Background Diagnostic biomarkers are decision-making tools in clinical lab routine and are of growing importance for clinical management of patients. In autoimmune diseases, one class of biomarkers are autoantibodies (AAB) directed against human autoantigens. Apart from diagnostic antigens used in clinical routine, additional AAB reactivities to more than 100 human antigens are described in literature. Obviously, the autoimmune profile of humans covers a huge number of AABs, which display an enormous resource to identify novel marker candidates. Objectives Here we describe a new screening platform SeroTag for screening of novel AABs in autoimmune diseases. By offering thousands of human antigens to serum samples of several autoimmune diseases a comprehensive landscape shall be drawn to identify diseases and disease subgroups according to their intrinsic, highly differentiated autoantibody pattern. Methods SeroTag utilizes over 7,000 human proteins as antigen collection in bead-based suspension arrays (Luminex FlexMap 3D) to allow for high throughput serum sample processing with high accuracy, followed by standard and advanced data mining procedures. We screened over 4,000 serum samples from patients with autoimmune diseases such as SLE (n= >500), SSc (n= >250), RA (n= >500), and healthy individuals (n= >350) to confirm known and to discover novel autoantibodies. Recombinant antigens were covalently coupled to magnetic, color coaded beads and serum samples were incubated with 20 multiplex bead mixes each representing hundreds of antigens. Univariate and multivariate statistical analyses were performed to reveal significant antigens and to define correlation of antigens with clinical parameters and amongst themselves. Results In SLE, SSc and RA novel autoantigens were discovered in independent discovery and validation studies. Antigens showing reproducible, significant reactivity compared to active and passive controls were selected in a stepwise marker refinement approach. Examples include BICD2 and KDM6B as novel antigens in SSc with 20–30% prevalence, TMPO and MVP in SLE with 15–25% prevalence, and several novel protein targets of anti-citrullinated peptide antibodies. Conclusions Discovery approaches for autoantigens in autoimmune diseases show great promise to further detail the autoimmune landscape. SeroTag screening is a valuable tool for “omics”-type biomarker discovery and verification. Novel autoantigens were discovered and validated in RA, SLE, SSc which show potential for improved and earlier diagnosis, differential diagnosis, and disease subgrouping. Disclosure of Interest P. Schulz-Knappe Employee of: Protagen AG, P. Budde Employee of: Protagen AG, H. Goehler Employee of: Protagen AG, H.-D. Zucht Employee of: Protagen AG, M. Schneider: None declared, S. Vordenbäumen: None declared, A. Lueking Employee of: Protagen AG, R. Brinks: None declared, J. Richter: None declared

Background Rheumatoid Factors (RF) and autoantibodies against citrullinated proteins (ACPA) are part of the diagnosis of rheumatoid arthritis (RA). Unfortunately these analytes lack sensitivity especially in early phases of RA. By way of screening proteins from a human protein library of over 7,000 proteins we discovered over 20 novel citrullinated antigens targeted by ACPAs in CCP positive as well as in 5–10% of CCP negative RA patients suggesting added diagnostic potential. Objectives To challenge novel human proteins as biomarkers for early RA which exhibited reactivities in citrullinated format against early and late RA patient serum samples we set to analyze sufficiently large, new cohorts of patients with early and established RA against the candidate proteins both in their citrullinated and non-citrullinated form. We also planned to veify single antigen performance by comparison to established benchmark proteins and marketed assays. Methods 34 novel citrullinated antigens were analyzed in serum samples from independent cohorts of 300 CCP positive and 151 CCP negative patients with early RA, 100 healthy volunteers and 50 patients with established RA. All samples were analyzed in a multiplexed, bead-based array on Luminex FlexMAP3D, compared to reactivities against known citrullinated proteins (vimentin, fibrinogen, enolase) and subsequently in 3 commercially available anti-CCP-assays. Results We confirmed novel citrullinated antigens targeted by ACPAs as markers of RA. The citrullinated antigens are highly reactive compared to their non-citrullinated forms in mature RA and in CCP positive samples of patients with early RA. 4 single antigens reach high overlap to established CCP-Assays each with >75% sensitivity at 97% specificity. Increased reactivity of novel antigens was identified in up to 10% of CCP negative samples suggesting the presence of additional citrullinated epitopes not present in commercial CCP assays used in this study. Conclusions Novel citrullinated antigens discovered by large scale “omics” screening via Luminex bead based arrays were confirmed in novel samples of early and established RA, especially in CCP positive samples. Reactivity in 10% CCP negative samples suggests added diagnostic value. Disclosure of Interest P. Schulz-Knappe Employee of: Protagen AG, P. Budde Employee of: Protagen AG, A. Lueking Employee of: Protagen AG, P. Schriek Employee of: Protagen AG, H. Goehler Employee of: Protagen AG, H.-D. Zucht Employee of: Protagen AG, J. Detert: None declared, G.-R. Burmester: None declared, M. Schneider: None declared, J. Richter: None declared, S. Vordenbäumen: None declared

Systemic lupus erythematosus (SLE) is a heterogeneous disease with respect to disease manifestations, disease progression and treatment response. Therefore, strategies to identify biomarkers that help distinguishing SLE subgroups are a major focus of biomarker research. We reasoned that a multiparametric autoantibody profiling approach combined with data mining tools could be applied to identify SLE patient clusters. We used a bead-based array containing 86 antigens including diverse nuclear and immune defense pathway proteins. Sixty-four autoantibodies were significantly (p < 0.05) increased in SLE (n = 69) compared to healthy controls (HC, n = 59). Using binary cut-off thresholds (95% quantile of HC), hierarchical clustering of SLE patients yields five clusters, which differ qualitatively and in their total number of autoantibodies. In two patient clusters the overall accumulated autoantibody reactivity of all antigens tested was 31% and 48%, respectively. We observed a positive association between the autoantibody signature present in these two patient clusters and the clinical manifestation of glomerulonephritis (GLMN). In addition, groups of autoantibodies directed against distinct intracellular compartments and/or biological motifs characterize the different SLE subgroups. Our findings highlight the relevant potential of multiparametric autoantibody detection and may contribute to a deeper understanding of the clinical and serological diversity of SLE.

Background The discovery of autoantibodies against citrullinated proteins (ACPA) and the consequent development of serological tests using cyclic citrullinated peptide (CCP) have improved diagnosis of rheumatoid arthritis (RA). ACPAs recognize specifically various citrullinated peptides that derive from different antigens. It has also been demonstrated that autoantibodies to different citrullinated peptides emerge at different stages of RA. However, by now the fine specificity against only a limited number of different peptides has been tested. Objectives To increase knowledge about ACPAs and their fine specificities in RA patients we aimed to identify novel citrullinated antigens and to characterize the ACPA profile in early and late RA. Methods 569 serum samples of CCP+ and CCP- RA patients with late RA, early RA or pre-RA and additional samples of volunteers and patients with early Arthritis (EA) but without RA at the time point tested have been analyzed in a multiplexed, bead-based, antigen array on the Luminex FlexMAP3D. Sera have been tested against a selection of 417 human proteins, including previously identified putative RA specific autoantigens and also known RA marker such as vimentin and fibrinogen. After coupling all proteins to individual color coded Luminex beads they were enzymatically citrullinated by peptidyl arginine deiminase and then tested against sera in the multiplex Luminex array. Complementarily all proteins were also measured in their native, unmodified form. Results We found novel citrullinated antigens targeted by ACPAs. The citrullinated antigens are highly reactive in mature RA whereas the unmodified proteins show only low reactivity. Generally, we observe an increase of the number of ACPA fine specificities with disease duration. In CCP negative tested RA patients, more than 10% of the patients recognize specifically several citrullinated antigens suggesting their diagnostic potential. Conclusions Novel citrullinated antigens targeted by ACPAs were identified. The identification of ACPA positive but CCP negative tested RA patients suggests that current CCP tests do not address the complete profile of ACPA fine specificities. Further studies are currently conducted to validate panels of citrullinated antigens in early RA patients. Disclosure of Interest A. Lueking Employee of: Protagen AG, P. Schriek Employee of: Protagen AG, H. Goehler Employee of: Protagen AG, M. Gamer Employee of: Protagen AG, K. Marquart Employee of: Protagen AG, A. Telaar: None declared, D. Chamrad Employee of: Protagen AG, P. Schulz-Knappe Employee of: Protagen AG, J. Richter: None declared, S. Vordenbaeumen: None declared, G. Burmester: None declared, M. Schneider: None declared

Background Rheumatoid arthritis (RA) is an autoimmune disease typically characterized by chronic inflammation, accumulation of self-reactive B-cells and production of autoantibodies of which anti–cyclic citrullinated peptide (anti-CCP) antibodies and rheumatoid factor (RF) have diagnostic utility. Despite these two well established markers up to 30% of RA patients remain sero-negative making an early diagnosis of RA more difficult. Although progress has been made to characterize risk factors for anti-CCP negative RA, studies on autoantibody profiles in CCP-negative RA are so far lacking. Objectives To improve the current diagnosis of RA, we aimed to determine whether the group of anti-CCP negative RA patients can be identified based on specific autoantibody profiles. Our major goal is to develop a novel autoantibody-based diagnostic test that allows to correctly diagnosing CCP- and RF-negative early RA patients. Methods In order to identify novel autoantigens characteristic for CCP-negative RA patients we performed a large-scale screen of 5800 proteins in a total of 150 serum samples of patients with stable RA and healthy volunteers. The major technology platform employed in this study is an automated bead-based Luminex xMAP technology which enables to measure the reactivity of autoantibodies to up to 500 different antigens in one single serum sample. All proteins are produced from E.coli, are highly purified by affinity capturing, sequenced by mass spectrometry, and each protein is coupled in optimised concentration to individual colour coded Luminex beads. Results Using both univariate and multivariate statistical algorithms we identified 144 novel antigens in RA patients. Furthermore, our data indicated heterogeneity in the autoantibody profile of RA patients and some overlap between the group of CCP-positive and CCP-negative patients. To address this complexity and heterogeneity we developed biomarker panels comprising five to ten antigens to identify CCP-negative RA patients. Notably, one panel with six antigens showed high specificity comparable to CCP. Another panel was able to detect about 60% of CCP-negative patients for whom currently no diagnostic marker is available. Conclusions CCP sero-negative RA patients can be identified based on specific set of autoantibodies. Further studies are currently conducted to validate the biomarker panels in early RA patients. Disclosure of Interest None Declared

In order to characterize the autoantibody repertoire in patients with RA, we performed in an earlier study a large-scale screen against 3,068 antigens using the bead-based Luminex xMAP technology. The autoantibody signature of 74 patients with an established RA was compared against 71 healthy controls. Antigens with high reactivity were selected and used to develop biomarker panels with improved sensitivity and specificity. Having defined this way the autoantibody repertoire of RA patients the goal of this study was to validate the autoantibody reactivity of RA patient samples analyzing 116 pre-treatment samples of early RA derived from the HITHARD treatment study (1).

Background SLE is a chronic multisystem autoimmune disease with yet unknown etiology. One hallmark of SLE is the B cell activation and the production of various autoantibodies. Differential diagnosis involves ANA screening which may yield also positive results in many connective tissue disorders and other autoimmune diseases, and may occur in normal individuals. More specific subtypes of antinuclear antibodies include anti-Smith and anti-double stranded DNA (dsDNA) antibodies. However, anti-dsDNA antibodies are present in just 70% SLE patients leaving a certain gap in diagnosis. Objectives In this study we characterized the autoimmune signatures of SLE patients and healthy blood donors in order to identify established plus novel autoantibodies with differential abundance in serum samples of SLE patients. Methods We used 5800 human proteins to investigate the autoantibodies present in more than 110 serum samples of SLE patients as well as healthy blood donors applying bead-based arrays (Luminex). Biostatistical analysis was performed using univariate as well as multivariate statistical algorithms. Results In addition to the well-established clinical markers Ro52/SS-A (TRIM21), ribosomal P0, ribosomal P1, ribosomal P2, SS-A/Ro60 (TROVE2), SSB and SmB/B´ new biomarkers were identified showing high discriminatory p-values and promising sensitivity and specificity. Consequently, a small protein antigen panel consisting of four new markers plus six well-established clinical markers (Ro52/SS-A, Ro60/SS-A, ribosomal P0, ribosomal P1, ribosomal P2, and SmB/B´protein) has been defined. This panel results in an improved AUC when compared to the classification performance of the established markers alone. Conclusions With novel biomarkers a large proportion of dsDNA AB-negative SLE patients can be addressed. The benefit of the identified marker panel for SLE diagnosis is currently evaluated in comparative analysis of other patient cohorts of ankylosing spondylitis, progressive systemic sclerosis, and rheumatoid arthritis. Disclosure of Interest None Declared

Huntington9s disease (HD) is a progressive neurodegenerative disorder, which is caused by expansion of a polyglutamine tract in the first exon of the huntingtin gene. Genetic testing allows the unambiguous diagnose of HD and the identification of individuals carrying the gene defect before onset of clinical symptoms. However, molecular markers that are suited to predict disease onset, monitor disease progression and asses the response to therapy are still not available. To identify HD-specific molecular markers we performed a comparative analysis of the autoimmune profile of symptomatic patients, presymptomatic gene carriers and a representative control cohort. Serum samples were tested for antigen/autoantibody interactions using a sequential arrangement of protein macroarrays and microarrays. The macroarrays contained E coli clones expressing approximately 10 000 human proteins and were used to characterise the autoimmune profile of 20 HD patients. Hereby, 273 human proteins were selected to generate a HD-specific protein microarray. Serum samples of 100 HD patients, 29 presymptomatic gene carriers and 100 healthy individuals were analysed using the HD-specific microarray. Thereby, several autoantigens were detected that represent putative molecular markers for HD. Further validation experiments are necessary to confirm the relevance of the identified molecular markers. The presence of HD-specific autoantibodies in serum samples supports the idea that progression of HD is associated with alterations of the immune system.

Protein Microarrays enable the analysis of multiple binding events in parallel. Based on patented UNIclone technology, we have established a reproducible protein biochip platform to determine specific binding profiles to multiple targets in a quantitative way. The UNIchip protein biochips provide a novel and unique tool for antibody characterization and ranking. Potential off-target activities can be detected by establishing a quantitative binding profile of an antibody to 400 different, unrelated targets. Together with on-chip determination of sensitivity, linearity and dynamic range the quantitative fingerprint enables a unique performance ranking and specificity analysis of antibodies. We have analyzed the performance of three block-buster biotherapeutics directed against TNF-alpha. The biotherapeutics show a clear differentiation both with regard to their binding profile to unrelated proteins as well as their binding affinity to the cognate target on the biochip. The quantitative fingerprint also offers a novel strategy for antibody pair selection in ELISA development.

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37 200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.

The human genome has been sequenced and the challenges of understanding the function of the newly discovered genes have been addressed. High-throughput technologies such as DNA microarrays have been developed for the profiling of gene expression patterns in whole organisms or tissues. Protein arrays are emerging to follow DNA chips as possible screening tools. Here, we review the generation and application of microarray technology to obtain more information on the regulation of proteins, their biochemical functions and their potential interaction partners. Already, a large variety of assays based on antibody-antigen interactions exists. In addition, the medical relevance of protein arrays will be discussed.

There is burgeoning interest in protein microarrays, but a source of thousands of nonredundant, purified proteins was not previously available. Here we show a glass chip containing 2413 nonredundant purified human fusion proteins on a polymer surface, where densities up to 1600 proteins/cm(2) on a microscope slide can be realized. In addition, the polymer coating of the glass slide enables screening of protein interactions under nondenaturing conditions. Such screenings require only 200-microl sample volumes, illustrating their potential for high-throughput applications. Here we demonstrate two applications: the characterization of antibody binding, specificity, and cross-reactivity; and profiling the antibody repertoire in body fluids, such as serum from patients with autoimmune diseases. For the first application, we have incubated these protein chips with anti-RGSHis(6), anti-GAPDH, and anti-HSP90beta antibodies. In an initial proof of principle study for the second application, we have screened serum from alopecia and arthritis patients. With analysis of large sample numbers, identification of disease-associated proteins to generate novel diagnostic markers may be possible.

Protein array technology has emerged as a new tool to enable ordered screening of proteins for expression and molecular interactions in high throughput. Besides classical solid-phase substrates, such as micro-titre plates and membrane filters, protein arrays have recently been devised with chip-sized supports. Several applications on protein chips have been described, but to our knowledge no studies using plant protein chips were published so far.

We describe the technical feasibility and methodology to characterize a protein by a minimal set of structural information generated by matrix assisted laser desorption/ionization (MALDI)-mass spectrometry, termed a "minimal protein Identifier" (MPI). MPIs can be determined for proteins from two-dimensional gels and recombinant proteins and can be used to compare and identify proteins from these sources.

We developed a high-throughput technique for the generation of cDNA libraries in the yeast Saccharomyces cerevisiae which enables the selection of cloned cDNA inserts containing open reading frames (ORFs). For direct screening of random-primed cDNA libraries, we have constructed a yeast shuttle/expression vector, the so-called ORF vector pYEXTSH3, which allows the enriched growth of protein expression clones. The selection system is based on the HIS3 marker gene fused to the C terminus of the cDNA insert. The cDNAs cloned in-frame result in histidine prototrophic yeast cells growing on minimal medium, whereas clones bearing the vector without insert or out-of-frame inserts should not grow on this medium. A randomly primed cDNA library from human fetal brain tissue was cloned in this novel vector, and using robot technology the selected clones were arrayed in microtiter plates and were analyzed by sequencing and for protein expression. In the constructed cDNA expression library, about 60% of clones bear an insert in the correct reading frame. In comparison to unselected libraries it was possible to increase the clones with inserts in the correct reading frame more than fourfold, from 14% to 60%. With the expression system described here, we could avoid time-consuming and costly techniques for identification of clones expressing protein by using antibody screening on high-density filters and subsequently rearraying the selected clones in a new "daughter" library. The advantage of this ORF vector is that, in a one-step screening procedure, it allows the generation of expression libraries enriched for clones with correct reading frames as sources of recombinant proteins.

We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.