Addressable Laser Bead Immunoassay Research Articles

Scurfy Mice Develop Features of Connective Tissue Disease Overlap Syndrome and Mixed Connective Tissue Disease in the Absence of Regulatory T Cells.

Due to a missense mutation in the Foxp3 gene, scurfy mice are deficient in functional regulatory T cells (Treg). The consequent loss of peripheral tolerance manifests itself by fatal autoimmune mediated multi-organ disease. Previous studies have outlined the systemic inflammatory disease and demonstrated production of anti-nuclear antibodies (ANA) in scurfy mice. However, specific autoantibody targets remained to be defined. ANA are immunological markers for several connective tissue diseases (CTD) and target a large number of intracellular molecules. Therefore, we examined scurfy sera for the presence of different ANA specificities and further assessed the organ involvement in these animals. Indirect immunofluorescence was used as a screen for ANA in the sera of scurfy mice and dilutions of 1/100 were considered positive. Addressable laser bead immunoassays (ALBIA) were used to detect specific autoantibody targets. Subsequent histological tissue evaluation was verified by hematoxylin and eosin (H&E) staining. In our study, we observed that nearly all scurfy mice produced ANA. The most prevalent pattern in scurfy sera was nuclear coarse speckled, also known as the AC-5 pattern according to the International Consensus on ANA Patterns. U1-ribonucleoprotein (U1RNP) was found to be the most common target antigen recognized by autoantibodies in scurfy mice. Additionally, scurfy mice exhibited a mild myositis with histological characteristics similar to polymyositis/dermatomyositis. Myopathy-specific autoantibody profile revealed significantly increased levels of anti-SMN (survival of motor neuron) as well as anti-Gemin3 antibodies in scurfy sera. Overall, we demonstrate that the impaired peripheral tolerance in the absence of regulatory T cells in scurfy mice is associated with features of mixed connective tissue disease (MCTD). This includes, along with our previous findings, very high titers of anti-U1RNP antibodies and an inflammatory myopathy.

Anti-NT5c1A Autoantibodies as Biomarkers in Inclusion Body Myositis.

Objective: Sporadic Inclusion Body Myositis (sIBM) is an inflammatory myopathy (IIM) without a specific diagnostic biomarker until autoantibodies to the cytosolic 5′-nucleotidase 1A (NT5c1A/Mup44) were reported. The objectives of our study were to determine the sensitivity and specificity of anti-NT5c1A for sIBM, demonstrate demographic, clinical and serological predictors for anti-NT5c1A positivity and determine if anti-nuclear antibody (ANA) indirect immunofluorescence (IIF) staining on HEp-2 cells is a reliable screening method for anti-NT5c1A.Methods: Sera from sIBM patients and controls were stored at −80°C until required for analysis. IgG antibodies to NT5c1A were detected by an addressable laser bead immunoassay (ALBIA) using a full-length human recombinant protein. Autoantibodies to other autoimmune myopathy antigens (Jo-1, OJ, TIF1y, PL-12, SAE, EJ, MDA5, PL7, SRP, NXP2, MI-2) were detected by line immunoassay (LIA), chemiluminescence immunoassay (CIA) or enzyme linked immunosorbent assay (ELISA) and ANA detected by IIF on HEp-2 substrate. Demographic, clinical and serological data were obtained by chart review.Results: Forty-three patients with sIBM, 537 disease control patients with other autoimmune, degenerative and neuromuscular diseases, and 78 healthy controls were included. 48.8% (21/43) of sIBM patients were positive for anti-NT5c1A. The overall sensitivity, specificity, positive predictive value, and negative predictive value of anti-NT5c1A for sIBM were 0.49, 0.92, 0.29, and 0.96, respectively. Compared to sIBM, the frequency of anti-NT5c1A was lower in both the disease control group (8.8%, OR 0.10 [95%CI: 0.05–0.20], p < 0.0001) and in the apparently healthy control group (5.1%, OR 0.06 [95%CI: 0.02–0.18], p < 0.0001). In the univariable analysis, sIBM patients with more severe muscle weakness were more likely to be anti-NT5c1A positive (OR 4.10 [95% CI: 1.17, 14.33], p = 0.027), although this was not statistically significant (adjusted OR 4.30 [95% CI: 0.89, 20.76], p = 0.069) in the multivariable analysis. The ANA of sIBM sera did not demonstrate a consistent IIF pattern associated with anti-NT5c1A.Conclusions: Anti-NT5c1A has moderate sensitivity and high specificity for sIBM using ALBIA. The presence of anti-NT5c1A antibodies may be associated with muscle weakness. Anti-NT5c1A antibodies were not associated with a specific IIF staining pattern, hence screening using HEp-2 substrate is unlikely to be a useful predictor for presence of these autoantibodies.

A case of aggressive atypical anti-GBM disease complicated by CMV pneumonitis

BackgroundAnti-glomerular basement membrane (anti-GBM) disease is characterized by circulating IgG glomerular basement membrane antibodies and is clinically expressed as a rapidly progressive crescentic glomerulonephritis (GN), with 30–60% of patients also developing pulmonary hemorrhage. Classically, the renal biopsy shows glomerular crescent formation, bright linear staining of glomerular basement membranes (GBM) for IgG on direct immunofluorescence (IF), and the serologic presence of circulating anti-GBM antibodies. Recently, patients with linear IgG IF staining, undetectable circulating anti-GBM antibodies and glomerular changes atypical for anti-GBM disease have been described as “atypical anti-GBM disease”, with a distinctly more benign clinical course than typical anti-GBM disease. We present a case report of a patient with negative anti-GBM serology but positive linear IgG staining by IF, severe diffuse crescentic and endocapillary proliferative glomerulonephritis, and renal failure, complicated by severe pulmonary hemorrhage after immunosuppression, likely due to cytomegalovirus (CMV) pneumonitis.Case presentationA 24-year-old man was admitted to hospital with hemoptysis and renal failure. Investigations for anti-GBM serology by addressable laser bead immunoassay (ALBIA) was negative for anti-GBM antibodies. Renal biopsy showed diffuse endocapillary proliferative glomerulonephritis with membranoproliferative features and diffuse circumferential crescents. Direct IF showed strong linear staining for IgG along GBMs. The patient’s hemoptysis improved with immunosuppression, but 1 month later he was readmitted with gross hemoptysis, which was refractory to further cyclophosphamide, plasma exchange and rituximab. Bronchoalveolar lavage (BAL) and blood work confirmed CMV pneumonitis, and the patient’s hemoptysis resolved with ganciclovir, though he became dialysis dependent.ConclusionsThis case demonstrates an atypical presentation of anti-GBM disease with both crescents and endocapillary hypercellularity and negative serology. The patient is dialysis dependent, unlike most previously described patients with atypical anti-GBM disease. The course was complicated by CMV pneumonitis, which contributed to the severity of the pulmonary manifestations and added diagnostic difficulty.

Practical Aspects of Introduction of Multiplex Bead Technology into the Routine Diagnostic Immunology Laboratory.

Multiplex bead assays, also known as addressable laser bead immunoassays (ALBIA) or Luminex® technology, have provided an alternative to enzyme-linked immunoassay, which is still the most widely utilized routine immunoassay for detection of specific autoantibodies. Our laboratory adopted the ALBIA technology early into its routine service. We report the performance and utility of measurement of three different autoantibody types tested using the FIDIS (BMD, Marne La Vallee, France) ALBIA system. The analytes discussed are thyroid antibodies (thyroglobulin [TG], thyroid peroxidase [TPO]), anti-neutrophil cytoplasmic antibodies (ANCA), and ribonucleo-protein (RNP) antibodies. In single antibody analysis, TPO antibody testing was superior to TG antibody in identifying patients with Graves' disease and Hashimoto's thyroiditis. However, testing only TPO antibody would result in missing 8.6% of Graves' and 11.9% of Hashimoto's thyroiditis patients, hence demonstrating an advantage for the multiplex TG plus TPO assay. With respect to ANCA, the FIDIS ALBIA produced an overall similar level of performance to our comparator method, the Phadia fluorescent enzyme linked immunoassay. Sensitivity of the ALBIA for RNP antibodies was low in comparison to countercurrent immunoelectrophoresis, but performance was improved by altering the cutoff value for the assay. ALBIA technology has many potential advantages in the routine laboratory, but as with any new assay, evaluation must be thorough and ongoing to ensure satisfactory clinical performance is obtained. Both false positive and false negative results have been reported in ALBIA studies. It may be necessary to re-evaluate assay performance and cutoff and consider further clinical correlation.

Evaluation of classical and novel autoantibodies for the diagnosis of Primary Biliary Cholangitis-Autoimmune Hepatitis Overlap Syndrome (PBC-AIH OS).

Background and aimsUp to 20% of Primary Biliary Cholangitis (PBC) patients are estimated to have features that overlap with Autoimmune Hepatitis (AIH). Patients with PBC-AIH overlap syndrome (PBC-AIH OS) have been reported to exhibit suboptimal responses to ursodeoxycholic acid therapy, and are more likely to progress to cirrhosis. Anti-double stranded DNA (anti-dsDNA) and anti-p53 have been previously suggested to be potential autoantibodies for identifying patients with PBC-AIH OS. In our well defined PBC patient cohorts, a comprehensive assessment of various classical and novel autoantibodies was evaluated for their utility in identifying PBC-AIH OS patients.MethodsPBC-AIH OS was classified according to the Paris criteria and PBC as per the European Association for the Study of the Liver guidelines. Biobanked serum samples from 197 patients at the University of Calgary Liver Unit and the University of Alberta were analyzed for classical and novel autoantibodies. Anti-dsDNA was measured by the Crithidia luciliae immunofluorescence (CLIFT) assay (1:20 dilution) and chemiluminescence (CIA: QUANTA Flash®, Inova Diagnostics, San Diego). Anti-p53, anti-Ro52/TRIM21, anti-YB 1, anti-GW182, anti-Ge-1, and anti-Ago 2 were measured by either an addressable laser bead immunoassay (ALBIA) or line immunoassay (LIA). Autoantibodies against MIT3, gp210, sp100, LKM1, SLA, and the novel autoantibodies Hexokinase-1 (HK-1), and Kelch like protein 12 (KLHL-12) were measured using QUANTA Lite® ELISA assays. We applied non-parametric methods to compare the biomarkers frequencies between study groups. We used multivariate adjusted models and AUROC to compare the diagnostic accuracy of the different autoantibodies alone or in combination with serum biochemistry.Results16 out of 197 PBC patients (8.1%) were classified as PBC-AIH OS. Compared to PBC patients, PBC-AIH OS patients were similar in age (median: 59 vs. 63, P = 0.21) and female predominance (94% vs. 89%, P = 1.00). Anti-dsDNA-by CLIFT (37.5% in PBC-AIH OS vs 9.9% in PBC alone, P <0.01) was the only autoantibody associated with PBC-AIH OS; a finding consistent with previous reports. Significant elevation in serum ALT (62 IU/L in PBC-AIH OS vs 37 IU/L in PBC alone, P < 0.01), and serum IgG (17.6 g/L in OS vs 12.1 g/L in PBC alone, P <0.01) were observed in patients with PBC-AIH OS receiving medical/immunosuppressive therapy. In a multivariate model, positive anti-dsDNA by CLIFT, ALT and IgG were significant predictors of PBC-AIH OS with an area under the receiver operator curve (AUROC) value of 0.84.ConclusionsConsistent with previous findings, the presence of anti-dsDNA by CLIFT is associated with PBC-AIH OS. Contrary to previous reports, anti-p53 was not associated with PBC-AIH OS. Our comprehensive evaluation of various classical and novel autoantibody biomarkers including Ro52/TRIM21, anti-p53, anti-KLHL-12 and anti-HK-1 were not significantly associated with PBC-AIH OS. Our findings highlight the ongoing need for the research and development of new autoantibody biomarkers to aid in the diagnosis of PBC-AIH OS.

Reference standards for the detection of anti-mitochondrial and anti-rods/rings autoantibodies.

AbstractBackground:Anti-mitochondrial antibodies (AMA) are found in &gt;90% of primary biliary cholangitis patients. Anti-rods/rings antibodies (anti-RR) are most commonly associated with interferon-α and ribavirin treatment in hepatitis C patients. Clinical laboratories routinely screen for AMA and anti-RR using indirect immunofluorescence on HEp-2 cells (HEp-2-IFA). Therefore, we sought to establish reference materials for use in AMA and anti-RR testing.Methods:AMA-positive and anti-RR-positive human plasma samples (AMA-REF and RR-REF), identified as potential reference materials based on preliminary data, were further validated by multiple laboratories using HEp-2-IFA, immunoprecipitation (IP), western blotting, IP-western, line immunoassay (LIA), addressable laser bead immunoassay (ALBIA) and enzyme-linked immunosorbent assay (ELISA).Results:AMA-REF showed a strong positive cytoplasmic reticular/AMA staining pattern by HEp-2-IFA to ≥1:1280 dilution and positive signal on rodent kidney/stomach/liver tissue. AMA-REF reacted with E2/E3, E3BP, E1α and E1β subunits of the pyruvate dehydrogenase complex by IP and western blotting and was positive for AMA antigens by LIA, ALBIA and ELISA. RR-REF showed a strong positive rods and rings staining pattern by HEp-2-IFA to ≥1:1280 dilution. RR-REF reacted with inosine monophosphate dehydrogenase by IP, IP-western and ALBIA. RR-REF also produced a nuclear homogenous staining pattern by HEp-2-IFA, immunoprecipitated proteins associated with anti-U1RNP antibody and reacted weakly with histones, nucleosomes, Sm and nRNP/Sm by LIA.Conclusions:AMA-REF and RR-REF are useful reference materials for academic or commercial clinical laboratories to calibrate and establish internal reference standards for immunodiagnostic assays. AMA-REF and RR-REF are now available for free distribution to qualified laboratories through Plasma Services Group.

Anti-HMGCR antibodies in systemic sclerosis.

The objective of this study was to investigate the frequency of autoantibodies to hydroxymethylglutaryl coenzyme A reductase (HMGCR) in systemic sclerosis (SSc) and associations with inflammatory myositis and statin exposure.This was a cross-sectional, multicenter study of 306 subjects from the Canadian Scleroderma Research Group cohort who had complete data on statin exposure and serology for anti-HMGCR antibodies assayed by an addressable laser bead immunoassay (ALBIA). Descriptive statistics were used to summarize the baseline characteristics of the patients and to compare subjects with and without anti-HMGCR antibodies.Four (1.3%) subjects had anti-HMGCR antibodies. None of the subjects with anti-HMGCR antibodies titers had a history of an inflammatory myositis or overlap with polymyositis/dermatomyositis, compared to 8.6% and 2.0% of those without anti-HMGCR antibodies, respectively. In addition, none of the subjects with anti-HMGCR antibodies had past or current exposure to statins compared to 12% of those with negative titers.Anti-HMGCR antibodies are rare in SSc and are not associated with inflammatory myopathy or statin exposure. Larger studies will be required to confirm these preliminary observations. Nevertheless, we conclude that anti-HMGCR antibodies are unlikely to play a major role in inflammatory myopathy in SSc and anti-HMGCR antibodies can be present in subjects without exposure to statins.

CE-21 The prevalence and determinants of ANTI-DFS70 antibodies in an international inception cohort of systemic lupus erythematosus (SLE) patients

Background When found in the absence of antibodies to extractable nuclear antigens (ENA) or anti-double-stranded DNA (dsDNA) (i.e., monospecific), autoantibodies to the nuclear autoantigen dense fine speckles 70 (DFS70) are purported to rule out SLE. The reported frequency of anti-DFS70 by chemiluminescence (CIA) in SLE is low compared to healthy individuals (0–5.7% vs. 1.3–23.2%), while the frequency of monospecific anti-DFS70 in SLE is even lower at 0–0.4%. There are no studies examining the frequency of anti-DFS70 in an early inception SLE cohort. This study determined the prevalence of anti-DFS70 in a multi-national, multi-ethnic early inception SLE cohort and examined demographic, clinical, and autoantibody associations. Materials and methods Patients fulfilling ACR Classification Criteria for SLE were enrolled in the Systemic Lupus International Collaborating Clinics (SLICC) inception cohort within 15 months of diagnosis. Demographic and clinical data were collected at enrollment. ANAs were detected by indirect immunofluorescence on HEp-2 cells (ImmunoConcepts, Sacramento) and ENAs and dsDNA by an addressable laser bead immunoassay (FIDIS Connective13, TheraDiag, Paris). Anti-DFS70 antibodies were measured by CIA (Inova Diagnostics, San Diego). The association between anti-DFS70 and baseline demographic, clinical, and autoantibody profiles was assessed using univariate and multivariate logistic regression. For the most informative model, only the remaining statistically significant predictors at the 95% CI: were included, after eliminating other potential predictors individually, starting with the least likely to be associated with the outcome. Results 1137 patients were included; 89.9% were female and 93.8% were ANA positive (Table 1). The frequency of anti-DFS70 was 7.1% [95% CI: 5.7–8.8%]. 13 of 1137 (1.1%) [95% CI: 0.6–1.9%] were positive for anti-DFS70 only (monospecific). In univariate analysis, patients with musculoskeletal activity (based on SLEDAI items) or anti-β−2 glycoprotein-1 (anti-β2GP1) were more likely to have anti-DFS70, whereas those with anti-dsDNA, anti-SSA/Ro60, anti-SSB/La, or anti-U1RNP were less likely to have anti-DFS70. In multivariate analysis, patients with musculoskeletal activity (Odd Ratio (OR) 1.25 [95% CI: 1.10, 1.41]) or anti-β2GP1 (OR 2.15, 95% CI: 1.21, 3.84) were more likely to have anti-DFS70, while those with anti-dsDNA (OR 0.53, 95% CI: 0.31, 0.92) or anti-SSB/La (OR 0.25, 95% CI:0.08, 0.82) were less likely to have anti-DFS70. Conclusions The prevalence of anti-DFS70 in newly diagnosed SLE patients was at the high end of the range previously ?published for SLE (7.1% vs. 0–5.7%) and was associated with musculoskeletal activity and anti-β2GP1. However, ‘monospecific’ anti-DFS70 was rare (1.1%) and is potentially useful to discriminate between ANA positive healthy individuals and SLE.

Comparison of two extractable nuclear antigen testing algorithms: ALBIA versus ELISA/line immunoassay

Extractable nuclear antigen (ENA) antibody testing is often requested in patients with suspected connective tissue diseases. Most laboratories in Australia use a two step process involving a high sensitivity screening assay followed by a high specificity confirmation test. Multiplexing technology with Addressable Laser Bead Immunoassay (e.g., FIDIS) offers simultaneous detection of multiple antibody specificities, allowing a single step screening and confirmation. We compared our current diagnostic laboratory testing algorithm [Organtec ELISA screen / Euroimmun line immunoassay (LIA) confirmation] and the FIDIS Connective Profile. A total of 529 samples (443 consecutive+86 known autoantibody positivity) were run through both algorithms, and 479 samples (90.5%) were concordant. The same autoantibody profile was detected in 100 samples (18.9%) and 379 were concordant negative samples (71.6%). The 50 discordant samples (9.5%) were subdivided into 'likely FIDIS or current method correct' or 'unresolved' based on ancillary data. 'Unresolved' samples (n=25) were subclassified into 'potentially' versus 'potentially not' clinically significant based on the change to clinical interpretation. Only nine samples (1.7%) were deemed to be 'potentially clinically significant'. Overall, we found that the FIDIS Connective Profile ENA kit is non-inferior to the current ELISA screen/LIA characterisation. Reagent and capital costs may be limiting factors in using the FIDIS, but potential benefits include a single step analysis and simultaneous detection of dsDNA antibodies.

Autoantibodies directed to centromere protein F in a patient with BRCA1 gene mutation

BackgroundAutoantibodies directed to centromere protein F were first reported in 1993 and their association with malignancy has been well documented.CaseWe present the case of a 48-year-old Caucasian female with a BRCA1 gene mutation associated with bilateral breast cancer. Antinuclear autoantibody immunofluorescence performed for workup of possible inflammatory arthropathy showed a high titre cell cycle related nuclear speckled pattern, with subsequent confirmation by addressable laser bead immunoassay of the target antigen as an immunodominant epitope at the C-terminus of centromere protein F.ConclusionHere we review the current literature on centromere protein F, its association with breast cancer and present the first case of this antibody being identified in a person with a BRCA1 gene mutation.

Autoantibodies directed to centromere protein F in a patient with BRCA1 gene mutation

Aim Autoantibodies directed to centromere protein F (CENP-F) were first reported in 1993 and their association with malignancy has been well documented. We present a case of this autoantibody detected in a 47-year-old female with BRCA1 gene mutation associated with bilateral breast cancer and ovarian cancer. Method and results Antinuclear autoantibody immunofluorescence carried out for possible inflammatory arthropathy showed high titre nuclear speckled-II (NSII) pattern consistent with CENP-F that was confirmed by addressable laser bead immunoassay (ALBIA) for the C-terminal p-F4, an immunodominant CENP-F peptide. Discussion We review the current literature on CENP-F, its association with breast cancer and present the first documented case of this antibody being identified in a person with BRCA1 gene mutation.

Absence of an association between anti-Ro antibodies and prolonged QTc interval in systemic sclerosis: A multicenter study of 689 patients

ObjectiveTo examine the association between anti-Ro antibodies, namely anti-Ro60/SS-A and anti-Ro52/TRIM21, together and separately, and a prolonged QT interval corrected for heart rate (QTc) in systemic sclerosis (SSc) patients. MethodsA total of 689 SSc patients enrolled in a multicenter cohort study underwent a 12-lead resting EKG at baseline. The QTc interval was measured, and a QTc ≥ 440ms was considered prolonged. Detailed clinical data and sera of these patients were collected and positivity for anti-Ro60/SS-A and anti-Ro52/TRIM21 antibodies was determined using an addressable laser bead immunoassay (ALBIA). ResultsQTc prolongation was common in this SSc cohort (25%). In a univariate analysis, Ro antibodies, together or separately, were not associated with prolongation of the QTc interval [mean difference in QTc in anti-Ro antibody positive versus negative subjects was −2.2ms (p = 0.5748), in anti-Ro60/SS-A antibody positive versus negative subjects was 1.3ms (p = 0.8616), and in anti-Ro52/TRIM21 antibody positive versus negative subjects was −3.3ms (p = 0.4106)]. In a multivariate logistic regression analysis adjusting for possible confounders, there was no association between prolonged QTc and anti-Ro antibodies [odds ratio (OR) = 0.74, 95% confidence interval (CI): 0.45, 1.22], anti-Ro60/SS-A antibodies (OR = 1.57, 95% CI: 0.72, 3.41), and anti-Ro52/TRIM21 antibodies (OR = 0.76, 95% CI: 0.46, 1.26). However, in both univariate and multivariate analyses, QTc prolongation was associated with longer disease duration, greater disease severity, and the presence of anti-RNA polymerase III antibodies. ConclusionsQTc prolongation is common in SSc, although anti-Ro antibodies do not seem to be associated with it as is the case in systemic lupus erythematosus. The reasons for this difference as well as the cause of abnormalities in cardiac repolarization in SSc will require additional studies.

Anti-HMGCR autoantibodies in European patients with autoimmune necrotizing myopathies: inconstant exposure to statin.

Necrotizing autoimmune myopathy (NAM) is a group of acquired myopathies characterized by prominent myofiber necrosis with little or no muscle inflammation. Recently, researchers identified autoantibodies (aAb) against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) in patients with NAM, especially in statin-exposed patients. Here we report what is to our knowledge the first European cohort of patients with NAM.The serum of 206 patients with suspicion of NAM was tested for detection of anti-HMGCR aAb using an addressable laser bead immunoassay. Forty-five patients were found to be anti-HMGCR positive. Their mean age was 48.9 ± 21.9 years and the group was predominantly female (73.3%). Statin exposure was recorded in 44.4% of patients. Almost all patients had a muscular deficit (97.7%), frequently severe (Medical Research Council [MRC] 5 ≤3 in 75.5%). Subacute onset (<6 mo) was noted for most of them (64.4%). Nevertheless, 3 patients (6.6%) had a slowly progressive course over more than 10 years. Except for weight loss (20%), no extramuscular sign was observed. The mean CK level was high (6941 ± 8802 IU/L) and correlated with muscle strength evaluated by manual muscle testing (r = −0.37, p = 0.03). Similarly, anti-HMGCR aAb titers were correlated with muscular strength (r = −0.31; p = 0.03) and CK level (r = 0.45; p = 0.01). Mean duration of treatment was 34.1 ± 40.8 months, and by the end of the study no patient had been able to stop treatment.This study confirms the observation and description of anti-HMGCR aAb associated with NAM. The majority of patients were statin naive and needed prolonged treatments. Some patients had a dystrophic-like presentation. Anti-HMGR aAb titers correlated with CK levels and muscle strength, suggesting their pathogenic role.

Antiphospholipase A₂ receptor autoantibodies: a comparison of three different immunoassays for the diagnosis of idiopathic membranous nephropathy.

Background. The recent identification of circulating autoantibodies directed towards the M-type phospholipase A2 receptor (PLA2R) has been a major advancement in the serological diagnosis of idiopathic membranous nephropathy (IMN), a common cause of nephrotic syndrome in adults. The goal of this study was to compare the performance characteristics of two commercial assays as well as the first addressable laser bead immunoassay (ALBIA) developed for the detection of anti-PLA2R antibodies. Methods. Serum samples of 157 IMN patients and 142 controls were studied. Samples were tested by a cell based immunofluorescence assay (CBA-IFA, Euroimmun, Germany), by ELISA (Euroimmun), and by a novel ALBIA employing an in vivo expressed recombinant human PLA2R. Results. Overall, the three assays showed significant qualitative and quantitative correlation. As revealed by receiver operating characteristic analysis, the ALBIA correlated better with the CBA-IFA than the ELISA (P = 0.0003). The clinical sensitivities/specificities for IMN were 60.0% (51.0–68.5%)/98.6% (95.0–99.8%) and 56.2% (47.2–64.8%)/100.0% (97.4–100.0%) for ALBIA and CBA-IFA, respectively. Conclusion. The ALBIA represents a promising assay for the detection of anti-PLA2R antibodies showing similar performance to the CBA-IFA and the advantage of ease of use and suitability for high throughput, rapid turnaround times, and multiplexing.

Exploring necrotizing autoimmune myopathies with a novel immunoassay for anti-3-hydroxy-3-methyl-glutaryl-CoA reductase autoantibodies

IntroductionNecrotizing autoimmune myopathies (NAM) have recently been defined as a distinct group of severe acquired myopathies, characterized by prominent myofiber necrosis without significant muscle inflammation. Because of the lack of appropriate biomarkers, these diseases have been long misdiagnosed as atypical forms of myositis. NAM may be associated to autoantibodies directed against signal recognition particle (SRP) or 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). The objective of this work was to quantify anti-HMGCR autoantibodies in patients with suspicion of NAM through the development of a new addressable laser bead immunoassay (ALBIA).MethodsRecombinant HMGCR C-domain was bound to fluorescent beads. After incubation with serum, autoantibodies were revealed using class- or subclass-specific anti-human immunoglobulin G (IgG) antibodies. Anti-HMGCR levels were assayed in 150 patients with suspicion of NAM, 142 controls with different inflammatory/autoimmune diseases and 100 healthy donors. Inhibition with free recombinant HMGCR and immunoprecipitation experiments confirmed test specificity. Reproducibility and repeatability were determined from sera with various levels of anti-HMGCR autoantibodies. A multiplex assay (ALBIA-NAM) was also developed to permit the simultaneous quantification of anti-HMGCR and anti-signal recognition particle autoantibodies.ResultsNo controls scored positive. Of 150 patients with suspicion of NAM, 24% were positive for anti-HMGCR autoantibodies with levels ranging from 24 to 2,656 AU/mL. Anti-HMGCR positivity could be associated to a cytoplasmic pattern in immunofluorescence assay on HEp-2 cells. Anti-HMGCR-positive patients had high creatine kinase (CK) levels (mean 6,630 IU/L) and only 40% of them had been exposed to statins. Multiplex ALBIA-NAM was equally as effective as monoplex anti-HMGCR and anti-SRP ALBIA.ConclusionsBoth monoplex ALBIA-HMGCR and multiplex ALBIA-NAM reliably detect and quantify anti-HMGCR autoantibodies. A positive result allows ascribing patients with a necrotizing myopathy to an autoimmune form. Anti-HMGCR autoantibodies may be found in patients who have not taken statins.

Reflections on Lupus 2013: butterflies, wolves and prophecies

The recently concluded Tenth International Congress on Systemic Lupus Erythematosus (SLE) held in Buenos Aires was a resounding success. This overview summarizes some of the origins of the First International Congress held in Calgary, Canada in 1986, predictions offered by past Congress Presidents, and a perspective on the trends in autoantibody testing, which remains one of the key approaches to the early and accurate diagnosis of SLE. The last few decades have witnessed a remarkable proliferation of new diagnostic technologies including addressable laser bead immunoassays and, more recently, chemiluminescence and lateral flow technologies that could find a clinical niche in point-of-care diagnostics. Against the backdrop of these constantly emerging technologies, indirect immunofluorescence has remained the platform of choice for many laboratories and diagnosticians. The notion that autoantibodies are pathogenic has been challenged by evidence that some autoantibodies are protective, some may have catalytic capacity while others may be neutral or have no function at all. The latter notion of functionless or "junk" autoantibodies needs to be taken under some advisement, because there was a time when a great proportion of the human genome was considered to include "junk DNA". The butterfly as a symbol of hope and progress in SLE research over the past 27 years since the First International Congress on SLE is almost certainly to be even more appropriate when future Congresses are held in Geneva (2015), Melbourne (2017) and eventually one in 2050.

P.14.11 Auto-immune necrotizing myopathies with anti-HMGCR antibodies are related to statin-exposure only for a minority of cases

Necrotizing autoimmune myopathy (NAM) is a group of severe acquired myopathies, characterized by prominent myofiber necrosis with little or no muscle inflammation. They can be misdiagnosed as muscular dystrophies, preventing initiation of appropriate therapy. Specific auto-antibody anti-Signal recognition protein is observed in 16% of cases. Recently, auto-antibodies against 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR) were identified in NAM patients by one group, especially in statin exposed patients. Here we report the first European series of patients. We detected and quantified anti-HMGCR auto-antibodies using addressable laser bead immunoassay that we developed. Patients (n = 38) were 44 ± 19 years old (3 pediatric cases) and sex ratio male: female was 0.22. Statin exposure was recorded in 40% of patients. Patients suffered from myalgia (44%), and had a muscular deficit (92%). Subacute onset (less than 6 months) was noted for most of them (n = 18), nevertheless 10 patients had a slow progressive muscular deficit (10 months to years). Severe proximal weakness was observed (72%) and three patients were bedridden at the diagnosis. All patients had increase CK level (6630 ± 5990 UI/L). CK level correlated with muscular strength (r2 = −0.64, p = 0.005) and to anti-HMGR titer (r2 = −0.5, p = 0.04). None patients presented signs of pulmonary involvement. Mean duration of treatments (steroids, immunosupressant and/or IgIV) was 30.1 ± 36.1 [3–42] months, and to date it was not possible to stop the treatment for any patient. This the first study confirming the observation and the description of anti-HMGCR NAM. The majority of patients we detected was statin-naive, had a severe weakness and need prolonged treatments. Anti-HMGCR auto-antibodies testing may be warranted in dystrophic like patients. Anti-HMGR titer correlates with CK level suggesting their role in physiopathogenesis.

AB1258 Characterization of the CDC anti-nuclear antibody (ANA) reference serum panel using multiplex and immunoprecipitation assays

BackgroundAnti-nuclear antibodies (ANA) represent serological hallmarks of systemic rheumatic diseases (SARD), and have been traditionally identified by indirect immunofluorescence (IIF) on HEp-2 cells, followed by antigen-specific confirmation assays. Several new...

An Anti-Phospholipase A2 Receptor Quantitative Immunoassay and Epitope Analysis in Membranous Nephropathy Reveals Different Antigenic Domains of the Receptor

The phospholipase A2 receptor (PLA2R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA2R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.

Is there an association between Ro52/TRIM21 antibodies and rheumatoid factor in systemic sclerosis?

Autoantibodies to Ro52/TRIM21 are among the most common autoantibodies detected in subjects with systemic autoimmune rheumatic autoimmune diseases (SARD), including systemic sclerosis (SSc) [1–3]. Ro52/TRIM21 is an immunoglobulin-binding protein [4]. It is also an extraordinarily prevalent and antigenic target of autoantibodies. It is part of the Ro/SSA ribonucleoprotein, which includes a single polypeptide and one of four small RNA molecules. Recently, it has been shown that Ro52/TRIM21 plays a major role in the destruction of misfolded IgG1. Its C-terminal PRYSPRY domain, also known as B30.2 domain, has been found to be of great importance for its function as a highly specific IgG receptor. Studies have suggested a possible association between anti-Ro52/ TRIM21 antibodies and rheumatoid factor (RF) in Sjogren’s syndrome (SjS) [5, 6]. In a cohort of 187 SjS patients, 37 % were anti-Ro52/TRIM21 positive and 74 % had significantly high RF titers [5]. Similarly, in another study, the prevalence of anti-Ro52/TRIM21 varied from 14.1 % (n = 106) in SSc patients to 97.5 % (n = 40) in SjS, and the mean anti-Ro52/TRIM21 signal intensity was significantly higher in SjS patients who were RF positive [6]. The presence of RF in SSc patients has been described in several studies with a frequency that varies from 12 to 37 % [7–9] and higher in selected SSc subjects (66 % in subjects with rheumatoid arthritis overlap and 59 % in subjects with SjS overlap) [10]. Taking these reports into account, we set out to determine if there was an association between Ro52/TRIM21 and RF in SSc. Sera from 89 anti-Ro52/TRIM21 antibody positive and 89 antibody negative SSc subjects from the Canadian Scleroderma Research Group cohort were selected consecutively. Anti-Ro52/TRIM21 antibodies were detected by an addressable laser bead immunoassay (ALBIA) utilizing a commercially available kit (QUANTAPlex ENA 8, INOVA Diagnostics Inc., San Diego, CA) in a Luminex 100 (Luminex Corp., Austin, TX) platform, the results of which were cross-validated on another kit (Connective13, BMD/FIDIS, Paris, France) in a Luminex 200 (Luminex Corp.) platform, according to protocols previously described [2]. The sera of all study subjects were tested for IgM RF by ELISA (INOVA, San Diego, CA) using a cutoff value for RF set according to the manufacturer’s recommendations at 6 IU/ml. Descriptive statistics and logistic regression were used to analyze the data, using SAS v.9.2 (SAS Institute, USA). After excluding 4 subjects with incomplete datasets, 86 Ro52/TRIM21 autoantibody positive and 88 Ro52/ TRIM21 antibody negative subjects were available for this analysis (Table 1). The overall prevalence of RF was high (45 %), but there was no difference in either Ro52/ The Investigators of the Canadian Scleroderma Research Group is given in ‘‘Appendix’’.