Clinical evaluation of a novel chemiluminescent immunoassay for the detection of anti-citrullinated peptide antibodies.

BACKGROUND: We evaluated the analytical and clinical performances of a novel, automated chemiluminescent immunoassay in comparison with several anti-citrullinated protein antibody (ACPA) assays (2nd and 3rd generations) based on various platforms and technologies. METHODS: Samples from rheumatoid arthritis (RA) patients (n=141) and controls (n=153) were collected based on an ordered ACPA test. All samples were tested with QUANTA Flash® CCP3, QUANTA Lite® CCP3, QUANTA Lite® CCP3.1, CCPlus and EliA® CCP assays. Rheumatoid factor (RF) was determined using Quantex RF(II). An additional cohort consisting of RA patients from three different sources (116, 79 and 50 samples), 61 juvenile idiopathic arthritis (JIA) patients and 233 controls were used in an extended evaluation on QUANTA Flash® CCP3 only. Precision and linearity of the QUANTA Flash® CCP3 were assessed according to CLSI guidelines. RESULTS: All ACPA assays showed good qualitative and quantitative agreements. The Quanta Flash CCP3 assay showed good analytical and clinical performance. Based on the extended cohort, the sensitivity, specificity and likelihood ratios of the novel Quanta Flash® CCP3 were defined as 70.2%, 97.4% and 27.3/0.31, respectively. CONCLUSION: Good agreements between different ACPA assays based on diverse platforms were found. Quanta Flash CCP3 is a reliable test for the fully automated and rapid detection of ACPA.

Clinical performance evaluation of a novel rapid response chemiluminescent immunoassay for the detection of autoantibodies to extractable nuclear antigens.

BACKGROUND: We analyzed the performance of a novel ENA screening chemiluminescent immunoassay (CIA) and the confirmation QUANTA Flash tests. METHODS: Sera (n=1079) from patients referred to a rheumatology clinic were screened by QUANTA Flash ENA7 (INOVA Diagnostics). All positive (n=89) and a matched control group (n=90) were reflexed for autoantibodies to the individual antigens. Moreover, sera from patients with systemic lupus erythematosus (SLE, n=252), systemic sclerosis (SSc, n=64), polymyositis/dermatomyositis (PM/DM, n=72), Sjögren's syndrome (SjS, n=39) as well as disease controls (n=605) were tested by ENA7 CIA and by Quanta Lite ENA6 ELISA (INOVA). RESULTS: 89/1079 (8.3%) samples were ENA7 CIA positive with the following reactivity profile: RNP (36.0%), Sm (13.5%), Scl-70 (9.0%), Jo-1 (0.0%), Ro60 (44.9%), Ro52 (39.3%) and SS-B (24.7%). In the negative group, the reactivity profile was: RNP (1.1%), Sm (1.1%), Scl-70 (2.2%) and 0.0% for Jo-1, Ro60, Ro52 and SS-B. The positive/negative/total agreements (ENA7 CIA vs. confirmation assays) were 95.3%/91.5%/93.3%. The sensitivity of the ENA7 CIA was 62.3% in SLE, 54.7% in SSc, 92.3% in SjS, 50.0% in PM/DM, and 61.8% in the total systemic autoimmune rheumatic disease (SARD) population (specificity 95.0%). CONCLUSION: The QUANTA Flash ENA7 CIA is a reliable screening test.

Rpp25 is a major target of autoantibodies to the Th/To complex as measured by a novel chemiluminescent assay.

INTRODUCTION: Autoantibodies to the Th/To antigen have been described in systemic sclerosis (SSc) and several proteins of the macromolecular Th/To complex have been reported to react with anti-Th/To antibodies. However, anti-Th/To has not been clinically utilized due to unavailability of commercial tests. The objective of the present study is to evaluate the newly developed ELISA and chemiluminescent immunoassay (CLIA) to measure autoantibodies to Rpp25 (a component of the Th/To complex) using immunoprecipitation (IP) as the reference method. METHODS: The first cohort consisted of 123 SSc patients including 7 anti-Th/To positive samples confirmed by IP. Additional seven anti-Th/To positive samples from non-SSc patients were also tested. For evaluation of the QUANTA Flash Rpp25 CLIA (research use only), 8 anti-Th/To IP positives, a cohort of 70 unselected SSc patients and sera from various disease controls (n = 357) and random healthy individuals (n = 10) were studied. RESULTS: Anti-Rpp25 antibodies determined by ELISA were found in 11/14 anti-Th/To IP positive but only in 1/156 (0.6%) negative samples resulting in a positive percent agreement of 78.6% (95% confidence interval [CI] 49.2, 95.3%) and a negative percent agreement of 99.4% (95% CI 96.4, 100.0%). To verify the results using a second method, 53 samples were tested by ELISA and CLIA for anti-Rpp25 reactivity and the results were highly correlated (rho = 0.71, 95% CI 0.56, 0.81; P < 0.0001). To define the cutoff of the CLIA, anti-Th/To IP positive and negative sera were tested using the anti-Rpp25 CLIA. At the cutoff selected by receiver operating characteristic (ROC) analysis 8/8 (100.0%) of the anti-Th/To positive sera but only 2/367 (0.5%) of the controls were positive for anti-Rpp25 antibodies. The positive and negative percent agreements were 100.0% (95% CI 63.1, 100.0%) and 99.5% (95% CI 98.0, 99.9%), respectively. In the disease cohorts 2/70 (2.9%) of the SSc patients were positive for anti-Rpp25 antibodies compared to 2/367 (0.5%) of the controls (P = 0.032). ROC analysis showed discrimination between SSc patients and controls with an area under the curve value of 0.732 (95% CI 0.655, 0.809). CONCLUSION: Rpp25 is a major target of autoantibodies to the Th/To autoantigen complex. Further studies are needed to evaluate the clinical utility of the new assays.

Third generation anti-citrullinated peptide antibody assay is a sensitive marker in rheumatoid factor negative rheumatoid arthritis.

INTRODUCTION: We compared 2 anti-citrullinated protein antibody (ACPA) assays using a routine patient cohort. METHODS: Two-hundred ninety-five sera were collected from patients for whom ACPA was ordered and tested for ACPA by QUANTA Lite® CCP 3 (INOVA Diagnostics, Inc., San Diego) and EliA® CCP (CCP, Phadia, Germany). Rheumatoid factor (RF) was determined using Quantex RF(II) (Biokit, Spain). RESULTS: Acceptable qualitative (96.6%, kappa=0.93) and quantitative agreements (Spearman rho=0.77; p<0.0001) were observed between the two ACPA assays. Nine samples were CCP3+/CCP2- and one sample was CCP2+/CCP3-. Of the 9 CCP3+/CCP2- patients, 6 (66.7%) had RA, one patient had ankylosing spondylitis, one osteoarthritis and one psoriatic arthritis. The CCP3-/CCP2+ patient had juvenile RA. At the manufacturer's cut-offs, the sensitivities and specificities were 77.3%/98.1% (CCP2), 81.6%/96.8% (CCP3) and 65.2%/89.6% (RF), respectively. At 98.7% specificity level, the sensitivities in the total cohort were 59.6% (CCP2) and 69.5% (CCP3) while the sensitivities in the RF-negative group were 49.0% (CCP2) and 57.1% (CCP3). In the RF-negative group, sensitivities for patients with a disease duration of ≤ 5years were 38.7% (CCP2) and 51.6% (CCP3). CONCLUSION: Discrimination between RA and non-RA patients was better using CCP3, most pronounced in RF-negative RA.

Anti-DFS70/LEDGF antibodies are more prevalent in healthy individuals compared to patients with systemic autoimmune rheumatic diseases.

OBJECTIVE: Antinuclear antibodies (ANA) are a serological hallmark of systemic autoimmune rheumatic diseases (SARD) such as systemic lupus erythematosus (SLE). While a number of ANA patterns detected by indirect immunofluorescence (IIF) have diagnostic significance, autoantibodies producing the dense fine speckled (DFS) pattern have been reported to be more prevalent in healthy individuals than in SARD. METHODS: Sequential samples submitted for ANA testing were screened for anti-DFS antibodies by IIF (n = 3263). Samples with the DFS pattern were tested for anti-DFS70/lens epithelium-derived growth factor (LEDGF) antibodies by ELISA and by a novel chemiluminescence assay (CIA, Quanta Flash DFS70). Sera from patients with various diseases and healthy individuals were tested for anti-DFS70/LEDGF antibodies by CIA. A cohort of 251 patients with SLE was used to analyze serological and clinical associations of anti-DFS70 antibodies. RESULTS: The frequency of anti-DFS antibodies by IIF was 1.62%. The prevalence of anti-DFS70/LEDGF antibodies as detected by CIA in the different cohorts was 8.9% in healthy individuals, 2.8% in SLE, 2.6% in rheumatoid arthritis, 4.0% in asthma, 5.0% in interstitial cystitis, 1.7% in Graves' disease, and 6.0% in Hashimoto's thyroiditis. Of note, the prevalence of anti-DFS70/LEDGF antibodies was significantly higher in healthy individuals compared to patients with SARD (p = 0.00085). In SLE results, anti-DFS70/LEDGF antibodies were not significantly associated with clinical features or other autoantibodies typically found in SLE. Only 1/7 SLE sera showed anti-DFS70/LEDGF, but no other autoantibody reactivity. CONCLUSION: "Monospecific" anti-DFS70/LEDGF antibodies may represent a biomarker for differentiating SARD from non-SARD individuals, but there is a need for a reliable assay to ensure reactivity to DFS70.

Clinical and serological evaluation of a novel CENP-A peptide based ELISA.

INTRODUCTION: Anti-centromere antibodies (ACA) are useful biomarkers in the diagnosis of systemic sclerosis (SSc). ACA are found in 20 to 40% of SSc patients and, albeit with lower prevalence, in patients with other systemic autoimmune rheumatic diseases. Historically, ACA were detected by indirect immunofluorescence (IIF) on HEp-2 cells and confirmed by immunoassays using recombinant CENP-B. The objective of this study was to evaluate a novel CENP-A peptide ELISA. METHODS: Sera collected from SSc patients (n=334) and various other diseases (n=619) and from healthy controls (n=175) were tested for anti-CENP-A antibodies by the novel CENP-A enzyme linked immunosorbent assay (ELISA). Furthermore, ACA were determined in the disease cohorts by IIF (ImmunoConcepts, Sacramento, CA, USA), CENP-B ELISA (Dr. Fooke), EliA CENP (Phadia, Freiburg, Germany) and line-immunoassay (LIA, Mikrogen, Neuried, Germany). Serological and clinical associations of anti-CENP-A with other autoantibodies were conducted in one participating centre. Inhibition experiments with either the CENP-A peptide or recombinant CENP-B were carried out to analyse the specificity of anti-CENP-A and -B antibodies. RESULTS: The CENP-A ELISA results were in good agreement with other ACA detection methods. According to the kappa method, the qualitative agreements were: 0.73 (vs. IIF), 0.81 (vs. LIA), 0.86 (vs. CENP-B ELISA) and 0.97 (vs. EliA CENP). The quantitative comparison between CENP-A and CENP-B ELISA using 265 samples revealed a correlation value of rho=0.5 (by Spearman equation). The receiver operating characteristic analysis indicated that the discrimination between SSc patients (n=131) and various controls (n=134) was significantly better using the CENP-A as compared to CENP-B ELISA (P<0.0001). Modified Rodnan skin score was significantly lower in the CENP-A negative group compared to the positive patients (P=0.013). Inhibition experiments revealed no significant cross reactivity of anti-CENP-A and anti-CENP-B antibodies. Statistically relevant differences for gender ratio (P=0.0103), specific joint involvement (Jaccoud) (P=0.0006) and anti-phospholipid syndrome (P=0.0157) between ACA positive SLE patients and the entire SLE cohort were observed. CONCLUSIONS: Anti-CENP-A antibodies as determined by peptide ELISA represent a sensitive, specific and independent marker for the detection of ACA and are useful biomarkers for the diagnosis of SSc. Our data suggest that anti-CENP-A antibodies are a more specific biomarker for SSc than antibodies to CENP-B. Furthers studies are required to verify these findings.