Nastya Kharlamova

Genetic and environmental determinants for disease risk in subsets of rheumatoid arthritis defined by the anticitrullinated protein/peptide antibody fine specificity profile

Objectives To increase understanding of the aetiology and pathogenesis of rheumatoid arthritis (RA), genetic and environmental risk factors for RA subsets, defined by the presence or absence of different anticitrullinated protein/peptide antibodies (ACPAs) targeting citrullinated peptides from α-enolase, vimentin, fibrinogen and collagen type II, were investigated. Methods 1985 patients with RA and 2252 matched controls from the EIRA case-control cohort were used in the study. Serum samples were assayed by ELISA for the presence of anticyclic citrullinated peptides (anti-CCP) antibodies and four different ACPA fine specificities. Cross-reactivity between ACPAs was examined by peptide absorption experiments. Genotyping was performed for HLA-DRB1 shared epitope (SE) alleles and the PTPN22 gene, while information regarding smoking was obtained by questionnaire. The association of genetic and environmental risk factors with different subsets of RA was calculated by logistic regression analysis. Results Limited cross-reactivity was observed between different ACPA fine specificities. In total, 17 RA subsets could be identified based on their different ACPA fine specificity profiles. Large differences in association with genetic and environmental determinants were observed between subsets. The strongest association of HLA-DRB1 SE, PTPN22 and smoking was identified for the RA subset which was defined by the presence of antibodies to citrullinated α-enolase and vimentin. Conclusion This study provides the most comprehensive picture to date of how HLA-DRB1 SE, PTPN22 and smoking are associated with the presence of specific ACPA reactivities rather than anti-CCP levels. The new data will form a basis for molecular studies aimed at understanding disease development in serologically distinct subsets of RA.

Antibodies to Porphyromonas gingivalis Indicate Interaction Between Oral Infection, Smoking, and Risk Genes in Rheumatoid Arthritis Etiology.

To investigate the role of the periodontal pathogen Porphyromonas gingivalis in the etiology of rheumatoid arthritis (RA) by analyzing the antibody response to the P gingivalis virulence factor arginine gingipain type B (RgpB) in relation to anti–citrullinated protein antibodies (ACPAs), smoking, and HLA–DRB1 shared epitope (SE) alleles in patients with periodontitis, patients with RA, and controls.

A6.8 Elevated antibody levels to porphyromonas gingivalis detected in rheumatoid arthritis patients with a specific anti-citrullinated protein/peptide antibody profile

Background and Objectives Porphyromonas gingivalis, a major cause of chronic periodontitis (PD), has been implicated also in the aetiology of rheumatoid arthritis (RA), specifically in anti-citrullinated protein/peptide antibody (ACPA) positive RA, based on its unique property among pathogens to express a citrullinating peptidylarginine deiminase enzyme. To investigate this hypothesis further, we have analysed the antibody response to P. gingivalis in relation to known risk factors for RA, including autoantibodies, smoking and HLA-DRB1 shared epitope (SE) alleles. Materials and Methods Serum from 66 patients with chronic PD and 60 individuals without (confirmed by dentists), as well as serum from the EIRA (Epidemiological Investigation of RA) cohort (n = 1985 RA cases and n = 400 controls), were screened by ELISA for presence of antibodies to the P. gingivalis-specific virulence factor arginine gingipainB (RgpB). Antibody levels were analysed in relation to the presence of anti-CCP antibodies, rheumatoid factor, anti-carbamylated protein antibodies and various ACPA fine-specificities, as well as smoking and SE. Differences in antibody levels were examined using Mann-Whitney U test for independent groups. Results Significantly higher anti-RgpB antibody levels were detected in the PD subset, compared to the non-PD control subset, and in EIRA RA cases, compared to EIRA controls. Among EIRA RA cases, anti-RgpB IgG levels were significantly increased in RF + as well as anti-CCP + patients. Interestingly, anti-RgpB IgG levels associated with the presence of some ACPA fine-specificities, but not others. There was no association with anti-carbamylated protein antibodies, smoking or HLA-DRB1 SE alleles. Conclusion Significantly increased anti-RgpB antibody levels in PD serum, compared to non-PD serum, may suggest that elevated anti-RgpB IgG levels can be used as a proxy for chronic PD, in studies where the periodontal status is of interest but unknown. A number of previous studies have demonstrated an association between anti-P. gingivalis antibody responses and ACPA positive RA. However, most of those studies have utilised bacterial lysates as capturing antigen, which are known to contain citrullinated epitopes. By using recombinant RgpB, we circumvent the potential problem of false positive results due to cross-reactive ACPAs, and we conclude that there is an association between elevated anti-RgpB IgG levels and a subset of RA that is characterised by a specific ACPA profile. Interestingly, we could not detect an association with smoking, a well-known risk factor for both PD and ACPA+ RA. These results may point to different aetiologies in different subsets of ACPA positive RA.

A2.11 Antibodies to carbamylated α-enolase epitopes in rheumatoid arthritis also bind citrullinated epitopes and are largely indistinct from anti-citrullinated protein antibodies

Objectives In addition to anti-citrullinated protein antibodies (ACPAs), antibodies targeting carbamylated (i.e. homocitrullinated) proteins (anti-CarP antibodies) have been described in rheumatoid arthritis (RA). However, the extent to which anti-CarP antibodies are truly distinct from ACPA remains unclear, and few studies have focused on specific autoantigens. Here, we examine cross-reactivity between ACPA and anti-CarP antibodies, in the context of the candidate autoantigen α-enolase. Methods Cross-reactivity was examined by immunoblotting of citrullinated and carbamylated proteins using purified ACPA; and by peptide absorption experiments, using the citrullinated α-enolase peptide CEP-1 and a homocitrulline-containing version (carb-CEP-1) in ELISA. The population-based case-control cohort EIRA (n = 2,836 RA; 373 controls) was screened for reactivity with CEP-1 and carb-CEP-1, using the ISAC multiplex array. Associations of RA subsets with smoking and genetic risk alleles were calculated using unconditional logistic regression models. Differences in antibody levels were examined using Mann-Whitney U test. Results Affinity-purified ACPA was found to bind carbamylated proteins and homocitrulline-containing peptides, demonstrating definitive cross-reactivity between ACPA and anti-CarP antibodies. Anti-carb-CEP-1 reactivity in EIRA was almost exclusively confined to the CEP-1-positive subset, and this group (21%) displayed a particularly strong ACPA response with marked epitope-spreading. The small RA subset (3%) with homocitrulline-reactivity in the absence of citrulline-reactivity did not associate with smoking or risk genes, and had significantly lower anti-carb-CEP-1 antibody levels. Conclusion Our data presented herein cast doubt on the specificity of anti-CarP antibodies in RA, which we posit may be a subset of cross-reactive ACPA.

THU0119 Rheumatoid Arthritis and Periodontal Disease: Association between Salivary Citrulline, ACPA Levels and Clinical Presentation

Background Periodontitis and other dental conditions have been associated with the pathogenesis of rheumatoid arthritis (RA). In addition, these dental pathologies are also important comorbidities for RA. Objectives We wished to evaluate the periodontal involvement of rheumatoid arthritis (RA) patients, and we correlated with various laboratory biomarkers including lipids, autoantibodies, serum vitamin D levels, markers of bone metabolism in relation with the periodontal condition and cariological indices; and also correlated them with salivary citrulline and anti citrullinated protein autoantibody levels with the above mentioned clinical and blood test findings. Methods Twenty-three RA patients were recruited for the study. Saliva samples were taken following whole scale periodontal and cariological examination. Protein concentration, peptidyl-citrulline and anti-cyclic citrullinated protein (anti-CCP) levels were measured from saliva samples. Blood test results were provided by rheumatologists. Citrullinated enolase protein-1 (CEP-1) level from serum was also measured. Results Periodontal diagnoses (scores) seem to have a positive dependency on LDL (R=0.722, p=0.008), PTH1 (R=0.586, p=0.022), D3 vitamin level (R=0.586, 0.022), the sum of D3/D2 (R=0.634, p=0.011) respectively, in these patients. Anti-CEP-1 positive patients had significantly higher periodontal scores (2.71±0.11 vs 2.50±0.09, p<0.05) compared to anti-CEP-1 negative subjects. Interestingly, anti-CEP-1 positive patients had significantly higher triglyceride levels compared to seronegative ones (1.81±0.17 vs 1.42±0.05 mmol/l; p<0.05). Salivary citrulline and salivary anti-CCP, (p=0.007, R: 0.583) level has a correlation with the maximum of clinical probing depth. Conclusions Our results may add further pieces to the mosaic of RA-periodontitis connection. The possible role of antimicrobial immunity, as well as the possible role of lipids and bone metabolism have also been delineated. Future therapies should aim the disruption of this framework. Disclosure of Interest None declared

FRI0048 Concentrations of Antibodies against Porphyromonas Gingivalis Are Increased before The Onset of Symptoms of Rheumatoid Arthritis

Background Citrullination by the periodontal pathogen Porphyromonas gingivalis has been hypothesized to play a role in RA aetiology, by contributing to the production of anti-citrullinated protein antibodies (ACPA). We have previously shown that the ACPA response precedes RA by several years, and we have recently demonstrated elevated anti-P. gingivalis antibody levels in patients with RA compared with controls (1–3). Objectives The aim of the present study was to investigate the link between P.gingivalis and RA further, by analysing whether anti-P. gingivalis antibodies predate RA diagnosis and ACPA production. Methods A case-control study was performed within the Medical Biobank and the Maternity cohort of Northern Sweden including 251 individuals who had donated 422 blood samples predating the onset of symptoms of RA, and 198 population-based controls from the same cohorts. The median (IQR) predating time until onset of symptoms was 5.2 (6.2) years. For 192 individuals diagnosed with RA, blood samples were available from the time of diagnosis. Samples were analysed using in-house ELISAs for presence of antibodies to the virulence factor arginine gingipainB (RgpB) purified from P. gingivalis, and to a citrullinated peptide (CPP3) derived from the P. gingivalis peptidylarginine deiminase enzyme P.PAD. Results The concentration of anti-RgpB IgG levels was significantly increased in pre-symptomatic individuals (mean±SEM; 152.7±14.8 AU/ml) and in RA patients (114.4±16.9 AU/ml), compared with controls (82.2±12.1 AU/ml, p<0.001 for both). Antibodies against CPP3 were detected in 5% of the pre-symptomatic individuals and in 8% of the RA-patients, with elevated levels in both pre-symptomatic individuals (4.33±0.59 AU/ml) and RA-patients (9.29±1.81 AU/ml) compared with controls (2.36±0.58 AU/ml, p<0.001 for both). The anti-CPP3 antibody response followed the ACPA response, with increasing antibody concentrations over time, whilst the anti-RgpB IgG response was elevated and stable in pre-symptomatic individuals during the pre-dating time and showed a trend towards lower levels after RA diagnosis. Conclusions Anti-P. gingivalis antibody concentrations were significantly increased in RA compared with controls and these antibodies were detected years before the onset of symptoms of RA, supporting an aetiological role for P. gingivalis in the development of RA. References Rantapaa-Dahlqvist, S., et al Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum 2003; 48(10): 2741–2749. Brink, M., M. Multiplex analyses of antibodies against citrullinated peptides in individuals prior to development of rheumatoid arthritis. 2013; Arthritis Rheum 65(4): 899–910. Kharlamova, N., et al Antibodies to Porphyromonas gingivalis indicate interaction between oral infection, smoking and risk genes in rheumatoid arthritis etiology.Arthritis Rheumatol. 2015 Nov 10. doi: 10.1002/art.39491 Disclosure of Interest None declared

Plasma Concentrations of Antibodies to Porphyromonas Gingivalis Are Increased before Onset of Symptom of Rheumatoid Arthritis

Are Ankylosing Spondylitis, Psoriatic Arthritis and Undifferentiated Spondylarthritis Associated with an Increased Risk of Cardiovascular Disease?For a searchable version of these abstracts, please visit www.acrabstracts.org. Please Note: It may take several minutes for this file to download.Background/Purpose: Person-centred care (PCC) is a holistic approach with respectful and individualized care allowing negotiation of care where persons with health problems are empowered to be involved in health decisions. Patients’ illness narratives constitute a starting point for building a collaboration with health care professionals and to empower them to play an active role in their health care. Little is known of the impact of PCC vs. regular care on patients’ skills as health care consumers. The aim was to study the impact on effective consumers’ skills over 6 and 12 months as measured by the Effective Consumer Scale (EC17) in patients undergoing biological therapy and randomly assigned to either a nurse-led rheumatology clinic (NLC) based on PCC or to a rheumatologist-led clinic (RLC) based on regular care.Methods: A 12 month RCT in 107 patients with chronic inflammatory arthritis1. Inclusion criteria were ongoing biological therapy and a DAS28 ≤3.2. All patients met a rheumatologist at inclusion and after 12 months, while the 6 month follow-up was randomized to either at an NLC (PCC) or at an RLC (regular care). Outcome measure was the EC17, developed and endorsed by the OMERACT, including five subscales; 1. Use of health information, 2. Clarifying personal priorities, 3. Communicating with others, 4. Negotiating roles and 5. Deciding and taking action. EC17 total score ranges from 0-100, worse to best. Differences between and within NLC and RLC were analyzed with Friedmans’ test or Mann Whitney U-test.Results: After 12 months 97 patients completed the RCT (NLC n=47, RLC n=50), mean (SD) age 55.4 (12.7) years, disease duration 16.7 (11.5) years, DAS28 2.1 (0.7), HAQ 0.54 (0.38), global health 20.4 (17.1), pain 21.1 (18.0) and 56% were women. There were no statistically significant differences within or between the two intervention groups at baseline nor in EC17 total score mean (SD) at baseline (NLC 83.5 (9.4) vs. RLC 83.2 (10.8), 6 months (NLC 85.4 (10.4) vs. RLC 82.9 (10.9) and 12 months (NLC 85.3 (11.1) vs. RLC 82.3 (10.9)). However, in NLC there was a statistically significant improvement in EC17 subscale “1. Use of health information” at both 6 and 12 months (p=0.041 and p=0.004 respectively).Conclusion: Replacing just one of three visits over 12 months to an NLC based on PCC instead of an RLC based on regular care resulted in more effective consumers concerning the use of health information. Larger studies over longer time frames focusing on PCC are needed to better understand its full impact on effective consumer skills measured by EC17.References:1. Larsson I, et al. Randomized controlled trial of a nurse-led rheumatology clinic for monitoring biological therapy. J Adv Nurs 2014;70:164-75.Background/Purpose: Chronic widespread pain (CWP), one of the hallmarks of fibromyalgia, is not uncommon in adolescents and it has previously been shown that adolescents with pain often become young adults with pain. CWP often co-varies with anxiety, depression, and stress symptoms in adults, but the knowledge regarding this is small in youth and young adults.The aim was to study the associations between CWP, anxiety, depression and stress in adolescents attending first year of high school.Methods: A computerized questionnaire to 296 adolescents attending Swedish high school, with validated questions regarding presence and distribution of pain (Epipain mannequin), stress symptoms (ELO question), anxiety and depression (Hospital Anxiety and Depression Scale – HADS), and health related quality of life (HRQL as measured by EQ5D). Pain was considered chronic when persistent for more than three months, and the subgroup CWP was defined according to the 1990 ACR criteria for fibromyalgia. Statistical analyses in SPSS v21 with comparison of means by Student’s t-test and proportions by chi2-test or Fischer’s exact test.Results: 257 (87%) out of 296 eligible students, mean (SD) age 16.1 (0.7) and 65.8% girls, responded to the questionnaire. Prevalence of chronic pain was 20.8% and that of the subgroup CWP was 4.7%, without any gender differences (boys 18.2% vs girls 22.2%; p=0.224, and 3.4% vs 5.4%; p=0.692). High level (4 or 5 on a 5 point scale) of stress symptoms were less common in boys (16.0% vs 28.2%; p=0.015), as was possible or probable anxiety (17.1% vs 44.4%; p<0.001), but not depression (10.3% vs 12.5%; p=0.764). Students with high level of stress reported CWP five times more often than those with less stress (30.4% vs 5.8%; p=0.001). Students with probable anxiety reported CWP ten times more often than students with no anxiety (17.6% vs 1.8%; p=0.001), and CWP was also more common, but not statistically significant, in students with probable depression (20.0% vs 3.1%; p=0.163). Those reporting CWP had significantly lower HRQL (0.58 vs 0.87; p=0.038) than students with no chronic pain.Conclusion: The high prevalence of chronic pain and the strong associations between CWP and reports of stress and anxiety in adolescents highlights that a multifactorial background to chronic pain must be considered early in life. An apparent lower score in EQ5D also indicates that the presence of CWP has an marked impact on HRQL also in adolescents.Background/Purpose: The treatment target for axial spondyloarthritis (SpA) is to maximize health-related quality of life (HRQoL) by controlling disease activity and improving functioning. The treatment cornerstones are a combination of patient education, pharmacological and non-pharmacological treatment. Health professionals are familiar with providing patient education but the knowledge is scarce concerning how this education is experienced by the patients.The aim was to describe patients’ experiences of education in SpA management.Methods: The study had a descriptive design with a qualitative conventional content analysis approach performed in seven steps in accordance with Graneheim & Lundman (1). The analysis aimed to describe and preserve contextual meanings. After coding and subgrouping meaningful parts of the text were merged into categories. Eleven interviews were conducted between 2014-2015 in patients with SpA based on a strategic sampling in order to achieve variation with regard to sex (7 men, 4 women), age (38-66 years), subdiagnoses (5 patients with AS, 6 with USpA), quality of life (EQ5D 0.29-1.0), disease activity (BASDAI 1-6), physical function (BASFI 0-5), and global health (BASG 0-7) .Results: Three categories representing patients’ experiences of patient education in disease management emerged; guiding education, reliable education and available education. Guiding education comprised SpA management including disease knowledge such as symptoms, prognosis, treatment, self-management, climate impact, heredity, and assisting devices. Reliable education meant how and by whom the education was communicated and was considered reliable if it was based on science and communicated by specialists, for example by physician, nurse, PT, dietician and senior patients with experience of rheumatic diseases. The patients experienced difficulties in assessing the large flow of education coming from various sources. Individualized education also increased the reliability. Available education meant that the education can and should be presented in varied formats, and that the amount of information could be chosen. The education could be given orally (through meetings, videos, lectures), in writing (by pamphlets, e-mails, journals, webpages) or obtained through own personal experiences. There were requests to utilize newer media like skype, video and chat forums. Furthermore, individual contacts with healthcare professionals when needed were of importance.Conclusion: This study highlights the importance of obtaining a guiding, reliable and available patient education for management of SpA. Health care professionals need to consider the importance of presenting varied formats of education based on patients’ experiences and expectations.References:1.Graneheim UH, Lundman B. Qualitative content analysis in nursing research: concepts, procedures and measures to achieve trustworthiness. Nurse education today 2004;24(2):105-12.PMN Reactivity Contribute to Acute Onset Joint Inflammation By Increasing CXCL8 Production in Joints of RA Patients with Anti-Collagen II AntibodiesBig Data International Primary Sjogren Syndrome Registry : Baseline Characterization and Diagnostic Approach in 6047 Patients Fulfilling the 2002 AE CriteriaThe Link Between DAS28 and the Short-Term Risk of Acute Coronary Syndrome in RA, and Its Driving FactorsHypomethylation in Enhancer and Promoter Regions of Interferon Regulated Genes in Multiple Tissues Is Associated with Primary Sjogrens SyndromeReceptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) and Sclerostin Are Related to Joint Destruction in Early Rheumatoid Arthritis Unrelated to Polymorphisms of the Genes

AB0043 Affinity purification and characterization of human acpas

Background Autoimmunity in rheumatoid arthritis (RA) is characterized by autoantibodies to citrullinated proteins/peptides (ACPA)1. These antibodies, present in 60-70% of patients, antedate clinical onset and associate with an erosive disease course, suggesting a direct pathogenic involvement in disease initiation and progression2. Objectives With this study, we aimed to develop an efficient method for the purification of human ACPA, and to characterize their frequency and fine-specificity pattern in synovial fluid (SF) and plasma of RA patients. Methods SF and plasma samples were collected with informed consent and ethical approval from patients (fulfilling the ACR criteria for RA) with high anti-CCP antibody levels. SF samples (n=36) were first treated with hyaluronidase to decrease viscosity, then proteins were precipitated with ammonium sulfate, dissolved and further dialysed against phosphate buffered saline (PBS), before the IgG fractions were purified on ProteinG columns (GE Healthcare, Uppsala, Sweden). Plasma samples (n=10) were diluted in PBS before applied to the ProteinG column. ACPAs were further purified using CCP2 affinity columns, kindly provided by Euro-Diagnostica. Recovery and purity of total IgG and anti-CCP IgG were analysed using SDS-PAGE, Nanodrop (Thermo Scientific, Wilmington, DE, USA) and the CCP2-ELISA kit. Fine-specificity of the purified ACPAs were investigated using in-house ELISAs, with peptides from citrullinated α-enolase (CEP-1), -vimentin (Cit-vim), -fibrinogen (Cit-fib) and -collagen type II (Cit-C1). Results Anti-CCP IgG could efficiently be purified from SF and plasma, using ProteinG-, followed by CCP2-, columns. No CCP IgG response could be detected in the flow-through fractions. Higher concentrations of total IgG were found in plasma (13,6 mg/ml) compared to SF (4,2 mg/ml), while a higher percentage of CCP-specific IgG was detected in SF (3%), compared to plasma (2%). The purified anti-CCP IgG fractions cross-reacted with CEP-1, Cit-vim, Cit-fib and Cit-C1, while no reactivity to these citrullinated antigens were detected in the IgG flow-through fractions. Anti-CCP IgG dilution curves (starting at 10 μg/ml of purified antibodies) demonstrated differences in affinity between patients, which may correspond to the different ACPA-fine specificity patterns seen in patients. Conclusions The described methodology efficiently purifies ACPAs with multiple specificities, which will allow for their use in in vivo and in vitro studies, to further elucidate their arthritogenic and pathogenic capacity. In addition, the ACPAs will be tools for future immunoprecipitation-, immunoblotting- and immunohistochemistry experiments. References Schellekens, G. A. et al., The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 43(1), 155 (2000). van der Helm-van Mil AH, Verpoort KN, Breedveld FC, Toes RE, Huizinga TW, Antibodies to citrullinated proteins and differences in clinical progression of rheumatoid arthritis. Arthritis Res Ther 7(5), R949 (2005). Disclosure of Interest None Declared

Affinity purification and characterisation of human ACPAs

Backgroundand objectives Autoimmunity in rheumatoid arthritis (RA) is characterised by autoantibodies to citrullinated proteins/peptides (ACPA). These antibodies, present in 60–70% of patients, antedate clinical onset and associate with an erosive disease course, suggesting a direct pathogenic involvement in disease initiation and progression. With this study, the authors aimed to develop an efficient method for the purification of human ACPA, and to characterise their frequency and fine-specificity pattern in synovial fluid (SF) and plasma of RA patients. Materials and methods SF and plasma samples were collected with informed consent and ethical approval from patients (fulfilling the American college of rheumatology criteria for RA) with high anti-CCP antibody levels. SF samples (n=36) were first treated with hyaluronidase to decrease viscosity, then proteins were precipitated with ammonium sulphate, dissolved and further dialysed against PBS, before the IgG fractions were purified on Protein G columns (GE Healthcare, Uppsala, Sweden).Plasma samples (n=10) were diluted in PBS before applied to the Protein G column. ACPAs were further purified using CCP2 affinity columns, kindly provided by Euro-Diagnostica. Recovery and purity of total IgG and anti-CCP immunoglobulin G (IgG) were analysed using SDS-PAGE, Nanodrop (Thermo Scientific, Wilmington, DE, USA) and the CCP2-ELISA kit. Fine-specificity of the purified ACPAs were investigated using inhouse ELISAs, with peptides from citrullinated α-enolase (CEP-1), -vimentin (Cit-vim), -fibrinogen (Cit-fib) and -collagen type II (Cit-C1). Results Anti-CCP IgG could efficiently be purified from SF and plasma, using ProteinG-, followed by CCP2-, columns. No CCP IgG response could be detected in the flow-through fractions. Higher concentrations of total IgG were found in plasma (13.6 mg/ml) compared to SF (4.2 mg/ml), while a higher percentage of CCP-specific IgG was detected in SF (3%), compared to plasma (2%). The purified anti-CCP IgG fractions cross-reacted with CEP-1, Cit-vim, Cit-fib and Cit-C1, while no reactivity to these citrullinated antigens were detected in the IgG flow-through fractions. Anti-CCP IgG dilution curves (starting at 10 µg/ml of purified antibodies), demonstrated differences in affinity between patients, which may correspond to the different ACPA-fine specificity patterns seen in patients. Conclusions The described methodology efficiently purifies ACPAs with multiple specificities, which will allow for their use in in vivo and in vitro studies, to further elucidate their arthritogenic and pathogenic capacity. In addition, the ACPAs will be tools for future immunoprecipitation-, immunoblotting- and immunohistochemistry experiments.