RA Sera Research Articles

Association Of Cellular Communication Network Factor 3 (CCN3) With Rheumatoid Arthritis Disease And It's Severity (Case-Control Study)

Background: An inflammatory condition that affects the joints in the limbs is rheumatoid arthritis. The most common symptom is chronic, recurrent joint inflammation. When patients are advanced, joint deformities and impairments may cause major impediments in the cardiac, skin, as well as other tissues and organs. A protein called cellular CCN3 is implicated in many biological processes such as cell adhesion, migration, as well as proliferation. Additionally, CCN3 has a role in a number of pathological processes, such as fibrosis, angiogenesis, and wound healing. CCN3 can be released from the cytoplasm. CCN3 may also be an inflammatory component in the course of RA. Future therapy approaches that focus on the activities and mechanisms of action of these proteins may result from better knowledge of how CCN proteins alter the pathophysiological processes underlying these types of arthritis and successfully reduce patients' pain (Wei et al., 2020). It was discovered that RA sera and tissues had increased levels of all CCN family members. IL6 production and CCN3 expression are correlated with disease activity (Giusti & Scotlandi, 2021).
 Methods: Between November 2021 and April 2022, a study including 70 patients with RA who met four or more 2010 American College of Rheumatology and European League Against Rheumatism (ACR/EULAR) classification criteria for the condition and 70 healthy persons serving as control groups was conducted. All subjects had their disease activity score (DAS28-ESR), clinical activity index (CDAI), and rheumatoid factors (RF) detected by latex agglutination. By using an enzyme-linked immunosorbent assay, the levels of anti-cyclic citrullinated peptide (ACCP) and CCN3 in human serum samples were determined (ELISA).The age group of both the patients and the controls included in this study was 20-70. RA was found to be high in individuals of the age group >40 years, at a percentage of 71.4%. The mean age was 46.2±10.3 years for patients and 1.7±0.5 for controls.Results: The results clearly showed high serum CCN3 levels in patient groups with RA compared to control (P = 0.0001). CCN3 and ACCP have a significantly positive association at control (P = 0.0001). According to DAS28-ESR, there were significantly increased concentrations of CCN3 in severe patients at P values of 0.0001 in comparison to mild patients.
 Conclusions: The biomarker CCN3 is a good prognostic marker for the severity of RA.

Human carbamylome description identifies carbamylated α2-macroglobulin and hemopexin as two novel autoantigens in early rheumatoid arthritis.

ObjectivesAnti-carbamylated protein antibodies (anti-CarPAs) are present in RA sera and have been associated with erosive disease. The exact targets of anti-CarPAs in vivo are currently not well known; we used a proteomic approach on serum and SF of RA patients to assess the human carbamylome and to identify carbamylated autoantigens as potential biomarkers in early RA.MethodsMass spectrometry was performed on SF and serum from RA patients. Carbamylated proteins present in both sample types were selected as candidate autoantigens for the establishment of ELISAs. A cohort of early RA patients was tested for positivity for specific anti-CarPAs.ResultsEleven novel carbamylated proteins were identified, and five were selected as potential autoantigens for detection of anti-CarPAs. Among them, antibodies against carbamylated hemopexin (anti-CaHPX) and alpha-2-macroglobulin (anti-CaA2M) showed comparable diagnostic value to the established carbamylated foetal calf serum–based ELISA. A cohort of 189 early RA patients was studied. The combination of these new biomarkers with anti-citrullinated protein antibodies and RF identified 89% of early RA patients in our cohort. There was little correlation between the tested biomarkers, and each one of the tested antigens could identify a different subset of seronegative RA patients. Anti-CaA2M positivity showed clinical potential, being associated with higher disease disability.ConclusionWe highlight the detection of novel carbamylated autoantigens in vivo using a combined proteomics approach in the SF and serum of RA patients. Anti-CaHPX and anti-CaA2M are promising clinical biomarkers, especially in seronegative RA.

The autoantibody response to cyclic citrullinated collagen type II peptides in rheumatoid arthritis.

The detection of anti-citrullinated peptide antibodies (ACPAs) is a serological hallmark of RA. Autoantibodies reactive with collagen type II (CII) are present in RA sera and synovial fluid and are potentially pathogenic. Here, we investigate the prevalence and specificity of the autoantibody responses to defined citrullinated cyclic peptides derived from CII in a China RA cohort. Using bead-based multiplex assay, we examined the presence of autoantibodies binding to 54 cyclic 17-mer citrullinated CII peptides, encompassing all citrullinate epitopes in CII, and the corresponding unmodified peptides in 415 RA patients, in addition to 304 patients with OA. Furthermore, the autoantibody responses to a selected set of 10 cyclic citrullinated peptides were also examined in 203 healthy individuals. Autoantibody responses to cyclic citrullinated CII peptides were higher in RA patients as compared with OA patients or healthy individuals, whereas little or negligible antibody responses to cyclic unmodified CII peptides were observed. Interestingly, several novel citrullinated CII epitopes were identified. Antibodies to these novel citrullinated CII epitopes showed not only substantial overlapping reactivities but also had unique specificities. We found a high prevalence of autoantibodies against cyclic citrullinated CII in the sera of patients in a China RA cohort. The present study revealed heterogeneous binding patterns against novel citrullinated CII epitopes, which may help to stratify RA patients into different subgroups.

Characterization of a Synovial B Cell–Derived Recombinant Monoclonal Antibody Targeting Stromal Calreticulin in the Rheumatoid Joints

Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from B cells isolated from ELS+ RA tissues react against NETs. Thus, alternative cellular sources of other potential autoantigens targeted by locally differentiated B cells remain undefined. RA fibroblast–like synoviocytes (FLS) have been implicated in the release of RA-associated autoantigens. In this study, we aimed to define stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Seventy-one RA-rmAbs were screened toward RA-FLS by living-cell immunofluorescence (IF). Western blotting was used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy, ELISA, immunoprecipitation assay, and surface plasmon resonance on unmodified/cit proteins. Serum immunoreactivity was tested in anti-citrullinated peptide/protein Abs (ACPA)+ versus ACPA− RA patients. Ten out of 71 RA-rmAbs showed clear reactivity toward RA-FLS in immunofluorescence with no binding to NETs. One stromal-reactive RA-rmAb (RA057/11.89.1) decorated a ∼58-kDa band that mass spectrometry and Western blotting with a commercial Ab identified as calreticulin (CRT). Confocal microscopy demonstrated significant cellular colocalization between anti-CRT RA057/11.89.1 in RA-FLS. RA057/11.89.1 was able to immunoprecipitate rCRT. Deimination of CRT to cit-CRT moderately increased RA057/11.89.1 immunoreactivity. cit-CRT displayed increased blocking capacity compared with unmodified CRT in competitive binding assays. Finally, anti–cit-CRT Abs were preferentially detected in ACPA+ versus ACPA− RA sera. We identified a synovial B cell–derived RA-rmAb locally differentiated within the ELS+ RA synovium reacting toward CRT, a putative novel autoantigen recently described in RA patients, suggesting that FLS-derived CRT may contribute to fuel the local autoimmune response.

Serum-soluble folate receptor β as a biomarker for the activity of rheumatoid arthritis synovitis and the response to anti-TNF agents.

This study aims to develop a sandwich ELISA system for the measurement of soluble folate receptor β (sFRβ) and evaluate whether base line levels of serum sFRβ are a biomarker for the activity of RA synovitis and the response to anti-TNF agents. Serum sFRβ from normal controls (41 samples), patients with OA (29 samples), and patients with RA (27 samples) and synovial fluid sFRβ from patients with RA (17 samples) were measured by sandwich ELISA, using anti-FRαβ and anti-FRβ antibodies as capture and detection antibodies, respectively. Baseline levels of serum sFRβ before therapy were evaluated in relation with DAS28-CRP or CRP and response to anti-TNF agents at 3-month follow-up. sFRβ levels in RA synovial fluids were higher than those in RA sera, and sFRβ levels in RA sera were higher than those in osteoarthritis and normal control sera. A significant relationship was observed between serum sFRβ levels and the DAS28-CRP scores or CRP values. The area under curve (AUC) values for receiver-operating characteristic curves defined using the serum sFRβ levels of RA patients before therapy had a higher predictive capacity than DAS28-CRP and CRP for the effective response of anti-TNF agents. The high serum sFRβ levels with a cutoff value of 8ng/mL were 100% specificity for the effective response of anti-TNF agents. The findings support that the serum sFRβ levels in patients with RA act as a disease activation biomarker and that high serum sFRβ levels act as a predictive biomarker for the response to anti-TNF agents.

Characterization of Autoantigens Targeted by Anti-Citrullinated Protein Antibodies In Vivo: Prominent Role for Epitopes Derived from Histone 4 Proteins.

Anti-citrullinated protein antibodies (ACPA) have become an integral part of the clinical definition of rheumatoid arthritis, and are hypothesized to be important in the immunopathogenesis of this autoimmune disease. Several citrullinated proteins have been demonstrated to serve as candidate autoantigens for the ACPA, based on in vitro immune reactions between citrullinated peptides/proteins and RA sera. Yet it remains unclear whether the autoantigens identified in vitro are indeed directly and specifically targeted by the ACPA in vivo. Moreover, it is unclear whether ACPA present in RA sera are directed towards the same spectrum of autoantigens as the ACPA present within the synovial compartment. In this study, we isolated ACPA immune complexes from RA synovial fluids (SF) and sera by using immobilized cyclic citrullinated peptides (CCP3) based immune affinity, and characterized the proteins that are directly and specifically associated with them by mass spectrometry. The results demonstrate that four histone proteins are prominent ACPA autoantigens, with the frequency of detection being histone H4 (89%), H2B (63%), H3 (63%), and H2A (58%) in ACPA positive RA SF. We further demonstrate that a histone 4 peptide containing citrulline at position Cit39 was recognized by 100% of ACPA positive RA SF. An adjacent citrulline residue at Cit40 was recognized by 34% of ACPA positive RA SF. An H4 peptide containing Cit39-40 was recognized in the serum of 94% ACPA positive RA, 77% ACPA positive first-degree relatives (FDR) of RA patients, and 2.5% of healthy controls. The Cit39-40 peptide substantially blocked the ACPA reactivity in both SF and serum. Although the spectrum of ACPA we identified was limited to those isolated using immobilized CCP3 peptides, the findings indicate that H4 is a widely recognized RA autoantigen in both the synovial and serum compartments. The identification of this immunodominant ACPA epitope may be valuable in designing approaches to immune tolerance induction in RA.

Relevance of Nitroxidation of Albumin in Rheumatoid Arthritis: A Biochemical and Clinical Study

Objective: To study the role of peroxynitrite-modified human serum albumin (nitroxidized-albumin) in rheumatoid arthritis. Methods: Human serum albumin was exposed to peroxynitrite and changes in albumin structure were monitored by UV-visible, fluorescence and circular dichroism spectroscopy, thioflavin T, Congo red binding and attenuated total reflection-Fourier transformed infrared spectroscopy (ATR-FTIR). Antioxidant properties of nitroxidized-albumin were evaluated by free radical induced RBC hemolysis test. Markers of protein oxidation like carbonyl, thiol, dityrosine and RBC hemolysis were evaluated in RA patients’ sera. Binding of autoantibodies in RA sera (n=50) with nitroxidized-albumin was studied by direct binding, inhibition ELISA and electrophoretic mobility shift gel assay. Results: The nitroxidized-albumin indicated the generation of nitrotyrosine, nitrotryptophan, carbonyl, dityrosine and reduction in tyrosine and tryptophan fluorescence and α-helicity. Fluorescence emission intensities of thioflavin T and Congo red got augmented upon binding with nitroxidized-albumin. Furthermore, secondary and tertiary structures of nitroxidized-albumin were altered as evident by ATR- FTIR, far and near-UV CD. Autoantibodies in RA sera (or IgG purified from sera) showed enhanced binding with nitroxidized-albumin as determined by direct binding and inhibition ELISA. Protein carbonyls, dityrosine and RBC hemolysis were significantly high, but thiol was significantly low in RA sera compared to age- and sex- matched control. Conclusion: Endogenously available peroxynitrite can nitrate and oxidize albumin, leading to protein nitration/ oxidation and subsequent formation of crosslinks, aggregates and immunogenic nitroxidized-albumin. Therefore, nitroxidized-albumin may be a potential trigger for autoantibodies in RA patients.

1.60 Autoantibodies to human citrullinated fibrinogen (AhFibA) and their subfamilies directed to the fibrin peptides α36-50Cit38,42 and β60-7460,72,74 are prognostic markers of radiographic damage in the very early arthritides of the French ESPOIR cohort

Background and ObjectiveIn established RA, autoantibodies (AAB) to human citrullinated fibrinogen (AhFibA) were demonstrated to be mainly composed of two subfamilies of AAB directed to immunodominant epitopes borne by the...

THU0093 Deiminated Histone 4 from Neutrophil Extracellular Traps is a Novel Autontigen in Rheumatoid Arthritis

BackgroundHistone deimination is a crucial event in cell biology, regulating gene function and contributing to antimicrobial response, through the formation of neutrophil extracellular traps (NETs.) Deiminated proteins are target of...

OP0120 Specific Identification of Anti-Citrullinated Glucose-6-Phospate Isomerase Peptide (CCG) Antibodies Associated with HLA-DRB1 SE and Disease Activity in Patients with RA

BackgroundAnti-citrullinated protein antibodies (Abs) (ACPA) are identified as a hallmark of diagnosis and disease progression in sera of patients with RA. Although several ACPA targeting citrullinated fibrinogen, collagen type II,...

Neutrophil extracellular traps are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis (P4061)

Abstract The early events leading to the development of rheumatoid arthritis remain unclear but formation of autoantibodies to citrullinated antigens is considered a key pathogenic phenomenon. Neutrophils isolated from patients with various autoimmune diseases display enhanced extracellular trap formation, a phenomenon that externalizes autoantigens and immunostimulatory molecules. We investigated if aberrant NETosis occurs in RA, its triggers and deleterious inflammatory consequences. Enhanced NETosis was observed in circulating and synovial fluid RA neutrophils. NETosis correlated with ACPA levels and with systemic inflammatory markers. RA sera and immunoglobulin fractions from RA patients with high levels of ACPA and/or rheumatoid factor significantly enhanced NETosis, and the NETs induced by these autoantibodies displayed distinct protein content. During NETosis, neutrophils externalized citrullinated autoantigens implicated in RA pathogenesis, while anti-citrullinated vimentin antibodies potently induced NET formation. In turn, NETs significantly augmented inflammatory responses in RA and OA synovial fibroblasts, including induction of IL-6, IL-8, chemokines and adhesion molecules. These observations implicate accelerated NETosis in RA pathogenesis, through externalization of citrullinated autoantigens and immunostimulatory molecules that may promote aberrant immune responses in the joint and in the periphery and perpetuate pathogenic mechanisms in this disease.

Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is characterised by autoimmunity to citrullinated proteins, and there is increasing epidemiologic evidence linking Porphyromonas gingivalis to RA. P gingivalis is apparently unique among periodontal pathogens in...

Absence of Autoantibodies Against Oral and Vascular-Related Cell Lines in the Sera of Patients with Behcet's Disease

Behcet's disease (BD) is an autoinflammatory disease with unclear pathogenesis. The oral and vascular tissue are the main target organs in BD. The role of humoral immunity in pathogenesis of oral and vascular lesions in BD patients has not been well studied. Therefore, the aim of this study was to investigate the presence of autoantibodies in the sera of BD patients using oral and vascular tissue related cell lines. Proteins from oral (KB, HGF-1) and vascular (HUVEC) related cell lines as well as C2C12 (a muscle myoblast cell line) were extracted as representatives of oral and vascular tissue antigens and the presence of autoantibodies in BD's sera were investigated using high throughput two dimensional electrophoresis (2DE) and immunoblotting techniques. Sera of other autoimmune diseases (RA and SLE) and normal individuals were used as controls. After silver staining of 2DE gels, 2831, 2195, 1732, and 1839 spots were detectable in the proteome map of HUVEC, KB, HGF-1, and C2C12 cell lines, respectively. The majority of spots were in the pH range of 5 - 8 and the molecular weight range of 14 - 66 Kd. The immunoreactivity of BD, RA, SLE, and normal sera were not different with separated proteins of the cell lines. According to our results, it seems that humoral immunity is not significantly involved in BD pathogenesis. Therefore, investigation of the role of cellular immunity, especially TH1 and TH17 cells and their cytokine profiles, in the pathogenesis of BD is recommended for future studies.

Prevalence of anti-toxoplasma antibodies in patients with autoimmune diseases

The identification of etiological factors in the induction of autoimmunity has remained elusive despite an enormous effort at dissection of the molecular structure of the target antigens and effector mechanisms. One characteristic feature of autoantigens is their repetitive structure as well as their conservation and evolution. Toxoplasma (T.) gondii is a primitive protozoan. We hypothesized that patients with autoimmune disease would have broad reactions against Toxoplasma antigens based on autoantigen conservation.To address this issue, we assessed serologic evidence of reactivity to Toxoplasma gondii along with a large profile of autoantibodies in patients with various autoimmune diseases (AID). We included sera of 1514 patients with 11 different AID collected from referral centers in Europe and Latin America as well as from 437 geographically matched controls, for the prevalence of anti Toxoplasma antibodies (ATxA) IgG and IgM and serum autoantibodies utilizing the BioPlex 2200 system (Bio- Rad Laboratories, USA).Serum ATxA IgG were positive in 42% of patients with AID versus 29% of controls (p < 0.0001). Among Europeans, ATxA IgG were associated with anti-phospholipid syndrome (APS; p < 0.0001), cryoglobulinemia (p < 0.0001), ANCA-associated vasculitides (p < 0.01), autoimmune thyroid diseases (p < 0.0001), systemic sclerosis (SSc; p < 0.0001) and rheumatoid arthritis (RA; p < 0.0001). Of note, Latin American RA sera exhibited similar frequency of ATxA IgG as controls. ATxA IgM were more prevalent in European patients with APS (p < 0.01), SSc (p < 0.05) and inflammatory bowel disease (IBD, p < 0.05) than in controls. Further, in AID patients the presence of ATxA correlated with autoantibodies characteristic of APS (anti- cardiolipin, B2GPI, complex of cardiolipin- B2GPI, prothrombin, phosphatydilethanolamine), and of SSc (anti-centromere, Scl-70).Our findings suggest that T. gondii may contribute to the pathogenesis of AID. This interaction may depend on or explain observed geoepidemiological variance in AID.

Anti-tissue transglutaminase antibodies in inflammatory and degenerative arthropathies

Recent studies identified tissue transglutaminase (tTG) as the antigen eliciting antiendomysial antibodies (EMA) in celiac disease (CD). Anti-tTG antibodies have therefore been proposed as a serological test for CD. Nevertheless, IgA anti-tTG but not EMA have also been found in inflammatory bowel disease patients, suggesting that these antibodies are linked to a tissue lesion rather than to an auto-immune component of CD. To confirm this hypothesis, we evaluated the presence of IgA anti-tTG in patients with inflammatory and degenerative diseases, in whom tissue lesions presented far away from the intestinal mucosa. The study was carried out on the serum and synovial fluid (SF) of 68 patients with rheumatoid arthritis (RA=33), psoriatic arthritis (PsA=26) and osteoarthritis (OA=9). In RA, PsA and OA sera, IgA anti-tTG were positive in 33%, 42% and 11% of patients, respectively. Serum anti-tTG levels were significantly higher in RA (p<0.0001), PsA (p<0.0001) and OA (p<0.02) with respect to healthy controls. SF anti-tTG levels were significantly higher in PsA (p<0.018) than in OA. A good correlation between serum and synovial fluid anti-tTG levels was found in all arthropathies This study suggests that tTG is not the only antigen of EMA and, furthermore, that IgA anti-tTG antibodies represent a general lesion-associated event. Moreover, the significant correlation between serum and synovial fluid anti-tTG levels allow us to hypothesise that these antibodies could be synthesized in the site of arthritic lesions.

Usefulness of anti-cyclic citrullinate peptide antibody determination in synovial fluid analysis of patients with rheumatoid arthritis

To assess the role of anti-cyclic citrullinated peptide (CCP) antibody detection in synovial fluid (SF) of RA patients compared to OA patients. We evaluated in 25 RA subjects and 14 OA patients, presenting a knee-joint effusion, the main clinical and laboratory parameters including the number of painful and/or swollen joints, Ritchie index, morning stiffness, ESR, CRP and analysis of SF obtained by therapeutic arthrocentesis. IgG anti-CCP (ELISA), rheumatoid factor (RF) and total IgG (nephelometry method) were measured in SF and paired serum samples. We found anti-CCP antibodies and RF in 64% (16/25) and 60% (15/25) of RA sera, respectively; 72% (18/25) of RA patients were positive for anti-CCP antibodies or RF. We found a higher SF/serum ratio for anti-CCP (p<0.004) compared to that for total IgG. The calculation of anti-CCP concentration as IgG anti-CCP (units)/total IgG (g L-1) revealed higher values in SF than in serum (p<0.046) in RA patients. Among these, correlation analysis showed that anti-CCP/total IgG values in SF correlated with the relative concentration of serum anti-CCP/total IgG (rs=0.842; p<0.00001) and serum anti-CCP antibody levels (rs=0.799; p<0.0001). We did not find any correlation between SF anti-CCP levels and the main characteristics of SF as well as the clinical or laboratory parameters. Our study give evidence for a preferential production of anti-CCP antibodies at RA joint level, confirming the pathogenetic role of these autoantibodies. Moreover, SF determination of anti-CCP, corrected for the total amount of the corresponding immunoglobulin, may be helpful as diagnostic tool in selected cases.

Targeted Synovial Fluid Proteomics for Biomarker Discovery in Rheumatoid Arthritis

AbstractObjectiveRheumatoid arthritis (RA) is an autoimmune disease that targets the synovium. The autoantigens involved in the autoantibody responses in RA are unknown. A targeted proteomics approach was used to identify proteins in RA synovial fluid (SF) that are recognized by autoantibodies in RA sera.MethodsRA SF, depleted of abundant proteins, was fractionated by two-dimensional liquid chromatography (chromatofocusing followed by reverse phase HPLC). Protein arrays constructed from these fractions were probed with RA and normal control sera, and proteins within reactive fractions were identified by mass spectrometry. The reactivity of RA sera to an identified peptide was confirmed by ELISA.ResultsRA sera specifically reacted to a SF fraction containing fibrin. Mass spectrometry analyses established the presence of a citrullinated arginine at position 271 of the fibrin fragment present in RA SF. A synthetic peptide corresponding to fibrin residues 259–287, containing the citrulline substitution at Arg 271, was recognized by 10 of 12 RA sera, but by two of 18 normal control sera and one of 10 systemic lupus erythematosus sera.ConclusionProteomics methodology can be used to directly characterize post-translational modifications in candidate autoantigens isolated from sites of disease activity. The finding that RA sera contain antibodies to the citrullinated fibrin 259–287 peptide may ultimately lead to improved diagnostic tests for RA and/or biomarkers for disease activity.

Comparison of Three Anti-CCP Antibody Tests and Rheumatoid Factor in RA and Control Patients

It was the aim of this study to compare the diagnostic properties of two second generation anti-CCP antibodies assays (Quanta Lite CCP, Inova and EliA CCP, Phadia) with an assay containing a new antigen mixture (Quanta Lite CCP 3, Inova) and RF IgM. Sera from 86 RA patients and 90 control patients with other diseases, such as infections, connective tissue diseases, or other rheumatic diseases, were analysed using the four different methods. Specificity (84.4%) and PPV (80.3%) were lowest for RF IgM and highest for EliA CCP (specificity 97.8%, PPV 96.7%), the other values being close to those of EliA CCP. Sensitivity was highest for Quanta Lite CCP (73.3%). Efficiency was highest for Quanta Lite CCP (84.1%) at the lower cut off followed by EliA CCP at both cut offs (83.0%). The discrimination between RA sera (mean value 407.67 U/ml) and control sera (4.54 U/ml) and the relative risk (23.34) were best for EliA CCP. The results of this study reveal that there was no advantage of the anti-CCP antibodies assay applying a new antigen mixture (Quanta Lite CCP 3 ELISA) compared to two second generation anti-CCP antibodies assays (Quanta Lite CCP ELISA and EliA CCP).

Development of immunoassays for serum tartrate-resistant acid phosphatase isoform 5a

Background Serum tartrate-resistant acid phosphatase (TRACP) consists of 2 structurally related isoforms, TRACP 5a and 5b. TRACP 5b is from bone-resorbing osteoclasts. TRACP 5a may be a macrophage product of inflammation. We used a novel antibody to TRACP 5a to standardize immunoassays for serum TRACP 5a activity and protein. Methods Biotinylated anti-TRACP antibodies were used to immobilize serum TRACP isoforms. TRACP activity was measured using 4-nitrophenyl phosphate as substrate. TRACP 5a protein was measured with an independent peroxidase-conjugated anti-TRACP antibody. Immunoassays were standardized for linearity of serum dose response, sensitivity and precision. Reference ranges for TRACP 5a were established from serum of 50 healthy males and 50 healthy age-matched females. Serum TRACP 5a activity and protein were determined in 29 cases of rheumatoid arthritis. Results Serum matrix interference in both TRACP 5a assays required dilution to 10% serum to approach linearity. Intra-assay and inter-assay CV% were <10%. Mean serum TRACP 5a activity and protein were significantly higher in healthy men than women. There was a slight, but significant age related increase in both serum TRACP 5a and 5b among females, but not males, from age 20 to 70 years. TRACP 5a activity was positively correlated to TRACP 5a protein in healthy sera. Neither TRACP 5a activity nor protein was correlated strongly to TRACP-5b activity. TRACP 5a protein was significantly increased in 8/29 RA sera, whereas TRACP 5a and 5b activities were not. TRACP 5a activity and protein were not significantly correlated in RA sera. Conclusions Although TRACP 5a and 5b are related biosynthetically, their circulating levels in healthy humans were independent, suggesting differential regulation of expression. In chronic diseases, increased TRACP 5a may represent pathological processes of inflammation unrelated to bone metabolism.

Anti-α-fodrin antibodies do not add much to the diagnosis of Sjögren's syndrome

The presence of anti-α-fodrin autoantibodies has been reported to be a highly specific and sensitive test for the diagnosis of Sjögren's syndrome (SjS). We looked (in Nijmegen) for anti-α-fodrin, anti-Ro60, and anti-La autoantibodies in a cohort of 51 patients with rheumatic diseases (primary SjS [21], secondary SjS [6], rheumatoid arthritis [RA] [12], systemic lupus erythematosus [SLE] [6], and scleroderma [6]) and in 28 healthy subjects, using ELISA, immunoblotting, and immunoprecipitation. The same samples were analyzed with an alternative anti-α-fodrin ELISA in Hanover. The Nijmegen ELISA of the sera from primary SjS showed sensitivities of 43% and 48% for IgA- and IgG-type anti-α-fodrin antibodies, respectively. The Hanover ELISA showed sensitivities of 38% and 10% for IgA- and IgG-type anti-α-fodrin antibodies, respectively. The ELISAs for α-fodrin showed six (Nijmegen) and four (Hanover) anti-α-fodrin-positive RA sera. IgA and IgG anti-fodrin antibodies were also present in four patients with secondary SjS. The sensitivities of Ro60 and La-antibodies in the Nijmegen ELISA were 67% and 62%, respectively. Unlike anti-α-fodrin antibodies, all anti-Ro60 and anti-La positive sera could be confirmed by immunoblotting or RNA immunoprecipitation. Thus, anti-Ro and anti-La autoantibodies were more sensitive than anti-α-fodrin autoantibodies in ELISA and were more frequently confirmed by other techniques. Anti-La antibodies appear to be more disease-specific than anti-α-fodrin antibodies, which are also found in RA sera. Therefore, the measurement of anti-α-fodrin autoantibodies does not add much to the diagnosis of Sjögren's syndrome.