Suspension Bead Research Articles

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.

Micro-Acoustic-Trap (µAT) for microparticle assembly in 3D.

Acoustic tweezers facilitate the manipulation of objects using sound waves. With the current state of the technology one can only control mobility for a single or few microparticles. This article presents a state of the art system where an Acoustic Lens was used for developing a Micro-Acoustic Trap for microparticle assembly in 3D. The model particles, 2 µm diameter polystyrene beads in suspension, were driven via acoustic pressure to form a monolayer at wavelength-defined distances above the substrate defined by the focal point of an Acoustic Lens The transducer was driven at 89 MHz, mixed with 100 ms pulses at a repetition rate of 2 Hz. Beyond a threshold drive amplitude sufficient to overcome Brownian motion, this led to 2D assembly of the microparticles into close-packed rafts >80 µm across (∼5 wavelengths of the carrier wave and >40 particles across). This methodology was further extended to manipulation of live Dictyostelium discoideum amoebae. This approach therefore offers maneuverability in controlling or assembling micrometer-scale objects using continuous or pulsed focused acoustic radiation pressure.

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.

Interleukin-15 and Interleukin-7 are the Major Cytokines to Maintain Endometriosis

Objective: The objective of this study was to evaluate cytokines related to natural killer and T-regulatory cells in endometriotic lesions, peritoneal fluid (PF) and the peripheral blood (PB) of patients with deep infiltrative endometriosis. Study Design: A case-control study was conducted in a tertiary referral hospital. Sixty-four consecutive patients after laparoscopy were divided into 2 groups: with endometriosis (Group A – n = 32) and without endometriosis (Group B – n = 32). Main Outcome Measures: Interleukin (IL)-2, IL-4, IL-7, IL-10, IL-12, IL-15, transforming growth factor β1, and IFNγ concentration was measured using a Luminex^TM multiplex suspension bead array. Tissues from endometriotic lesions of patients with endometriosis and from eutopic endometrium were evaluated, as well as PF and PB of all patients. Results: Compared to the other analyzed groups, IL-15 concentration was significantly higher in the ectopic endometrium and IL-7 in the eutopic endometrium of the endometriosis group (p < 0.05). Compared to endometriosis group, IFNγ, IL-7, and IL-15 were observed to be significantly higher in the PF of the control group, and IL-10 was lower in the control group (p < 0.05). In PB, compared to endometriosis group, IL-4, IL-10, IL-12, IL-15, and IFNγ concentrations were significantly higher in the control group (p < 0.05). Conclusions: Our hypothesis is that deep endometriosis is a disease out of control. This disease’s nature is of progression and invasion of adjacent structures, and proof of this disease state is the disorganized secretion of cytokine regulation and inflammation, which seem to be among the factors responsible for the maintenance of the disease.

The antimicrobial protein S100A12 identified as a potential autoantigen in a subgroup of atopic dermatitis patients

BackgroundAtopic dermatitis (AD) is a complex heterogeneous chronic inflammatory skin disease. Specific IgE antibodies against autoantigens have been observed in a subgroup of AD patients, however, little is known about IgG-auto-reactivity in AD. To investigate the presence of autoreactive IgG antibodies, we performed autoantibody profiling of IgG in patients with AD of different severities and in healthy controls (HC).MethodsFirst, we performed an untargeted screening in plasma samples from 40 severe AD (sAD) patients and 40 HC towards 1152 protein fragments on planar antigen microarrays. Next, based on the findings and addition of more fragments, a targeted antigen suspension bead array was designed to profile a cohort of 50 sAD patients, 123 patients with moderate AD (mAD), and 84 HC against 148 protein fragments representing 96 unique proteins.ResultsForty-nine percent of the AD patients showed increased IgG-reactivity to any of the four antigens representing keratin associated protein 17-1 (KRTAP17-1), heat shock protein family A (Hsp70) member 4 (HSPA4), S100 calcium binding proteins A12 (S100A12), and Z (S100Z). The reactivity was more frequent in the sAD patients (66%) than in those with mAD (41%), whereas only present in 25% of the HC. IgG-reactivity to S100A12, a protein including an antimicrobial peptide, was only observed in AD patients (13/173).ConclusionsAutoantibody profiling of IgG-reactivity using microarray technology revealed an autoantibody-based subgroup in patients with AD. The four identified autoantigens and especially S100A12 could, if characterized further, increase the understanding of different pathogenic mechanisms behind AD and thereby enable better treatment.

Validation of monoplex assays detecting antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus and parvovirus B19 for incorporation into Multiplex Serology.

Serological assays detecting antibodies in serum or plasma samples are useful and versatile instruments to investigate an individual's infection and vaccination history, e.g. for clinical diagnosis, personal risk evaluation, and seroepidemiological studies. Multiplex Serology is a suspension bead array-based high-throughput methodology for simultaneous measurement of antibodies against multiple pathogens in a single reaction vessel, thus economizing sample volume, measurement time, and costs. We developed and validated bead-based pathogen-specific Monoplex Serology assays, i.e. assays including only antigens for the respective pathogen, to detect antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus and parvovirus B19. The developed assays expand the portfolio of existing pathogen-specific bead-based serology assays and can be efficiently incorporated into larger Multiplex Serology panels. The newly developed Monoplex Serology assays consist of only one antigen per infectious agent, expressed as Glutathione S-transferase-fusion proteins in E. coli. Specificity, sensitivity and Cohen's kappa statistics in comparison with routine clinical diagnostic assays were calculated for serum dilutions 1:100 and 1:1000. All pathogen-specific assays were successfully validated at both serum dilutions with the exception of rubella Monoplex Serology which showed impaired sensitivity (57.6%) at dilution 1:1000. Specificities of successfully validated Monoplex Serology assays ranged from 85.6% to 100.0% (median: 91.7%), and sensitivities from 81.3% to 95.8% (median: 90.9%); agreement with the reference assays ranged from substantial to almost perfect (kappa: 0.66-0.86, median: 0.78). Statistical performance and slim assay design enable efficient incorporation of the developed assays into Multiplex Serology.

M71 - HIGH-THROUGHPUT ANTIBODY-BASED PROFILING OF SERUM IN SCHIZOPHRENIA AND BIPOLAR DISORDER PATIENTS: AN INTEGRATIVE GENOMICS-PROTEOMICS PILOT STUDY

Background The identification of biological markers in the peripheral blood for the prediction of the individual clinical outcome has an enormous potential in the frame of personalized medicine. Previous studies have already tried to create predictive models for major psychiatric disorders based on serum protein profiles though with inconclusive results. This study leverages the rich repertoire of biosamples available in the KFO241/PsyCourse cohort to perform a high-throughput antibody-based serum protein profiling. The aim of this pilot study is to establish the feasibility of such an analysis in the serum of these patients. This study also capitalizes on the availability of genomic data to carry out an integrative (genomics/proteomics) profiling. Methods Serum samples of 113 Schizophrenia (SCZ) and 125 Bipolar Disorder (BD) patients belonging to the German KFO241/PsyCourse cohort (www.kfo241.de; www.PsyCourse.de) were included in this study. All biomaterials in this cohort (blood, serum, plasma, DNA, RNA), available at different time points given its longitudinal design, are part of a high-end biobank. The expression of a selected panel of ~100 serum proteins was determined using a set of 155 antibodies in a high-throughput antibody-based assay. This suspension bead array technology enables a multiplexed protein profiling and it leverages the information generated by the Human Protein Atlas (www.proteinatlas.org). Median fluorescent intensities were log-transformed / standardized for downstream analyses. DNA samples were genotyped (Infinium PsychArray) and underwent genotype imputation (1000 Genomes Phase 3 ref. panel). Polygenic Risk Scores (PRS) were calculated by summing up the weighted effect of each SNP contributing to the PRS to obtain an individual estimate of SCZ genetic risk burden. Age, sex and ancestry principal components were used as covariates. Results Multivariate profiles based on the whole panel of quantified proteins did not significantly discriminate between both diagnoses (SCZ and BD). Single-protein ANCOVA analyses revealed, after Bonferroni correction, significant differences in C4B, CFB, VWF, NRG1, BACE1 and other proteins between SCZ and BD samples. High-risk versus Low-risk patients (according to PRS thresholds 5E-8, 5E-2 and 1) did not show a differential level of expression of these proteins. Likewise, polygenic risk did not correlate with the serum level of any of the proteins. Suggestive serum pQTL loci were identified. Discussion Our results confirm the feasibility and potential of this approach using serum samples of psychiatric patients. The observed differences in protein levels between diagnoses warrant replication in independent samples controlling the effect of medication. These results lay the groundwork for large-scale analyses for the discovery of predictive biomarkers of course, outcome and treatment response in SCZ and BD. Such an approach, joining the longitudinal phenotyping and biosample availability at four different time points in the KFO241/PsyCourse cohort with state-of-the-art biomarker quantification methodologies, holds promise for the generation of clinically-relevant predictive models in complex psychiatric disorders.

Array-Based Profiling of Proteins and Autoantibody Repertoires in CSF.

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.

Levels of human proteins in plasma associated with acute paediatric malaria

BackgroundThe intimate interaction between the pathophysiology of the human host and the biology of the Plasmodium falciparum parasite results in a wide spectrum of disease outcomes in malaria. Development of severe disease is associated with a progressively augmented imbalance in pro- and anti-inflammatory responses to high parasite loads and sequestration of parasitized erythrocytes. Although these phenomena collectively constitute common denominators for the wide variety of discrete severe malaria manifestations, the mechanistic rationales behind discrepancies in outcome are poorly understood. Exploration of the human pathophysiological response by variations in protein profiles in plasma presents an excellent opportunity to increase the understanding. This is ultimately required for better prediction, prevention and treatment of malaria, which is essential for ongoing elimination and eradication efforts.ResultsAn affinity proteomics approach was used to analyse 541 paediatric plasma samples collected from community controls and patients with mild or severe malaria in Rwanda. Protein profiles were generated with an antibody-based suspension bead array containing 255 antibodies targetting 115 human proteins. Here, 57 proteins were identified with significantly altered levels (adjusted p-values < 0.001) in patients with malaria compared to controls. From these, the 27 most significant proteins (adjusted p-values < 10−14) were selected for a stringent analysis approach. Here, 24 proteins showed elevated levels in malaria patients and included proteins involved in acute inflammatory response as well as cell adhesion. The remaining three proteins, also implicated in immune regulation and cellular adhesivity, displayed lower abundance in malaria patients. In addition, 37 proteins (adjusted p-values < 0.05) were identified with increased levels in patients with severe compared to mild malaria. This set includes, proteins involved in tissue remodelling and erythrocyte membrane proteins. Collectively, this approach has been successfully used to identify proteins both with known and unknown association with different stages of malaria.ConclusionIn this study, a high-throughput affinity proteomics approach was used to find protein profiles in plasma linked to P. falciparum infection and malaria disease progression. The proteins presented herein are mainly involved in inflammatory response, cellular adhesion and as constituents of erythrocyte membrane. These findings have a great potential to provide increased conceptual understanding of host-parasite interaction and malaria pathogenesis.

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.

Rolling circle amplification integrated with suspension bead array for ultrasensitive multiplex immunodetection of tumor markers

Multiplex detection of ultra-low abundant tumor markers is extremely important for early diagnosis and therapy evaluation. Herein, an ultrasensitive multiplex immunoassay was developed by combination of rolling circle amplification (RCA) and suspension bead array (SBA) technology. Based on a conventional sandwich-type immunoreaction on beads, the detection antibodies were conjugated with DNA primers, so RCA could be implemented to generate long-stranded DNA with abundant repeated sequences allowing for hybridization with fluorochrome-labeled oligonucleotide probes. Thus the fluorescence signal of immunocomplexes on the encoded beads can be greatly enhanced. Using the as-developed immuno-RCA suspension bead array (iRCA-SBA), simultaneous analysis of multiple tumor markers was achieved with the limits of detection of 3.1 pg/mL (∼0.1 pM) for prostate specific antigen (PSA), 9.1 pg/mL (∼50 fM) for carcinoembryonic antigen (CEA), and 0.66 pg/mL (∼9 fM) for α-fetoprotein (AFP), which are two to three orders of magnitude lower than those obtained by the conventional SBA method. The dynamic range were 4.5, 4.7, and 5.5 orders of magnitude for PSA, CEA, and AFP, respectively. Tests on clinical serum samples demonstrate that the tumor marker concentrations measured by the newly developed iRCA-SBA assay agreed well with those obtained by the conventional SBA method. These results indicate that the iRCA-SBA assay significantly increased the detection sensitivity and dynamic range without sacrificing the reliability and accuracy of conventional SBA. Upon the integration with iRCA, SBA could find more applications in the detection of low abundance protein biomarkers for early diagnosis of cancer and other diseases.

Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer

ObjectivesCancer-testis antigens (CTAs) are defined as proteins that are specifically expressed in testis or placenta and their expression is frequently activated in cancer. Due to their ability to induce an immune response, CTAs may serve as suitable targets for immunotherapy. The aim of this study was to evaluate if there is reactivity against CTAs in the plasma of non-small cell lung cancer (NSCLC) patients through the detection of circulating antibodies. Materials and methodsTo comprehensively analyze autoantibodies against CTAs the multiplexing capacities of suspension bead array technology was used. Bead arrays were created with 120 protein fragments, representing 112 CTAs. Reactivity profiles were measured in plasma samples from 133 NSCLC patients and 57 cases with benign lung diseases. ResultsAltogether reactivity against 69 antigens, representing 81 CTAs, was demonstrated in at least one of the analyzed samples. Twenty-nine of the antigens (45 CTAs) demonstrated exclusive reactivity in NSCLC samples. Reactivity against cancer-testis antigen family 47; member A (CT47A) genes, P antigen family member 3 (PAGE3), variable charge X-linked (VCX), melanoma antigen family B1 (MAGEB1), lin-28 homolog B (LIN28B) and chromosome 12 open reading frame 54 (C12orf54) were only found in NSCLC patients at a frequency of 1%–4%. The presence of autoantibodies towards these six antigens was confirmed in an independent group of 34 NSCLC patients. ConclusionWe identified autoantibodies against CTAs in the plasma of lung cancer patients. The reactivity pattern of autoantibodies was higher in cancer patients compared to the benign group, stable over time, but low in frequency of occurrence. The findings suggest that some CTAs are immunogenic and that these properties can be utilized as immune targets.

Xenobiotic Metabolism in Alginate-Encapsulated Primary Human Hepatocytes Over Long Timeframes

Introduction: Application of in vitro-to-in vivo extrapolation to chemical safety assessment has gained significant attention in the past few years. Accurate prediction of hepatic metabolic clearance from in vitro data is critical in this regard as it determines compound pharmacokinetics and exposure at sites of action. To estimate hepatic metabolic clearance, intrinsic clearance (CLint) is typically measured using in vitro systems such as primary hepatocytes or subcellular fractions derived from liver tissues. However, currently available in vitro systems used in these studies often lack long-term metabolic competence, making it challenging to estimate CLint for poorly metabolized and slowly cleared chemicals. Materials and Methods: To address this challenge, we developed a three-dimensional (3D) primary human hepatocyte cell culture system using alginate hydrogels (“alginate bead culture”) with extended viability and metabolic competence. Results: Alginate bead culture generates comparable estimates of metabolism to other in vitro and in vivo systems. In addition, expression of several xenobiotic metabolizing enzymes over long timeframes in beads is comparable to freshly isolated hepatocytes in suspension culture. Discussion: We compare CLint estimated from traditional suspension versus alginate bead culture for two chemicals (S-warfarin and coumarin) and show that alginate bead culture holds promise to increase confidence in estimates of CLint for slowly cleared chemicals. Conclusions: In addition to providing an improved in vitro tool to estimate metabolic clearance for a broad range of chemicals, this simple 3D hepatocyte culture can be adapted to a continuous flow-based bioreactor system allowing repeated exposure experiments over long periods in vitro, tackling an important challenge in in vitro-based chemical safety assessment.

Analysis of factors affecting the variability of a quantitative suspension bead array assay measuring IgG to multiple Plasmodium antigens.

Reducing variability of quantitative suspension array assays is key for multi-center and large sero-epidemiological studies. To maximize precision and robustness of an in-house IgG multiplex assay, we analyzed the effect of several conditions on variability to find the best combination. The following assay conditions were studied through a fractional factorial design: antigen-bead coupling (stock vs. several), sample predilution (stock vs. daily), temperature of incubation of sample with antigen-bead (22°C vs. 37°C), plate washing (manual vs. automatic) and operator expertise (expert vs. apprentice). IgG levels against seven P. falciparum antigens with heterogeneous immunogenicities were measured in test samples, in a positive control and in blanks. We assessed the variability and MFI quantification range associated to each combination of conditions, and their interactions, and evaluated the minimum number of samples and blank replicates to achieve good replicability. Results showed that antigen immunogenicity and sample seroreactivity defined the optimal dilution to assess the effect of assay conditions on variability. We found that a unique antigen-bead coupling, samples prediluted daily, incubation at 22°C, and automatic washing, had lower variability. However, variability increased when performing several couplings and incubating at 22°C vs. 37°C. In addition, no effect of temperature was seen with a unique coupling. The expertise of the operator had no effect on assay variability but reduced the MFI quantification range. Finally, differences between sample replicates were minimal, and two blanks were sufficient to capture assay variability, as suggested by the constant Intraclass Correlation Coefficient of three and two blanks. To conclude, a single coupling was the variable that most consistently reduced assay variability, being clearly advisable. In addition, we suggest having more sample dilutions instead of replicates to increase the likelihood of sample MFIs falling in the linear part of the antigen-specific curve, thus increasing precision.

Affinity proteomic profiling of plasma for proteins associated to pancreatic cancer reveal candidates for patient survival

Cancer-testis antigens (CTAs) are defined as proteins that are specifically expressed in testis or placenta and their expression is frequently activated in cancer. Due to their ability to induce an immune response, CTAs may serve as suitable targets for immunotherapy. The aim of this study was to evaluate if there is reactivity against CTAs in the plasma of non-small cell lung cancer (NSCLC) patients through the detection of circulating antibodies.To comprehensively analyze autoantibodies against CTAs the multiplexing capacities of suspension bead array technology was used. Bead arrays were created with 120 protein fragments, representing 112 CTAs. Reactivity profiles were measured in plasma samples from 133 NSCLC patients and 57 cases with benign lung diseases.Altogether reactivity against 69 antigens, representing 81 CTAs, was demonstrated in at least one of the analyzed samples. Twenty-nine of the antigens (45 CTAs) demonstrated exclusive reactivity in NSCLC samples. Reactivity against cancer-testis antigen family 47; member A (CT47A) genes, P antigen family member 3 (PAGE3), variable charge X-linked (VCX), melanoma antigen family B1 (MAGEB1), lin-28 homolog B (LIN28B) and chromosome 12 open reading frame 54 (C12orf54) were only found in NSCLC patients at a frequency of 1%–4%. The presence of autoantibodies towards these six antigens was confirmed in an independent group of 34 NSCLC patients.We identified autoantibodies against CTAs in the plasma of lung cancer patients. The reactivity pattern of autoantibodies was higher in cancer patients compared to the benign group, stable over time, but low in frequency of occurrence. The findings suggest that some CTAs are immunogenic and that these properties can be utilized as immune targets.

Immunoprofiling of Chlamydia trachomatis using whole-proteome microarrays generated by on-chip in situ expression

Using Chlamydia trachomatis (Ct) as a complex model organism, we describe a method to generate bacterial whole-proteome microarrays using cell-free, on-chip protein expression. Expression constructs were generated by two successive PCRs directly from bacterial genomic DNA. Bacterial proteins expressed on microarrays display antigenic epitopes, thereby providing an efficient method for immunoprofiling of patients and allowing de novo identification of disease-related serum antibodies. Through comparison of antibody reactivity patterns, we newly identified antigens recognized by known Ct-seropositive samples, and antigens reacting only with samples from cervical cancer (CxCa) patients. Large-scale validation experiments using high-throughput suspension bead array serology confirmed their significance as markers for either general Ct infection or CxCa, supporting an association of Ct infection with CxCa. In conclusion, we introduce a method for generation of fast and efficient proteome immunoassays which can be easily adapted for other microorganisms in all areas of infection research.

Multiple optical trapping assisted bead-array based fluorescence assay of free and total prostate-specific antigen in serum

Although suspension bead-based assay technology has been widely used owing to its advantages of high-throughput and microvolume detection, its sensitivity is greatly limited because it detects the fluorescence signal emitted by microbeads for a short time in the flowing fluid. In this work, we present the approach for prostate-specific antigen (PSA) detection of both free PSA (fPSA) and total PSA (tPSA) based on bead-array based fluorescence imaging by combining multiple optical trapping and bead-based bioassays. The polystyrene beads were employed to enrich the targets using the classic sandwich immuno-binding and tagged with fluorescent quantum dots (QDs), and the QDs-tagged beads in suspension were trapped array-by-array using multiple optical tweezers constructed with a diffraction optical element and excited with a 405 nm fiber laser for wide-field fluorescence imaging. The distinctive size information from the image of the trapped beads enabled the sorting of different targets. Moreover, the limits of detection for fPSA and tPSA are 3.8 pg/mL and 2.5 pg/mL respectively with good specificity. More importantly, this strategy was successfully used to detect fPSA and tPSA simultaneously in real serum samples. The high sensitivity, good selectivity, and tiny sample volume make this strategy a promising method for life sciences and clinical applications.

F13. TOWARDS MOLECULAR INSIGHTS INTO PSYCHIATRIC DISORDERS USING AFFINITY PROTEOMICS

BackgroundNumerous studies have shown a correlation between high autoantibody titers and subsequent autoimmune disease in patients with psychiatric disorders compared to healthy individuals. In this study we used a targeted affinity proteomics approach to investigate these autoantibody repertoires. We therefore obtained serum samples from patients diagnosed with various psychiatric disorders and compared these with samples of healthy volunteers. Additionally we used our approach to identify autoantibodies in post mortem brain tissue from patients diagnosed with schizophrenia.MethodsIn this study we utilized a well characterized cohort of patients with psychiatric disorder to study the autoantibody repertoire. From this sample set we analysed more than 600 serum in a first discovery approach. Based on the in-house screening and previous external published studies of autoantibodies within psychiatry we selected a set of 231 protein fragments from the Human Protein Atlas with a length of roughly 80 amino acids. With this selected panel we screened additional 130 post mortem brain tissue samples. Autoantibody profiling was performed using suspension bead array technology and IgG reactivity was measured in patients and controls.ResultsOur findings could indicate altered immune response in patients with psychiatric disorders compared to healthy controls. In our study we identified potential predictive autoimmune signatures. Those were presented with higher IgG reactivity in patients compared to healthy control samples.DiscussionWith our approach we were able to profile autoantibody repertoires in patients with psychiatric disorders. Additionally, we were able to use our approach to profile brain tissue using a multiplexed affinity proteomics approach. By further validating these putative autoimmunity targets, we could gain insights into the autoantigens associated to chronical mental illnesses.

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.

A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common tumors within the oral cavity. Early diagnosis and prognosis tools are urgently needed. This study aimed to investigate the activation of the complement system in OSCC patients as potential biomarker. Therefore, an innovative complement activation array was developed.Characterized antibodies detecting the complement activation specific epitopes C3a, C5a and sC5b-9 along with control antibodies were implemented into a suspension bead array. Human serum from a healthy (n = 46) and OSCC patient (n = 57) cohort were used to investigate the role of complement activation in oral tumor progression. The novel multiplex assay detected C3a, C5a and sC5b-9 from a minimal sample volume of human tears, aqueous humor and blood samples. Limits of detection were 0.04 ng/mL for C3a, 0.03 ng/mL for C5a and 18.9 ng/mL for sC5b-9, respectively. Biological cut-off levels guaranteed specific detections from serum. The mean serum concentration of a healthy control cohort was 680 ng/mL C3a, 70 ng/mL C5a and 2247 ng/mL sC5b-9, respectively. The assay showed an intra-assay precision of 2.9–6.4% and an inter-assay precision of 9.2–18.2%.Increased systemic C5a (p < 0.0001) and sC5b-9 (p = 0.01) concentrations in OSCC patients were determined using the validated multiplex complement assay. Higher C5a concentrations correlated with tumor differentiation and OSCC extension state. Systemic sC5b-9 determination provided a novel biomarker for infiltrating tumor growth and C3a levels were associated with local tumor spreading.Our study suggests that systemic complement activation levels in OSCC patients may be useful to assess disease progression.