Serum reactivity in dermatitis herpetiformis: an international multicentre study.

BACKGROUND: Dermatitis herpetiformis (DH) is a rare gluten-induced skin disorder characterized predominantly by IgA autoantibodies against endomysium, tissue transglutaminase (TG2/tTG), epidermal transglutaminase (TG3/eTG) and deamidated gliadin. To date, circulating autoantibody reactivity has not been systematically described. OBJECTIVES: Characterization of serum reactivities in DH. METHODS: This multicentre international study analysed sera from 242 patients with DH taken at the time of initial diagnosis. DH-specific IgA and IgG serum autoantibodies were analysed by indirect immunofluorescence (IF) on monkey oesophagus, and by enzyme-linked immunosorbent assay (ELISA) based on recombinant TG2/tTG, TG3/eTG and deamidated gliadin (GAF3X). RESULTS: IgA indirect IF microscopy on monkey oesophagus revealed the highest reactivity (84.3%; specificity 100%) followed by IgA TG2/tTG ELISA (78.5%, specificity 99.0%), IgA TG3/eTG ELISA (72.7%, specificity 95.0%) and IgA GAF3X ELISA (69.0%, specificity 98.5%). CONCLUSIONS: Serum IgA and IgG autoantibodies against endomysium, TG2/tTG, TG3/eTG and deamidated gliadin are highly prevalent in DH. Indirect IF microscopy on monkey oesophagus (IgA) provides the highest diagnostic accuracy that can be further enhanced by 4.5% when combined with IgA TG2/tTG ELISA.

Is periodontitis a prognostic factor in order to indicate antibodies against citrullinated peptides in patients with rheumatoid arthritis?

OBJECTIVES: In this cross-sectional study we investigated antibody titres against cyclic citrullinated peptides derived from filaggrin (anti-CCP) and citrullinated α-enolase (anti-CEP-1) among patients with RA as a function of periodontal findings. METHODS: 107 patients with RA (median age 56 years, 75% females) were included. For periodontal diagnoses missing teeth, periodontal epithelial surface area, periodontal inflamed surface area and periodontal diagnosis according to the working group's guidelines of the Center for Disease Control and Prevention were determined. Subgingival bacterial DNA of five periodontopathic bacteria was assessed by PCR with sequence-specific oligonucleotides. Anti-CCP and anti-CEP-1 antibodies in plasma samples were investigated using enzyme-linked immunosorbent assays. Low resolution human leukocyte antigen (HLA) typing was carried out using PCR with sequence-specific primers. RESULTS: PESA was found associated with a low adjusted odds ratio for anti-CCP positivity (OR=1.002, p=0.040). All patients who were infected with Aggregatibacter actinomycetemcomitans were simultaneously anti-CCP positive (p=0.043). HLA-DRB1*13 lowered the adjusted odds ratio for anti-CCP (OR=0.073, p=0.002) and anti-CEP-1 (OR=0.068, p=0.018) positivity whereas HLA-DRB1*07 indicated a lower risk only for demonstrable anti-CCP antibodies (OR=0.079, p=0.004). HLA-DRB1*04 was associated with increased adjusted odds ratio for anti-CEP-1 positivity (OR=4.154, p=0.005) and the simultaneous proof of both investigated autoantibodies (OR=3.725, p=0.011). CONCLUSIONS: Among patients with RA periodontitis may be a minor risk factor for anti-CCP positivity. Our data first provide evidence that an infection with A. actinomycetemcomitans is associated with an increased formation of anti-CCP. HLA phenotype proved to be a significant risk indicator for both investigated antibodies.

What is the clinical significance of anti-Sm antibodies in systemic lupus erythematosus? A comparison with anti-dsDNA antibodies and C3.

OBJECTIVES: To investigate the clinical value of anti-Sm antibodies in diagnosis and monitoring of systemic lupus erythematosus (SLE) and their ability to predict lupus flares compared with that of anti-dsDNA antibody and complement (C3) assays. METHODS: Autoantibodies against Smith antigen (Sm) and double-stranded DNA (dsDNA) in sera from SLE (n=232), myositis (n=26), systemic sclerosis (n=81), Sjögren's syndrome (n=88), and rheumatoid arthritis patients (n=165) and healthy donors (n=400) were determined by using enzyme-linked immunosorbent assays (both from Euroimmun). New thresholds for both autoantibodies were calculated by receiver operating characteristics (ROC) curve analysis. Cross-sectional, longitudinal and predictive analyses of anti-Sm and disease activity were also performed. RESULTS: Sensitivities of 25.9% for anti-Sm (cut-off: 3.6 relative units/ml) and 30.2% for anti-dsDNA (cut-off 157.4 international units/ml) were obtained at a specificity of 99%. 14.8% of anti-dsDNA-negative patients were positive for anti-Sm, and more than half (51.4%) of anti-dsDNA-positive patients were also positive for anti-Sm. Anti-Sm antibodies were associated with age (p=0.0174), the number of ACR criteria (p=0.0242), the ACR criteria renal (p=0.0350) and neurologic disorder (p=0.0239), the BILAG category constitutional symptoms (p=0.0227), fatigue (p=0.0311) and cross-sectional disease activity (r=0.2519, p=0.0224). Although no correlations with lupus activity were observed in the longitudinal and predictive analysis, a remarkable association was found between anti-Sm and proteinuria. CONCLUSIONS: Anti-Sm antibodies are essential for diagnosis of SLE, especially in anti-dsDNA-negative patients. However, our data suggest that anti-Sm monitoring is only helpful in SLE patients with active lupus nephritis.

Prospective studies on the routine use of a novel multivariant enzyme-linked immunosorbent assay for the diagnosis of autoimmune bullous diseases.

BACKGROUND: Serologic diagnosis of autoimmune blistering disease (AIBD) usually follows a sophisticated multistep algorithm. OBJECTIVE: We sought validation of a multivariant enzyme-linked immunosorbent assay (ELISA) in the routine diagnosis of AIBD. METHODS: The multivariant ELISA comprising 6 recombinant immunodominant forms of major AIBD target antigens, ie, desmoglein 1, desmoglein 3, envoplakin, BP180, BP230, and type VII collagen was applied in: (1) a cohort of well-characterized AIBD (n = 173) and control sera (n = 130), (2) a prospective multicenter study with 204 sera from patients with newly diagnosed AIBD with positive direct immunofluorescence microscopy, and (3) a prospective monocenter study with 292 consecutive sera from patients with clinical suspicion of AIBD in comparison with the conventional multistep diagnostic algorithm. RESULTS: Concordant results in the multivariant ELISA compared with direct immunofluorescence microscopy were seen in 94% of patients with pemphigus and 71% of patients with pemphigoid (Cohen κ value, 0.95 and 0.66) and with the conventional multistep diagnostic approach in 91% of patients with pemphigus and 88% of patients with bullous pemphigoid and 93% of autoantibody-negative sera (Cohen κ, 0.95, 0.84, and 0.78). LIMITATIONS: IgA autoantibodies and less common target antigens were not analyzed. CONCLUSIONS: The multivariant ELISA is a practical, highly standardized, and widely available novel diagnostic tool for the routine diagnosis of AIBD.

Association of levels of antibodies against citrullinated cyclic peptides and citrullinated α-enolase in chronic and aggressive periodontitis as a risk factor of Rheumatoid arthritis: a case control study.

BACKGROUND: Periodontal disease could be a risk factor for rheumatoid arthritis (RA). It is assumed that the bacterial strain Porphyromonas gingivalis mediates citrullination of host peptides and thereby the generation of RA-associated autoantibodies in genetically predisposed individuals. For that reason non-RA individuals who suffered from generalized aggressive (GAgP, N = 51) and generalized chronic periodontitis (GChP, N = 50) were investigated regarding the occurrence of antibodies against citrullinated cyclic peptides (anti-CCP) and citrullinated α-enolase peptide-1 (anti-CEP-1) in comparison to non-RA non-periodontitis controls (N = 89). Furthermore, putative associations between infections with five periodontopathic bacteria or expression of certain human leucocyte antigens (HLA) to these autoantibodies were investigated. METHODS: The presence of anti-CCP and anti-CEP-1 in plasma samples was conducted with enzyme linked immunosorbent assay. Subgingival plaque specimens were taken from the deepest pocket of each quadrant and pooled. For detection of DNA of five periodontopathic bacteria PCR with sequence specific oligonucleotides was carried out. Low resolution HLA typing was carried out with PCR with sequence specific primers. Differences between patients and controls were assessed using Chi square test with Yates correction or Fisher`s exact test if the expected number n in one group was <5. RESULTS: Two patients with GAgP (3.9%), no patient with GChP and two controls (2.2%, pFisher = 0.662) were positive for anti-CEP-1 whereas no study participant was anti-CCP positive. Individuals with P. gingivalis were slightly more often anti-CEP-1 positive in comparison to individuals without P. gingivalis (3.2 vs. 1.1%, pFisher = 0.366). Carrier of HLA-DQB1*06 or the HLA combination DRB1*13; DRB3*; DQB1*06 were slightly more anti-CEP-1 positive (6.1 and 4.3%) than no carriers (0.7 and 0%, pFisher 0.053). CONCLUSIONS: GAgP and GChP and the presence of periodontopathic bacteria are not associated with an increased risk for occurrence of anti-CCP and anti-CEP-1 autoantibodies. The putative relationship between periodontitis and RA should be investigated in further studies.

IFNα and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus.

OBJECTIVES: To evaluate and compare the clinical efficacy of three biomarkers for interferon (IFN) activity (measured directly and indirectly) and six traditional biomarkers in indicating current and prospective disease activity (DA) in systemic lupus erythematosus (SLE). METHODS: IFNα (dissociation-enhanced lanthanide fluorescent immunoassay), IFNγ-inducible protein 10 (IP-10) (ELISA) and sialic acid-binding Ig-like lectin 1 (SIGLEC-1) (flow cytometry) were measured in 79 accurately characterised patients with lupus and compared with serum titres of Anti-dsDNA (ELISA and radioimmunoassay), Anti-dsDNA-NcX ELISA, Anti-Nuc ELISA, and complement C3 and C4. DA was evaluated using the British Isles Lupus Assessment Group 2004 Index (BILAG-2004) and a modified SLE Disease Activity Index-2000 (mSLEDAI-2K). In addition, 31 clinically quiescent patients were monitored for flares over the course of 180 days. RESULTS: Increased levels of IFNα, IP-10 and SIGLEC-1 were found in 32%, 50% and 86%, respectively, of 66 patients with active SLE. IFNα (r=0.45; p<0.0001) and SIGLEC-1 (r=0.54; p<0.0001) correlated better with BILAG-2004 than did IP-10 (r=0.38; p=0.0002), Farr assay (r=0.40; p=0.0001), Anti-dsDNA-NcX ELISA (r=0.28; p=0.0061), Anti-dsDNA ELISA (r=0.31; p=0.0025), Anti-Nuc ELISA (r=0.25; p=0.0121), C3 (r=-0.43; p<0.0001) and C4 (r=-0.33; p=0.0013). Predictors of SLE flares were disease duration ≤92 months, mild clinical activity (in contrast with no activity), complement C3≤89 mg/dl and IFNα≥20 pg/ml, while only lymphocyte count and age were independent predictors in multivariate analysis. CONCLUSIONS: IFNα, IP-10 and SIGLEC-1 emerged as beneficial biomarkers of DA in patients with SLE. Therefore the implementation of IFN biomarkers in standard lupus diagnostics should be reappraised, especially in view of emerging anti-IFN-directed therapies.

Development of a recombinant cell-based indirect immunofluorescence assay for the determination of autoantibodies against soluble liver antigen in autoimmune hepatitis.

Autoantibodies against soluble liver antigen (SLA) are specific markers for autoimmune hepatitis (AIH) type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA), detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA) to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors). Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases.

Significance of antibodies against the native ribosomal P protein complex and recombinant P0, P1, and P2 proteins in the diagnosis of Chinese patients with systemic lupus erythematosus.

BACKGROUND: This study aimed to evaluate autoantibodies against the native ribosomal P complex (anti-Rib-P(C)) and recombinant ribosomal P proteins (anti-Rib-P0, anti-Rib-P1, anti-Rib-P2) for their prevalence, diagnostic relevance and clinical associations in a Chinese cohort with systemic lupus erythematosus (SLE). METHODS: Anti-Rib-P, anti-dsDNA and anti-Smith antigen (Sm) antibodies were analyzed in sera from 198 patients with SLE, 33 with rheumatoid arthritis, 61 with Sjögren's syndrome and 70 healthy individuals by means of ELISA. RESULTS: Antibody prevalences were 29.8% (anti-Rib-P(C)), 33.3% (anti-Rib-P0), 42.9% (anti-Rib-P1) and 34.3% (anti-Rib-P2), at a specificity of 99%. Among SLE patients lacking anti-dsDNA and anti-Sm, 27.8% showed positive for at least one of the investigated anti-Rib-P types. The serological hit rate provided by anti-dsDNA/anti-Sm detection (72.7%) was increased upon parallel testing for anti-Rib-P(C) (77.3%) or anti-Rib-P0/P1/P2 (80.3%). Anti-Rib-P positivity was associated with disease activity, neuropsychiatric events, lupus nephritis, skin rash, lymphocytopenia, increased erythrocyte sedimentation rates, decreased complement C3/C4 and elevated IgA/IgG levels. CONCLUSION: Based on these results, antibodies against ribosomal P proteins are important complementary parameters to anti-dsDNA and anti-Sm, and should be considered for inclusion in the classification criteria for SLE. The diagnostic value of anti-Rib-P0/P1/P2 is diagnostically superior to that of anti-Rib-P(C).

Novel assay for detecting celiac disease-associated autoantibodies in dermatitis herpetiformis using deamidated gliadin-analogous fusion peptides.

BACKGROUND: Dermatitis herpetiformis (DH) is a cutaneous manifestation of celiac disease (CD), the latter being identified by circulating autoantibodies against native gliadin (nGli), tissue transglutaminase (tTG), endomysium, or a combination of these. Recently, a novel serologic assay using deamidated gliadin-analogous fusion peptides (GAF3X) showed high diagnostic sensitivity in patients with CD. OBJECTIVE: The aim of this study was to explore the anti-GAF3X enzyme-linked immunosorbent assay (ELISA) and to compare it with a panel of classic CD-related serologic tests in patients with DH. METHODS: Antibodies against nGli, GAF3X, and tTG were determined by separate ELISA tests and against endomysium by indirect immunofluorescence microscopy in 45 patients with DH and 52 control patients (30 patients with bullous pemphigoid and 22 patients with linear IgA disease). A total of 24 patients with DH underwent intestinal biopsies to confirm underlying CD. RESULTS: Antibodies to nGli were detected in 26 (58%) (IgA) and 24 (53%) (IgG), to GAF3X in 38 (84%) (IgA) and 36 (80%) (IgG), to tTG in 35 (78%) (IgA), and to endomysium in 32 (71%) (IgA) patients with DH. Combined testing of IgA and IgG antibodies to GAF3X detected 7 of 10 (70%) IgA-anti-tTG-negative patients with DH and together with the IgA-anti-tTG ELISA showed the highest serologic hit rate (93%) for CD. LIMITATIONS: Intestinal biopsies were not performed in all patients with DH. CONCLUSION: The novel anti-GAF3X ELISA shows a higher sensitivity to detect CD-associated autoantibodies in patients with DH compared with tests using nGli, tTG, or endomysium as substrates.

Determination of IgG and IgA antibodies against native gliadin is not helpful for the diagnosis of coeliac disease in children up to 2 years old.

OBJECTIVE: Assays for antibodies against native gliadin (anti-nGli) are still often assumed to perform better in the diagnosis of coeliac disease in young children than tests for antibodies to deamidated gliadin (anti-dGli), tissue transglutaminase (anti-tTG), and endomysium (EmA). We compared the performance of assays for anti-nGli, anti-dGli, anti-tTG, and EmA in this age group. METHODS: We investigated retrospectively 184 children (42 with coeliac disease under normal diet and 142 controls) up to 2 years of age. Immunoglobulin (Ig) A- and IgG-anti-dGli, IgA- and IgG-anti-nGli, IgA- and IgG-anti-tTG, and IgA-EmA were measured in serum. Areas under receiver operating characteristics curves, sensitivities, specificities, positive and negative predictive values, positive and negative likelihood ratios, as well as diagnostic odds ratios were calculated. RESULTS: From all of the tests investigated, only assays for IgG-anti-dGli, IgA-anti-tTG, and IgA-EmA had high specificity (≥ 0.96) connected with high sensitivity (≥ 0.86), with high positive predictive values (≥ 0.52 and ≥ 0.69 at pretest probabilities of 0.05 and 0.1, respectively) and negative predictive values (≥ 0.99 and ≥ 0.98 at pretest probabilities of 0.05 and 0.1, respectively). These assays also showed high positive likelihood ratio (≥ 24) at low negative likelihood ratio (≤ 0.15) and high diagnostic odds ratios (≥ 136). CONCLUSIONS: Our results do not support the use of assays of anti-nGli to diagnose coeliac disease in young children. IgA-anti-tTG, IgA-EmA, and IgG-anti-dGli perform better than anti-nGli.

Serodiagnosis of Anti-glomerular Basement Membrane Disease Using a Newly Developed Chemiluminescence Immunoassay.

Circulating autoantibodies directed against the kidney glomerular basement membrane (GBM) antigens are important markers in the diagnosis and monitoring of autoimmune glomerulonephritides, including the classic Goodpasture's syndrome. Rapid and reliable diagnostic tools for the detection of anti-GBM autoantibodies are crucial as anti-GBM disease can progress rapidly and, if too late or incorrectly diagnosed, can have serious, even fatal consequences. The performance of the newly developed standardized chemiluminescence immunoassay (ChLIA) was evaluated in comparison with the established Anti-GBM ELISA (IgG) (EUROIMMUN). For the assessment of its diagnostic performance, sera from 67 clinically characterized anti-GBM disease patients and 221 disease controls were analyzed. The clinical sensitivity of the Anti-GBM ChLIA (IgG) reached 100% at a specificity of 98.6%. The Anti-GBM ELISA (IgG) performance was less sensitive (89.6%) without any positive findings in the control group, indicating a specificity of 100%. Both methods were homogeneous (κ = 0.901). The Anti-GBM ChLIA (IgG) represents a promising alternative tool for accurate anti-GBM assessment in routine diagnostic settings with the advantage of rapid turnaround time and fully automated random-access processing.

A New Chemiluminescence Immunoassay for Phospholipase A2 Receptor 1 Autoantibodies Allows Early Identification of Autoantibody Recurrence in Patients With Membranous Nephropathy.

BACKGROUND: Circulating autoantibodies against the M-type phospholipase A2 receptor 1 (PLA2R1) are important biomarkers in membranous nephropathy (MN), supporting the diagnosis and the clinical monitoring of patients. Standardized recombinant cell-based indirect immunofluorescence assay (RC-IFA) and enzyme-linked immunosorbent assay (ELISA) are widely established for the detection of anti-PLA2R1 autoantibodies (PLA2R1-ab). The RC-IFA provides higher sensitivity than the ELISA, but lacks exact graduated quantification of antibody levels. In this study, we evaluated the diagnostic performance of a novel PLA2R1-ab immunoassay based on chemiluminescence (ChLIA) by comparing it to RC-IFA and ELISA in samples from patients with MN with different diagnostic scenarios. METHODS: Serum samples from patients with biopsy-proven MN and disease controls were analyzed for PLA2R1-ab by ChLIA, ELISA, and RC-IFA. RESULTS: The ChLIA demonstrated almost perfect agreement with RC-IFA for the identification of patients with PLA2R1-associated MN, while additionally allowing fine-graduated quantification of PLA2R1-ab levels. In patients with a relapse of MN, the ChLIA allowed an earlier detection of PLA2R1-ab recurrence by at least 3 months in 63% of cases compared with the ELISA. CONCLUSIONS: The PLA2R1-ab ChLIA had the same excellent diagnostic performance as the RC-IFA and outperformed the ELISA in the diagnosis of MN and the early identification of relapses. It thus presents a favorable tool for accurate PLA2R1-ab assessment in routine diagnostic settings, while enabling fast processing and fully automated random-access implementation.

Novel ELISA systems for antibodies to desmoglein 1 and 3: correlation of disease activity with serum autoantibody levels in individual pemphigus patients.

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are intraepidermal blistering skin diseases. PV is characterised by autoantibodies directed against desmoglein (Dsg) 3 and in patients with the mucocutaneous variant also against Dsg 1, whereas in PF, only Dsg 1 is targeted. Here, ectodomains of Dsg 3 and Dsg 1 were recombinantly expressed in a human cell line (HEK293) and applied as authentic solid phases in ELISA test systems. Autoantibodies against Dsg 3 and/or Dsg 1 could be detected in 71 (100%) of 71 PV sera and against Dsg 1 in 48 (96%) of 50 PF sera. Control sera showed reactivity with Dsg 3 and Dsg 1 in 0.2% and 0.7%, respectively, of 401 healthy blood donors and in 2.1% of 48 randomly selected patients with bullous pemphigoid. No reactivity with Dsg 1 and 3 was detected in 21 patients with linear IgA disease. For both pemphigus variants, a statistically significant correlation between clinical severity and autoantibody levels was observed as demonstrated for 10 PV and 5 PF patients. In conclusion, the use of the ectodomains of Dsg 3 and 1 as target antigens expressed in a human cell line resulted in sensitive and specific ELISA systems for both diagnosis and monitoring of PV and PF.

Development of a Standardized Chemiluminescence Immunoassay for the Detection of Autoantibodies Against Human M-Type Phospholipase A2 Receptor in Primary Membranous Nephropathy.

INTRODUCTION: Autoantibodies against the M-type phospholipase A2 receptor (PLA2R) are important markers in the diagnosis and monitoring of primary membranous nephropathy (pMN). For the detection of anti-PLA2R autoantibodies, a standardized recombinant cell-based indirect immunofluorescence assay (RC-IFA) and enzyme-linked immunosorbent assay (ELISA) are widely used, the former providing higher sensitivity but lacking a finely graduated quantification of antibody titers. In this study, we evaluated the diagnostic performance characteristics of a novel standardized chemiluminescence immunoassay (ChLIA) by comparison with the established anti-PLA2R test systems. METHODS: Sera from 155 patients with biopsy-proven pMN and 154 disease controls were analyzed for autoantibodies against PLA2R by the novel ChLIA as well as by ELISA and RC-IFA. RESULTS: The clinical sensitivity of the ChLIA (83.9%) was higher compared with ELISA (73.5%) and equaled that of RC-IFA (83.2%), at similar specificities (≥99.4%). Among ELISA-negative pMN samples, ChLIA and RC-IFA yielded positive results in 39.0% and 36.6%, respectively. The qualitative agreement amounted to 94.5% (ChLIA vs. ELISA) and 99.4% (ChLIA vs. RC-IFA). CONCLUSION: The novel anti-PLA2R ChLIA outperforms the ELISA in detecting patients with pMN and demonstrates almost perfect agreement with RC-IFA. It thus presents a promising alternative tool for accurate anti-PLA2R testing, with the advantage of rapid turnaround times and fully automated random-access processing.

New ELISA for detecting primary biliary cirrhosis-specific antimitochondrial antibodies.

BACKGROUND: Antimitochondrial antibodies specific for primary biliary cirrhosis (PBC) target the E2 subunits of 2-oxo acid dehydrogenase complexes, in particular the pyruvate dehydrogenase complex (PDC)-E2. Their antigen-specific detection relies on conventional ELISA using purified PDC. More recent assays have employed a hybrid containing the 3 E2-subunits (MIT3). Some PBC sera react with one or the other preparation, suggesting the presence of nonoverlapping epitopes. METHODS: We have developed an ELISA (anti-M2-3E) using a mixture of purified PDC and MIT3 as antigenic targets. We compared this assay to anti-MIT3 alone, conventional anti-PDC, and indirect immunofluorescence using 173 PBC and 247 disease controls. RESULTS: The anti-M2-3E ELISA showed a 93.6% diagnostic sensitivity compared with 91.3%, 83.8%, and 87.3% for MIT3, purified PDC, or indirect immunofluorescence, respectively, when all specificities are set to 98.8%. By immunoblotting, anti-M2-3E-positive sera unreactive to purified PDC recognized recombinant E2-subunits of the other 2 complexes, whereas those with no reactivity to MIT3 immunofixed PDC subunits E1alpha or E1beta. CONCLUSIONS: The diagnostic accuracy of the anti-M2-3E ELISA for detection of antibodies to 2-oxo acid dehydrogenase complexes exceeds that of conventional ELISA and IFL; its novelty derives from the combination of the MIT3 hybrid and purified PDC.

Serological Diagnosis of Autoimmune Bullous Skin Diseases.

Autoimmune bullous dermatoses (AIBD) encompass a variety of organ-specific autoimmune diseases that manifest with cutaneous and/or mucosal blisters and erosions. They are characterized by autoantibodies targeting structural proteins of the skin, which are responsible for the intercellular contact between epidermal keratinocytes and for adhesion of the basal keratinocytes to the dermis. The autoantibodies disrupt the adhesive functions, leading to splitting and blister formation. In pemphigus diseases, blisters form intraepidermally, whereas in all other disease types they occur subepidermally. Early identification of autoimmune bullous dermatoses is crucial for both treatment and prognosis, particularly as regards tumor-associated disease entities. The diagnosis is based on clinical symptoms, histopathology, direct immunofluorescence to detect antibody/complement deposits, and the determination of circulating autoantibodies. The identification of various target antigens has paved the way for the recent development of numerous specific autoantibody tests. In particular, optimized designer antigens and multiplex test formats for indirect immunofluorescence and ELISA have enhanced and refined the laboratory analysis, enabling highly efficient serodiagnosis and follow-up. This review elaborates on the current standards in the serological diagnostics for autoimmune bullous dermatoses.

Autoantibodies to Cytosolic 5'-Nucleotidase 1A in Primary Sjögren's Syndrome and Systemic Lupus Erythematosus.

Introduction: Autoantibodies to cytosolic 5'-nucleotidase 1A (cN-1A; NT5C1A) have a high specificity when differentiating sporadic inclusion body myositis from polymyositis and dermatomyositis. In primary Sjögren's syndrome (pSS) and systemic lupus erythematosus (SLE) anti-cN-1A autoantibodies can be detected as well. However, various frequencies of anti-cN-1A reactivity have been reported in SLE and pSS, which may at least in part be explained by the different assays used. Here, we determined the occurrence of anti-cN-1A reactivity in a large number of patients with pSS and SLE using one standardized ELISA. Methods: Sera from pSS (n = 193) and SLE patients (n = 252) were collected in five European centers. Anti-cN-1A, anti-Ro52, anti-nucleosome, and anti-dsDNA reactivities were tested by ELISA (Euroimmun AG) in a single laboratory. Correlations of anti-cN-1A reactivity with demographic data and clinical data (duration of disease at the moment of serum sampling, autoimmune comorbidity and presence of muscular symptoms) were analyzed using SPSS software. Results: Anti-cN-1A autoantibodies were found on average in 12% of pSS patients, with varying frequencies among the different cohorts (range: 7-19%). In SLE patients, the anti-cN-1A positivity on average was 10% (range: 6-21%). No relationship was found between anti-cN-1A reactivity and the presence or absence of anti-Ro52, anti-nucleosome, and anti-dsDNA reactivity in both pSS and SLE. No relationship between anti-cN-1A reactivity and duration of disease at the moment of serum sampling and the duration of serum storage was observed. The frequency of muscular symptoms or viral infections did not differ between anti-cN-1A-positive and -negative patients. In both disease groups anti-cN-1A-positive patients suffered more often from other autoimmune diseases than the anti-cN-1A-negative patients (15 versus 5% (p = 0.05) in pSS and 50 versus 30% (p = 0.02) in SLE). Conclusion: Our results confirm the relatively frequent occurrence of anti-cN-1A in pSS and SLE patients and the variation in anti-cN-1A reactivity between independent groups of these patients. The explanation for this variation remains elusive. The correlation between anti-cN-1A reactivity and polyautoimmunity should be evaluated in future studies. We conclude that anti-cN-1A should be classified as a myositis-associated-, not as a myositis-specific-autoantibody based on its frequent presence in SLE and pSS.

Development and evaluation of a standardized ELISA for the determination of autoantibodies against cN-1A (Mup44, NT5C1A) in sporadic inclusion body myositis.

PURPOSE: Sporadic inclusion body myositis (sIBM) is an autoimmune degenerative disease of the muscle, with inflammatory infiltrates and inclusion vacuoles. Its pathogenesis is not fully understood and the diagnosis is hampered by its imprecise characteristics, at times indistinguishable from other idiopathic inflammatory myopathies such as polymyositis and dermatomyositis. The diagnosis may be assisted by the detection of autoantibodies targeting Mup44, a skeletal muscle antigen identified as cytosolic 5'-nucleotidase 1A (cN-1A, NT5C1A). A novel standardized anti-cN-1A IgG ELISA was developed and its diagnostic performance was evaluated by two reference laboratories. METHODS: Recombinant human full-length cN-1A was expressed and purified, and subsequently utilized to set up a standardized ELISA. To evaluate the novel assay, laboratory A examined sera from North American patients with clinically and pathologically diagnosed definite sIBM (n = 17), suspected sIBM (n = 14), myositis controls (n = 110), non-myositis autoimmune controls (n = 93) and healthy subjects (n = 52). Laboratory B analyzed a Dutch cohort of definite sIBM patients (n = 51) and healthy controls (n = 202). RESULTS: Anti-cN-1A reactivity was most frequent in definite sIBM (39.2-47.1%), but absent in biopsy-proven classic polymyositis or dermatomyositis. Overall diagnostic sensitivity and specificity amounted to 35.5 and 96.1% (laboratory A) and 39.2 and 96.5% (laboratory B). CONCLUSIONS: Anti-cN-1A autoantibodies were detected by ELISA with moderate sensitivity, but high specificity for sIBM and may therefore help diagnose this infrequent and difficult-to-diagnose myopathy. The novel anti-cN-1A IgG ELISA can improve and accelerate the diagnosis of sIBM using sera where muscle biopsy is delayed or unfeasible.

Neuronal Na+/K+ ATPase is an autoantibody target in paraneoplastic neurologic syndrome.

OBJECTIVES: To identify an autoreactivity in a 66-year-old woman who presented with combined brainstem and cerebellar syndrome including vertical gaze palsy, severe progressive ataxia, and spastic tetraparesis, an acute deterioration of vision, dysarthria, and dysphagia with concurrent diagnosis of a colon adenocarcinoma. METHODS: Patient's serum and CSF underwent comprehensive autoantibody screening by indirect immunofluorescence assay and immunoblot. For autoantigen purification, a histo-immunoprecipitation technique was developed followed by mass spectrometrical analysis. Recombinant candidate antigens were expressed in HEK293 and used to verify the identification. RESULTS: Indirect immunofluorescence assay screening revealed strong immunoglobulin G reactivity with neural tissues in serum and CSF, but not with a panel of 28 recombinantly expressed established neural autoantigens. The hitherto unknown target antigen was identified as the neuronal Na(+)/K(+) ATPase. Epitope mapping and competitive inhibition experiments showed that the autoantibodies were directed against the membrane-spanning alpha 3 subunit (ATP1A3) of the enzyme but did not bind to extracellular epitopes. Immunohistochemical analysis revealed overexpression of this subunit in the patient's tumor. CONCLUSIONS: We describe a case of an anti-ATP1A3-associated neurologic disorder. Mutations in the gene encoding this neuronal surface protein have already been recognized as the cause of infantile alternating hemiplegia, rapid-onset dystonia parkinsonism, and CAPOS syndrome. Although the autoantibodies are unlikely to be pathogenic, they are likely to be rare biomarkers for the apparently paraneoplastic neurologic syndrome or for the tumor itself.