoimmunity within AD. In the present study, a proteomic approach was used to determine the autoantibody profiles within plasma and cerebrospinal fluid (CSF) within AD patients and healthy controls.Paired plasma and CSF samples from 23 healthy controls and 49 patients were included in the present study. In addition, 2 plasma samples and 18 CSF samples from patients were included (not paired). One 380-plex and one 314-plex targeted suspension bead array (SBA), each consisting of color-coded magnetic microspheres with immobilized antigens, were used to analyze autoantibody profiles in all samples. The resulting data revealed an increased autoantibody response towards anti-gens SLC17A6 (Solute Carrier Family 17 Member 6), MAP1A (Microtubule Associated Protein 1A), and MAP2 (Microtubule Associated Protein 2) in patients compared to healthy controls. However, as these antigens have displayed wide reactivities in previous, unpublished studies, they require further investigation to determine their role in AD.Furthermore, the paired CSF and plasma samples were used to investigate the correlation of autoantibody profiles within patients. The correlation was found to follow a normal distribution, with correlation being higher in antigens displaying stronger autoantibody reactivity. This work represents one of the first large-scale studies on the correlation of autoantibody profiles in plasma and CSF.

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. / <p>QC 20170302</p>

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP. / QC 20100719

Small intestine neuroendocrine tumors (SI-NETs) and lung carcinoids (LCs) are relatively indolent tumors, which originate from neuroendocrine (NE) cells of the diffuse NE system. Metastases can spread before diagnosis. Thus, potential cures become unavailable, which entitles new biomarker development. Indeed, we aimed at developing Ma2 autoantibodies and olfactory receptor 51E1 (OR51E1) as potential novel biomarkers and exploring other candidate protein markers in patients’ serum. First, we established a sensitive, specific and reliable anti-Ma2 indirect ELISA to distinguish SI-NET patients from healthy controls. We detected longer progression-free and recurrence-free survivals in patients expressing low anti-Ma2 titers. Moreover, a high anti-Ma2 titer was more sensitive than chromogranin A for the risk of recurrence after radical operation of SI-NET patients. We then investigated OR51E1 expression in SI-NETs and LCs. OR51E1 mRNA expression, analyzed by quantitative real-time PCR, was high in microdissected SI-NET cells, in LC cell lines and in frozen LC specimens. Immunohistochemistry (IHC) showed abundant OR51E1 protein expression in SI-NETs. OR51E1 co-expressed with vesicular-monoamine-transporter-1 in the majority of normal and neoplastic enterochromaffin cells. Furthermore, the study on LCs revealed that OR51E1, somatostatin receptor (SSTR) 2, SSTR3, and SSTR5 are expressed in 85%, 71%, 25% and 39% of typical carcinoids (TCs), whereas in 86%, 79%, 43% and 36% of atypical carcinoids (ACs). Based on the proposed IHC scoring system, in the LC cases, where all SSTR subtypes were absent, membrane OR51E1 expression was detected in 10 out of 17 TCs and 1 out of 2 ACs. Moreover, higher OR51E1 scores were detected in 5 out of 6 OctreoScan-negative LC lesions. In addition, the last presented study used a novel suspension bead array, which targeted 124 unique proteins, by using Human Protein Atlas antibodies, to profile biotinylated serum samples from SI-NET patients and healthy controls. We showed 9 proteins, IGFBP2, IGF1, SHKBP1, ETS1, IL1α, STX2, MAML3, EGR3 and XIAP as significant contributors to tumor classification. In conclusion, we proposed Ma2 autoantibodies as a sensitive circulating marker for SI-NET recurrence; OR51E1 as a candidate therapeutic target for SI-NETs; whereas as a novel diagnostic marker for LCs and 9 serum proteins as novel potential SI-NET markers. / 小肠神经内分泌肿瘤(SI-NET)和肺类癌(LC)是起源于不同神经内分泌细胞的生长缓慢的肿瘤。肿瘤往往于诊断前已经转移。这导致目前缺乏有效的治疗方法，同时也使得对于新的生物标记物的研发变得有意义。因此，我们在本论文中分别研究了Ma2自身抗体(抗Ma2)，以及潜在的新型生物标记物嗅觉受体51E1(OR51E1)。我们还探讨了患者血清中的其他候选蛋白标记物。 首先，我们建立了一个灵敏特异而可靠的抗Ma2间接酶联免疫吸附试验，用以区分SI-NET患者组和健康对照组。在表达低滴度抗Ma2的患者中，我们检测到了较长的病情无恶化存活率以及肿瘤无复发存活率。此外，高滴度抗Ma2比嗜铬粒蛋白A更为灵敏地检测到了SI-NET患者根治手术后复发的风险。     接下来，我们研究了SI-NET和LC患者肿瘤中的OR51E1受体蛋白的表达。我们用实时定量PCR技术检测到了OR51E1信使核糖核酸在显微切除的SI-NET肿瘤细胞中，以及在LC细胞系和冷冻LC标本中的高度表达。免疫组化结果显示出OR51E1蛋白在SI-NET肿瘤组织中的高度表达。OR51E1与囊泡单胺转运蛋白1在大多数正常和肿瘤的肠嗜铬细胞中可共表达。 另外，我们针对LC患者的研究显示，OR51E1受体蛋白以及促生长素抑制素受体(SSTR)2，SSTR3和SSTR5分别在85％，71％，25％和39％的典型性肺类癌(TC)，以及86％，79％，43％和36的非典型性肺类癌(AC)中表达。基于我们我提出的免疫组化结果得分系统，在无SSTR表达的LC中，OR51E1蛋白在17个TC中的10个以及2个AC中的1个中呈细胞膜表达。而且，在6个OctreoScan显象呈阴性的LC中，有5个OR51E1免疫组化得分很高。     此外，在本论文最后的一项研究中，我们采用了一种新型的悬浮磁珠阵列技术，通过使用来自于人类蛋白质图谱项目的针对124种独特蛋白质的抗体，对SI-NET患者和健康对照组的用生物素标记过的血清样本进行了分析。结果显示，通过利用9种蛋白，即IGFBP2，IGF1，SHKBP1，ETS1，STX2，IL1α，MAML3，EGR3和XIAP，我们可以显著的对肿瘤进行分类。     综上所述，我们提出Ma2自身抗体可作为一个体液中灵敏的生物标记物用以暗示SI-NET肿瘤的复发; OR51E1受体蛋白可作为一个在SI-NET治疗中所能用及的候选生物靶分子，并在LC中作为一种新型的潜在生物标记物。此外，我们在SI-NET患者血清中检测到了9种新的候选标记物蛋白。

Affinity-based proteomics utilizes affinity binders to detect target proteins in a large-scale manner. This thesis describes a high-throughput method, which enables the search for biomarker candidates in human plasma and serum. A highly multiplexed antibody-based suspension bead array is created by coupling antibodies generated in the Human Protein Atlas project to color-coded beads. The beads are combined for parallel analysis of up to 384 analytes in patient and control samples. This provides data to compare protein levels from the different groups. In paper I osteoporosis patients are compared to healthy individuals to find disease-linked proteins. An untargeted discovery screening was conducted using 4608 antibodies in 16 cases and 6 controls. This revealed 72 unique proteins, which appeared differentially abundant. A validation screening of 91 cases and 89 controls confirmed that the protein autocrine motility factor receptor (AMFR) is decreased in the osteoporosis patients. Paper II investigates the risk proteome of inflammatory bowel disease (IBD). Antibodies targeting 209 proteins corresponding to 163 IBD genetic risk loci were selected. To find proteins related to IBD or its subgroups, sera from 49 patients with Crohn’s disease, 51 with ulcerative colitis and 50 matched controls were analyzed. From these targeted assays, the known inflammation-related marker serum amyloid protein A (SAA) was shown to be elevated in the IBD cases. In addition, the protein laccase (multi-copper oxidoreductase) domain containing 1 (LACC1) was found to be decreased in the IBD subjects. In conclusion, assays using affinity-based bead arrays were developed and applied to screen human plasma and serum samples in two disease contexts. Untargeted and targeted screening strategies were applied to discover disease-associated proteins. Upon further validation, these potential biomarker candidates could be valuable in future disease studies. / <p>QC 20180412</p>

Chronic pain affects 20 % of the global population, causes suffering, is difficult to treat, and constitutes a large economic burden for society. So far, the characterization of molecular mechanisms of chronic pain-like behaviors in animal models has not translated into effective treatments. In this thesis, consisting of five studies, pain patient biofluids were analyzed with modern proteomic methods to identify biomarker candidates that can be used to improve our understanding of the pathophysiology chronic pain and lead to more effective treatments. Paper I is a proof of concept study, where a multiplex solid phase-proximity ligation assay (SP-PLA) was applied to cerebrospinal fluid (CSF) for the first time. CSF reference protein levels and four biomarker candidates for ALS were presented. The investigated proteins were not altered by spinal cord stimulation (SCS) treatment for neuropathic pain. In Paper II, patient CSF was explored by dimethyl and label-free mass spectrometric (MS) proteomic methods. Twelve proteins, known for their roles in neuroprotection, nociceptive signaling, immune regulation, and synaptic plasticity, were identified to be associated with SCS treatment of neuropathic pain. In Paper III, proximity extension assay (PEA) was used to analyze levels of 92 proteins in serum from patients one year after painful disc herniation. Patients with residual pain had significantly higher serum levels of 41 inflammatory proteins. In Paper IV, levels of 55 proteins were analyzed by a 100-plex antibody suspension bead array (ASBA) in CSF samples from two neuropathic pain patient cohorts, one cohort of fibromyalgia patients and two control cohorts. CSF protein profiles consisting of levels of apolipoprotein C1, ectonucleotide pyrophosphatase/phosphodiesterase family member 2, angiotensinogen, prostaglandin-H2 D-isomerase, neurexin-1, superoxide dismutases 1 and 3 were found to be associated with neuropathic pain and fibromyalgia. In Paper V, higher CSF levels of five chemokines and LAPTGF-beta-1were detected in two patient cohorts with neuropathic pain compared with healthy controls. In conclusion, we demonstrate that combining MS proteomic and multiplex antibody-based methods for analysis of patient biofluid samples is a viable approach for discovery of biomarker candidates for the pathophysiology and treatment of chronic pain. Several biomarker candidates possibly reflecting systemic inflammation, lipid metabolism, and neuroinflammation in different pain conditions were identified for further investigation. / Uppsala Berzelii Technology Centre for Neurodiagnostics