Antibody Suspension Bead Arrays Research Articles

The cerebrospinal fluid proteome of preterm infants predicts neurodevelopmental outcome.

BackgroundSurvival rate increases for preterm infants, but long-term neurodevelopmental outcome predictors are lacking. Our primary aim was to determine whether a specific proteomic profile in cerebrospinal fluid (CSF) of preterm infants differs from that of term infants and to identify novel biomarkers of neurodevelopmental outcome in preterm infants.MethodsTwenty-seven preterm infants with median gestational age 27 w + 4 d and ten full-term infants were enrolled prospectively. Protein profiling of CSF were performed utilizing an antibody suspension bead array. The relative levels of 178 unique brain derived proteins and inflammatory mediators, selected from the Human Protein Atlas, were measured.ResultsThe CSF protein profile of preterm infants differed from that of term infants. Increased levels of brain specific proteins that are associated with neurodevelopment and neuroinflammatory pathways made up a distinct protein profile in the preterm infants. The most significant differences were seen in proteins involved in neurodevelopmental regulation and synaptic plasticity, as well as components of the innate immune system. Several proteins correlated with favorable outcome in preterm infants at 18–24 months corrected age. Among the proteins that provided strong predictors of outcome were vascular endothelial growth factor C, Neurocan core protein and seizure protein 6, all highly important in normal brain development.ConclusionOur data suggest a vulnerability of the preterm brain to postnatal events and that alterations in protein levels may contribute to unfavorable neurodevelopmental outcome.

Proteomic profiles in cerebrospinal fluid predicted death and disability in term infants with perinatal asphyxia: A pilot study.

AimPerinatal asphyxia, resulting in hypoxic‐ischaemic encephalopathy (HIE), has been associated with high mortality rates and severe lifelong neurodevelopmental disabilities. Our aim was to study the association between the proteomic profile in cerebrospinal fluid (CSF) and the degree of HIE and long‐term outcomes.MethodsWe prospectively enrolled 18‐term born infants with HIE and 10‐term born controls between 2000 and 2004 from the Karolinska University Hospital. An antibody suspension bead array and FlexMap3D analysis was used to characterise 178 unique brain‐derived and inflammation associated proteins in their CSF.ResultsIncreased CSF concentrations of several brain‐specific proteins were observed in the proteome of HIE patients compared with the controls. An upregulation of neuroinflammatory pathways was also noted and this was confirmed by pathway analysis. Principal component analysis revealed a gradient from favourable to unfavourable HIE grades and outcomes. The proteins that provided strong predictors were structural proteins, including myelin basic protein and alpha‐II spectrin. The functional proteins included energy‐related proteins like neuron‐specific enolase and synaptic regulatory proteins. Increased CSF levels of 51 proteins correlated with adverse outcomes in infants with HIE.ConclusionBrain‐specific proteins and neuroinflammatory mediators in CSF may predict HIE degrees and outcomes after perinatal asphyxia.

Plasma Protein Levels Analysis in Multiple Sclerosis Sardinian Families Identified C9 and CYP24A1 as Candidate Biomarkers.

Here we investigate protein levels in 69 multiple sclerosis (MS) cases and 143 healthy controls (HC) from twenty Sardinian families to search for promising biomarkers in plasma. Using antibody suspension bead array technology, the plasma levels of 56 MS-related proteins were obtained. Differences between MS cases and HC were estimated using Linear Mixed Models or Linear Quantile Mixed Models. The proportion of proteins level variability, explained by a set of 119 MS-risk SNPs as to the literature, was also quantified. Higher plasma C9 and CYP24A1 levels were found in MS cases compared to HC (p < 0.05 after Holm multiple testing correction), with protein level differences estimated as, respectively, 0.53 (95% CI: 0.25, 0.81) and 0.42 (95% CI: 0.19, 0.65) times plasma level standard deviation measured in HC. Furthermore, C9 resulted in both statistically significantly higher relapsing-remitting MS (RRMS) and secondary-progressive MS (SPMS) compared to HC, with SPMS showing the highest differences. Instead, CYP24A1 was statistically significantly higher only in RRMS as compared to HC. Respectively, 26% (95% CI: 10%, 44%) and 16% (95% CI: 9%, 39%) of CYP24A1 and C9 plasma level variability was explained by known MS-risk SNPs. Our results highlight C9 and CYP24A1 as potential biomarkers in plasma for MS and allow us to gain insight into molecular disease mechanisms.

Serum Proteomic Profiling in Rheumatoid Arthritis by Antibody Suspension Bead Arrays.

The versatility of protein microarrays provides researchers with a wide variety of possibilities to address proteomic studies. Therefore, protein microarrays are becoming very useful tools to identify candidate biomarkers in human body fluids for disease states such as rheumatoid arthritis (RA). In RA serum, there is a high prevalence of rheumatoid factor (RF), which is an antibody with high specificity against Fc portion of IgG. The presence of RF, in particular RF-IgM, has the great potential to interfere with antibody-based immunoassays by nonspecifically binding capture antibodies. Because of this concern, we describe a procedure to reduce the interference of RF-IgM on RA serum protein profiling approaches based on multiplexed antibody suspension bead arrays.

CSF levels of apolipoprotein C1 and autotaxin found to associate with neuropathic pain and fibromyalgia.

ObjectiveNeuropathic pain and fibromyalgia are two common and poorly understood chronic pain conditions that lack satisfactory treatments, cause substantial suffering and societal costs. Today, there are no biological markers on which to base chronic pain diagnoses, treatment choices or to understand the pathophysiology of pain for the individual patient. This study aimed to investigate cerebrospinal fluid (CSF) protein profiles potentially associated with fibromyalgia and neuropathic pain.MethodsCSF samples were collected from 25 patients with neuropathic pain (two independent sets, n=14 patients for discovery, and n=11 for verification), 40 patients with fibromyalgia and 134 controls without neurological disease from two different populations. CSF protein profiling of 55 proteins was performed using antibody suspension bead array technology.ResultsWe found increased levels of apolipoprotein C1 (APOC1) in CSF of neuropathic pain patients compared to controls and there was a trend for increased levels also in fibromyalgia patients. In addition, levels of ectonucleotide pyrophosphatase family member 2 (ENPP2, also referred to as autotaxin) were increased in the CSF of fibromyalgia patients compared to all other groups including patients with neuropathic pain.ConclusionThe increased levels of APOC1 and ENPP2 found in neuropathic pain and fibromyalgia patients may shed light on the underlying mechanisms of these conditions. Further investigation is required to elucidate their role in maintaining pain and other main symptoms of these disorders.

Plasma protein profiling reveals candidate biomarkers for multiple sclerosis treatment.

Multiple sclerosis (MS) treatment options have improved significantly over the past decades, but the consequences of MS can still be devastating and the needs for monitoring treatment surveillance are considerable. In the current study we used affinity proteomics technology to identify potential biomarkers which could ultimately be used to as facilitate treatment decisions. We profiled the intra-individual changes in the levels of 59 target proteins using an antibody suspension bead array in serial plasma samples from 44 MS patients during treatment with natalizumab followed by fingolimod. Nine proteins showed decreasing plasma levels during natalizumab treatment, with PEBP1 and RTN3 displaying the most significant changes. Protein levels remained stable during fingolimod treatment for both proteins. The decreasing PEBP1 levels during natalizumab treatment could be validated using ELISA and replicated in an independent cohort. These results support the use of this technology as a high throughput method of identifying potentially useful biomarkers of MS treatment.

Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann–Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10−9, 3 × 10−6, and 5 × 10−6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.

Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease

Detailed knowledge of protein changes in cerebrospinal fluid (CSF) across healthy and diseased individuals would provide a better understanding of the onset and progression of neurodegenerative disorders.In this study, we selected 20 brain-enriched proteins previously identified in CSF by antibody suspension bead arrays (SBA) to be potentially biomarkers for Alzheimer's disease (AD) and verified these using an orthogonal approach. We examined the same set of 94 CSF samples from patients affected by AD (including preclinical and prodromal), mild cognitive impairment (MCI), non-AD dementia and healthy individuals, which had previously been analyzed by SBA. Twenty-eight parallel reaction monitoring (PRM) assays were developed and 13 of them could be validated for protein quantification.Antibody profiles were verified by PRM. For seven proteins, the antibody profiles were highly correlated with the PRM results (r > 0.7) and GAP43, VCAM1 and PSAP were identified as potential markers of preclinical AD. In conclusion, we demonstrate the usefulness of targeted mass spectrometry as a tool for the orthogonal verification of antibody profiling data, suggesting that these complementary methods can be successfully applied for comprehensive exploration of CSF protein levels in neurodegenerative disorders.

Targeted Analysis of Serum Proteins Encoded at Known Inflammatory Bowel Disease Risk Loci

BackgroundFew studies have investigated the blood proteome of inflammatory bowel disease (IBD). We characterized the serum abundance of proteins encoded at 163 known IBD risk loci and tested these proteins for their biomarker discovery potential.MethodsBased on the Human Protein Atlas (HPA) antibody availability, 218 proteins from genes mapping at 163 IBD risk loci were selected. Targeted serum protein profiles from 49 Crohn’s disease (CD) patients, 51 ulcerative colitis (UC) patients, and 50 sex- and age-matched healthy individuals were obtained using multiplexed antibody suspension bead array assays. Differences in relative serum abundance levels between disease groups and controls were examined. Replication was attempted for CD-UC comparisons (including disease subtypes) by including 64 additional patients (33 CD and 31 UC). Antibodies targeting a potentially novel risk protein were validated by paired antibodies, Western blot, immuno-capture mass spectrometry, and epitope mapping.ResultsBy univariate analysis, 13 proteins mostly related to neutrophil, T-cell, and B-cell activation and function were differentially expressed in IBD patients vs healthy controls, 3 in CD patients vs healthy controls and 2 in UC patients vs healthy controls (q < 0.01). Multivariate analyses further differentiated disease groups from healthy controls and CD subtypes from UC (P < 0.05). Extended characterization of an antibody targeting a novel, discriminative serum marker, the laccase (multicopper oxidoreductase) domain containing 1 (LACC1) protein, provided evidence for antibody on-target specificity.ConclusionsUsing affinity proteomics, we identified a set of IBD-associated serum proteins encoded at IBD risk loci. These candidate proteins hold the potential to be exploited as diagnostic biomarkers of IBD.

Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density

BackgroundMammographic breast density is one of the strongest risk factors for breast cancer, but molecular understanding of how breast density relates to cancer risk is less complete. Studies of proteins in blood plasma, possibly associated with mammographic density, are well-suited as these allow large-scale analyses and might shed light on the association between breast cancer and breast density.MethodsPlasma samples from 1329 women in the Swedish KARMA project, without prior history of breast cancer, were profiled with antibody suspension bead array (SBA) assays. Two sample sets comprising 729 and 600 women were screened by two different SBAs targeting a total number of 357 proteins. Protein targets were selected through searching the literature, for either being related to breast cancer or for being linked to the extracellular matrix. Association between proteins and absolute area-based breast density (AD) was assessed by quantile regression, adjusting for age and body mass index (BMI).ResultsPlasma profiling revealed linear association between 20 proteins and AD, concordant in the two sets of samples (p < 0.05). Plasma levels of seven proteins were positively associated and 13 proteins negatively associated with AD. For eleven of these proteins evidence for gene expression in breast tissue existed. Among these, ABCC11, TNFRSF10D, F11R and ERRF were positively associated with AD, and SHC1, CFLAR, ACOX2, ITGB6, RASSF1, FANCD2 and IRX5 were negatively associated with AD.ConclusionsScreening proteins in plasma indicates associations between breast density and processes of tissue homeostasis, DNA repair, cancer development and/or progression in breast cancer. Further validation and follow-up studies of the shortlisted protein candidates in independent cohorts will be needed to infer their role in breast density and its progression in premenopausal and postmenopausal women.

Affinity Proteomics Exploration of Melanoma Identifies Proteins in Serum with Associations to T-Stage and Recurrence

BACKGROUND: Blood-based proteomic profiling may aid and expand our understanding of diseases and their different phenotypes. The aim of the presented study was to profile serum samples from patients with malignant melanoma using affinity proteomic assays to describe proteins in the blood stream that are associated to stage or recurrence of melanoma. MATERIAL AND METHODS: Multiplexed protein analysis was conducted using antibody suspension bead arrays. A total of 232 antibodies against 132 proteins were selected from (i) a screening with 4595 antibodies and 32 serum samples from melanoma patients and controls, (ii) antibodies used for immunohistochemistry, (iii) protein targets previously related with melanoma. The analysis was performed with 149 serum samples from patients with malignant melanoma. Antibody selectivity was then assessed by Western blot, immunocapture mass spectrometry, and epitope mapping. Lastly, indicative antibodies were applied for IHC analysis of melanoma tissues. RESULTS: Serum levels of regucalcin (RGN) and syntaxin 7 (STX7) were found to be lower in patients with both recurring tumors and a high Breslow's thickness (T-stage 3/4) compared to low thickness (T-stage 1/2) without disease recurrence. Serum levels of methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) were instead elevated in sera of T3/4 patients with recurrence. The analysis of tissue sections with S100A6 and MTHFD1L showed positive staining in a majority of patients with melanoma, and S100A6 was significantly associated to T-stage. CONCLUSIONS: Our findings provide a starting point to further study RGN, STX7, MTHFD1L and S100A6 in serum to elucidate their involvement in melanoma progression and to assess a possible contribution to support clinical indications.

Discovery of circulating proteins associated to knee radiographic osteoarthritis

Currently there are no sufficiently sensitive biomarkers able to reflect changes in joint remodelling during osteoarthritis (OA). In this work, we took an affinity proteomic approach to profile serum samples for proteins that could serve as indicators for the diagnosis of radiographic knee OA. Antibody suspension bead arrays were applied to analyze serum samples from patients with OA (n = 273), control subjects (n = 76) and patients with rheumatoid arthritis (RA, n = 244). For verification, a focused bead array was built and applied to an independent set of serum samples from patients with OA (n = 188), control individuals (n = 83) and RA (n = 168) patients. A linear regression analysis adjusting for sex, age and body mass index (BMI) revealed that three proteins were significantly elevated (P < 0.05) in serum from OA patients compared to controls: C3, ITIH1 and S100A6. A panel consisting of these three proteins had an area under the curve of 0.82 for the classification of OA and control samples. Moreover, C3 and ITIH1 levels were also found to be significantly elevated (P < 0.05) in OA patients compared to RA patients. Upon validation in additional study sets, the alterations of these three candidate serum biomarker proteins could support the diagnosis of radiographic knee OA.

Multidimensional Normalization to Minimize Plate Effects of Suspension Bead Array Data.

Enhanced by the growing number of biobanks, biomarker studies can now be performed with reasonable statistical power by using large sets of samples. Antibody-based proteomics by means of suspension bead arrays offers one attractive approach to analyze serum, plasma, or CSF samples for such studies in microtiter plates. To expand measurements beyond single batches, with either 96 or 384 samples per plate, suitable normalization methods are required to minimize the variation between plates. Here we propose two normalization approaches utilizing MA coordinates. The multidimensional MA (multi-MA) and MA-loess both consider all samples of a microtiter plate per suspension bead array assay and thus do not require any external reference samples. We demonstrate the performance of the two MA normalization methods with data obtained from the analysis of 384 samples including both serum and plasma. Samples were randomized across 96-well sample plates, processed, and analyzed in assay plates, respectively. Using principal component analysis (PCA), we could show that plate-wise clusters found in the first two components were eliminated by multi-MA normalization as compared with other normalization methods. Furthermore, we studied the correlation profiles between random pairs of antibodies and found that both MA normalization methods substantially reduced the inflated correlation introduced by plate effects. Normalization approaches using multi-MA and MA-loess minimized batch effects arising from the analysis of several assay plates with antibody suspension bead arrays. In a simulated biomarker study, multi-MA restored associations lost due to plate effects. Our normalization approaches, which are available as R package MDimNormn, could also be useful in studies using other types of high-throughput assay data.

Elevated levels of circulating CDH5 and FABP1 in association with human drug-induced liver injury.

Background & AimsThe occurrence of drug‐induced liver injury (DILI) is a major issue in all phases of drug development. To identify novel biomarker candidates associated with DILI, we utilised an affinity proteomics strategy, where antibody suspension bead arrays were applied to profile plasma and serum samples from human DILI cases and controls.MethodsAn initial screening was performed using 4594 randomly selected antibodies, representing 3450 human proteins. Resulting candidate proteins together with proposed DILI biomarker candidates generated a DILI array of 251 proteins for subsequent target analysis and verifications. In total, 1196 samples from 241 individuals across four independent cohorts were profiled: healthy volunteers receiving acetaminophen, patients with human immunodeficiency virus and/or tuberculosis receiving treatment, DILI cases originating from a wide spectrum of drugs, and healthy volunteers receiving heparins.ResultsWe observed elevated levels of cadherin 5, type 2 (CDH5) and fatty acid‐binding protein 1 (FABP1) in DILI cases. In the two longitudinal cohorts, CDH5 was elevated already at baseline. FABP1 was elevated after treatment initiation and seemed to respond more rapidly than alanine aminotransferase (ALT). The elevations were verified in the DILI cases treated with various drugs. In the heparin cohort, CDH5 was stable over time whereas FABP1 was elevated.ConclusionsThese results suggest that CDH5 may have value as a susceptibility marker for DILI. FABP1 was identified as a biomarker candidate with superior characteristics regarding tissue distribution and kinetics compared to ALT but likely with limited predictive value for the development of severe DILI. Further studies are needed to determine the clinical utility of the proposed markers.

Identification of a serum protein biomarker panel for the diagnosis of knee osteoarthritis

s / Osteoarthritis and Cartilage 24 (2016) S8eS62 S23 knee criteria and included features related to osteophytes, cartilage damage, bone marrow lesions, subchondral cysts and labral lesions. Logistic regression analysis was used to evaluate the association of FAI with moderate cartilage damage ( 2), moderate BML ( 2) and MRI-OA. Analyses were adjusted for age, sex, body mass index and family history of OA and, in secondary analyses, also for physical activity. Results: Subjects in the MRI study (n 1⁄4 182) had mean age 43.5 years, mean BMI 26.8, mean WOMAC pain 14.4 and 65.4% were female. FAI was present in 48.9% and hip pain in 51.7%. Cartilage score 2, BML 2 and MRI-OA were present in 11.0%, 9.9% and 8.9%, respectively. FAI was not significantly associated with cartilage damage 2 (OR 2.0, 95% CI 0.7e5.5) and this relationship remained non-significant after adjustment for physical activity (OR 1.8, 95% CI 0.6e5.2). There was a statistically significant association of FAI with BML 2 (OR 4.7, 95% CI 1.4e16.0) and with MRI-OA (OR 3.4, 95% CI 1.1e12.3). Additional adjusting for physical activity did not change the findings for BML (OR 4.2, 95% CI 1.4e14.7) and MRI-OA (OR 2.9, 95% CI 0.9e10.6), although the latter was only of borderline statistical significance. Conclusions: In this population-based cross-sectional study of young adults, the risk of moderate BML and MRI-defined OA was significantly increased in subjects with radiographic FAI compared to those without radiographic FAI. No association was found for FAI with moderate cartilage damage. These findings suggest that FAI is linked to early osteoarthritic changes onMRI in young adults. Whether BML is a precursor to future cartilage damage and radiographic OA requires longitudinal evaluation of this cohort. 27 IDENTIFICATION OF A SERUM PROTEIN BIOMARKER PANEL FOR THE DIAGNOSIS OF KNEE OSTEOARTHRITIS L. Lourido y, B. Ayoglu z, J. Fern andez-Tajes x, F. Henjes z, J.M. Schwenk z, C. Ruiz-Romero y, P. Nilsson z, F.J. Blanco y. yRheumatology Div., ProteoRed/ISCIII Proteomics Group, INIBIC e Hospital Universitario de A Coru~ na, Spain; zAffinity Proteomics, SciLifeLab, Sch. of Biotechnology, KTH-Royal Institute of Technology, Stockholm, Sweden; xWellcome Trust Ctr. For Human Genetics, McCarthy's Group, University of Oxford, Oxford, United Kingdom Purpose: Osteoarthritis (OA) is the most common rheumatic disease. Despite the active research in the OA biomarker field, no single protein is sufficiently reliable for its use in OA diagnosis. This is mainly due to the lack of validation studies in large populations, making the findings questionable to be considered as robust biomarkers for the OA joint turnover. In this study we addressed a large-scale study with the aim of search for proteins that could serve as potential indicators of knee OA. Methods: Antibody suspension bead arrays were applied to profile serum samples from patients with knee OA (n 1⁄4 288) with different Kellgren-Lawrence (K/L) scores and compare to patients with rheumatoid arthritis (RA, n 1⁄4 288), psoriatic arthritis (PsA, n 1⁄4 288) and healthy control (n 1⁄4 96) subjects with non-radiographic knee osteoarthritis. The serum protein content was labelled and protein profiles obtained using 174 antibodies from the Human Protein Atlas targeting 78 different proteins. A focused bead array was then built to further profile 46 selected protein targets as a replication of the first findings and also to validate these results in an independent cohort of serum samples composed by patients with OA (n 1⁄4 196), RA (n 1⁄4 192), PsA (n1⁄4 192) and healthy control individuals (n1⁄4 92). The panel of proteins proposed and analysed for this study was selected based on previous internal protein profiling studies in the context of rheumatic diseases. For the statistical analysis, a lineal regression analysis adjusting by sex, age and body mass index (BMI) was applied to observe differences in protein profiles between compared groups and these were denoted statistically significant if they concordantly revealed p.values 2-fold increase in the risk of incident ROA, while sarcopenia alone had no increased risk, compared with those who were non-obese non-sarcopenic (Table 1). This association of obesity persisted after adjustment for body weight. Similar results were

Plasma profiling reveals three proteins associated to amyotrophic lateral sclerosis.

ObjectiveAmyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease leading to muscular paralysis and death within 3–5 years from onset. Currently, there are no reliable and sensitive markers able to substantially shorten the diagnosis delay. The objective of the study was to analyze a large number of proteins in plasma from patients with various clinical phenotypes of ALS in search for novel proteins or protein profiles that could serve as potential indicators of disease.MethodsAffinity proteomics in the form of antibody suspension bead arrays were applied to profile plasma samples from 367 ALS patients and 101 controls. The plasma protein content was directly labeled and protein profiles obtained using 352 antibodies from the Human Protein Atlas targeting 278 proteins. A focused bead array was then built to further profile eight selected protein targets in all available samples.ResultsDisease-associated significant differences were observed and replicated for profiles from antibodies targeting the proteins: neurofilament medium polypeptide (NEFM), solute carrier family 25 (SLC25A20), and regulator of G-protein signaling 18 (RGS18).InterpretationUpon further validation in several independent cohorts with inclusion of a broad range of other neurological disorders as controls, the alterations of these three protein profiles in plasma could potentially provide new molecular markers of disease that contribute to the quest of understanding ALS pathology.

Affinity proteomics reveals elevated muscle proteins in plasma of children with cerebral malaria.

Systemic inflammation and sequestration of parasitized erythrocytes are central processes in the pathophysiology of severe Plasmodium falciparum childhood malaria. However, it is still not understood why some children are more at risks to develop malaria complications than others. To identify human proteins in plasma related to childhood malaria syndromes, multiplex antibody suspension bead arrays were employed. Out of the 1,015 proteins analyzed in plasma from more than 700 children, 41 differed between malaria infected children and community controls, whereas 13 discriminated uncomplicated malaria from severe malaria syndromes. Markers of oxidative stress were found related to severe malaria anemia while markers of endothelial activation, platelet adhesion and muscular damage were identified in relation to children with cerebral malaria. These findings suggest the presence of generalized vascular inflammation, vascular wall modulations, activation of endothelium and unbalanced glucose metabolism in severe malaria. The increased levels of specific muscle proteins in plasma implicate potential muscle damage and microvasculature lesions during the course of cerebral malaria.

Biomarkers of reactive resistance and early disease progression during chemotherapy plus bevacizumab treatment for colorectal carcinoma.

Molecular markers for predicting or monitoring the efficacy of bevacizumab in patients with metastatic colorectal cancer (mCRC) remain to be identified. We have now measured the serum concentrations of 25 angiogenesis-related molecules with antibody suspension bead array systems for 25 mCRC patients both before and during treatment in a previously reported phase II trial of FOLFIRI chemotherapy plus bevacizumab. The serum concentration of vascular endothelial growth factor-A (VEGF-A) decreased after the onset of treatment (P < 0.0001), whereas that of placental growth factor increased (P < 0.0001). Significant differences in the levels of several factors (such as VEGF-A, soluble VEGF receptor-2, and interleukin-8) were apparent between responders and nonresponders during treatment. The rapid and pronounced decrease in serum VEGF-A level after treatment onset was apparent in all subjects and was independent of the baseline concentration. However, four of nine nonresponders showed a subsequent early increase in the serum VEGF-A level. Our results thus suggest that an early increase in the serum VEGF-A concentration after the initial decrease is a potential predictive marker of a poor response and reactive resistance to bevacizumab plus chemotherapy.

Selectivity analysis of single binder assays used in plasma protein profiling

The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries.

Antibody‐based profiling of cerebrospinal fluid within multiple sclerosis

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.