Receiver-operating characteristic analysis of two SmD peptide-based anti-Sm antibody assays

<p><b>Copyright information:</b></p><p>Taken from "Effect of dsDNA binding to SmD-derived peptides on clinical accuracy in the diagnosis of systemic lupus erythematosus"</p><p>http://arthritis-research.com/content/9/4/R68</p><p>Arthritis Research & Therapy 2007;9(4):R68-R68.</p><p>Published online 18 Jul 2007</p><p>PMCID:PMC2206372.</p><p></p> The results of this comparative study were used to generate receiver-operating characteristic curves. The discrimination between systemic lupus erythematosus patient samples and control samples was similar for both SmD immunoassays

Table_1_Comparison of Serological Biomarkers in Rheumatoid Arthritis and Their Combination to Improve Diagnostic Performance.docx

Introduction<p>The diagnosis of rheumatoid arthritis (RA) is based on a combined approach that includes serological markers such as rheumatoid factor (RF) and anti-citrullinated peptide/protein antibodies (ACPA). The goal of this study was to evaluate the clinical performance of several RF and ACPA immunoassays for the diagnosis of RA, as well as the diagnostic value of a combinatory approach with these markers.</p>Methods<p>The study cohort included 1,655 patients from the Swiss Clinical Quality Management registry with sera from 968 patients with RA and 687 disease controls, including patients with axial spondyloarthritis (n = 450) and psoriatic arthritis (n = 237). ACPA were determined by anti-CCP2 IgG enzyme-linked immunosorbent assay (ELISA), QUANTA Flash^® CCP3 IgG [chemiluminescent immunoassay (CIA)], and QUANTA Lite^® CCP3 IgG ELISA. RF was determined by ELISA (QUANTA Lite^® RF IgM, RF IgA, and RF IgG) and with two research use only CIAs (QUANTA Flash^® RF IgM and RF IgA).</p>Results<p>All three ACPA assays showed good discrimination between RA patients and controls and good clinical performance. Overall, CCP3 performed better than CCP2. More pronounced differences were observed between the RF assays. We observed that CIA platforms for both RF IgM and RF IgA showed better performance than the ELISA platforms. Excellent and good total agreements were found between ELISA and CIA for CCP3 (total agreement 95.3%, kappa = 0.90), and between CCP2 and CCP3 ELISA (total agreement 86.6%, kappa = 0.73), respectively. RF IgM CIA and ELISA had a good qualitative agreement (86.5%, kappa = 0.73); RF IgA CIA and ELISA showed a moderate total agreement (78.5%, kappa = 0.53). When combinatory analyses were performed, the likelihood of RA increased with dual positivity and triple positivity and combining different markers resulted in higher odds ratio than the individual markers in all cases.</p>Conclusion<p>ACPA and RF showed good clinical performance in this large Swiss cohort of RA patients and controls. Overall, the performance of CCP3 was superior to CCP2. The combination of these biomarkers in an interval model represents a potential tool for the diagnosis of RA patients.</p

image_1_Comparison of Serological Biomarkers in Rheumatoid Arthritis and Their Combination to Improve Diagnostic Performance.jpeg

Introduction<p>The diagnosis of rheumatoid arthritis (RA) is based on a combined approach that includes serological markers such as rheumatoid factor (RF) and anti-citrullinated peptide/protein antibodies (ACPA). The goal of this study was to evaluate the clinical performance of several RF and ACPA immunoassays for the diagnosis of RA, as well as the diagnostic value of a combinatory approach with these markers.</p>Methods<p>The study cohort included 1,655 patients from the Swiss Clinical Quality Management registry with sera from 968 patients with RA and 687 disease controls, including patients with axial spondyloarthritis (n = 450) and psoriatic arthritis (n = 237). ACPA were determined by anti-CCP2 IgG enzyme-linked immunosorbent assay (ELISA), QUANTA Flash^® CCP3 IgG [chemiluminescent immunoassay (CIA)], and QUANTA Lite^® CCP3 IgG ELISA. RF was determined by ELISA (QUANTA Lite^® RF IgM, RF IgA, and RF IgG) and with two research use only CIAs (QUANTA Flash^® RF IgM and RF IgA).</p>Results<p>All three ACPA assays showed good discrimination between RA patients and controls and good clinical performance. Overall, CCP3 performed better than CCP2. More pronounced differences were observed between the RF assays. We observed that CIA platforms for both RF IgM and RF IgA showed better performance than the ELISA platforms. Excellent and good total agreements were found between ELISA and CIA for CCP3 (total agreement 95.3%, kappa = 0.90), and between CCP2 and CCP3 ELISA (total agreement 86.6%, kappa = 0.73), respectively. RF IgM CIA and ELISA had a good qualitative agreement (86.5%, kappa = 0.73); RF IgA CIA and ELISA showed a moderate total agreement (78.5%, kappa = 0.53). When combinatory analyses were performed, the likelihood of RA increased with dual positivity and triple positivity and combining different markers resulted in higher odds ratio than the individual markers in all cases.</p>Conclusion<p>ACPA and RF showed good clinical performance in this large Swiss cohort of RA patients and controls. Overall, the performance of CCP3 was superior to CCP2. The combination of these biomarkers in an interval model represents a potential tool for the diagnosis of RA patients.</p

Influence of Tail Groups during Functionalization of ZnO Nanoparticles on Binding Enthalpies and Photoluminescence

We report on the tailoring of ZnO nanoparticle (NP) surfaces by catechol derivatives (CAT) with different functionalities: <i>tert</i>-butyl group (tertCAT), hydrogen (pyroCAT), aromatic ring (naphCAT), ester group (esterCAT), and nitro group (nitroCAT). The influence of electron-donating/-withdrawing properties on enthalpy of ligand binding (Δ<i>H</i>) was resolved and subsequently linked with optical properties. First, as confirmed by ultraviolet/visible (UV/vis) and Fourier transform infrared (FT-IR) spectroscopy results, all CAT molecules chemisorbed to ZnO NPs, independent of the distinct functionality. Interestingly, the ζ-potentials of ZnO after functionalization shifted to more negative values. Then, isothermal titration calorimetry (ITC) and a mass-based method were applied to resolve the heat release during ligand binding and the adsorption isotherm, respectively. However, both heat- and mass-based approaches alone did not fully resolve the binding enthalpy of each molecule adsorbing to the ZnO surface. This is mainly due to the fact that the Langmuir model oversimplifies the underlying adsorption mechanism, at least for some of the tested CAT molecules. Therefore, a new, fitting-free approach was developed to directly access the adsorption enthalpy per molecule during functionalization by dividing the heat release measured via ITC by the amount of bound molecules determined from the adsorption isotherm. Finally, the efficiency of quenching the visible emission caused by ligand binding was investigated by photoluminescence (PL) spectroscopy, which turned out to follow the same trend as the binding enthalpy. Thus, the functionality of ligand molecules governs the binding enthalpy to the particle surface, which in turn, at least in the current case of ZnO, is an important parameter for the quenching of visible emission. We believe that establishing such correlations is an important step toward a more general way of selecting and designing ligand molecules for surface functionalization. This allows developing strategies for tailored colloidal surfaces beyond empirically driven formulation on a case by case basis

PLA₂R schematic plot of the seven potential antigen determinants identified by epitope mapping.

<p>All of the determinants identified by epitope mapping were located in the extracellular domain of PLA₂R and are ∼10 to 25 aa long. Only one epitope is not in the C-type lectin like domains of the receptor. <i>[C-R,cysteine-rich region; FNII, fibronectin type II domain; CTLDs, C-type lectin like domains; N, N-terminal end; C, C-terminal end].</i></p

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

<div><p>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.</p></div

Illustrative Inhibition studies of anti-PLA₂R antibodies using synthetic peptides with an anti-PLA₂R positive serum sample.

<p><i>Panel A:</i> Different concentrations of a mixture of PLA₂R derived peptides were used to inhibit the reactivity to the PLA₂R whole molecule in an addressable laser bead assay. The reactivity showed a significant, dose dependent inhibition. The inhibition with a control peptide (GE-1) was significantly lower. All values are expressed as residual reactivity after inhibition (in %) compared to the sample without inhibitor or control. <i>Panel B:</i> The peptide mixture together with seven individual PLA₂R peptides and a control peptide were used at a concentration of 126 µg/mL. Besides the peptide mixture, peptide 3 and peptide 4 showed inhibition of anti-PLA₂R antibodies. All values are expressed as residual reactivity after inhibition (in %) compared to the sample without inhibitor or control. <i>Panel C:</i> The peptide mixture together with seven individual PLA₂R peptides and a control peptide were used at a concentration of 126 µg/mL. Besides the peptide mixture, peptide 3 and peptide 4 showed inhibition of anti-PLA₂R antibodies. All values are expressed as ALBIA median fluorescence intensities (MFI) or titer by indirect immunofluorescence on cell-based assay (IIF-CBA).</p

ELISA of synthesized PLA₂R peptides.

<p>To verify potential epitopes, synthetic peptides (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061669#pone-0061669-t001" target="_blank">Table 1</a>) were tested by ELISA. Absorbance of patient samples tested positive on the CB-IIF assay was higher than of patient samples tested negative and normal healthy control samples but the difference was not statistically significant (p>0.05).</p

Epitope Mapping of PLA₂R Peptides with anti-human IgG₄.

<p>Grey scale heat map representation of results from SPOT used to detect PLA₂R epitopes. Peptide membranes were probed with 10 randomly selected IMN samples that had previously been tested by IIF-CBA for anti-PLA₂R antibodies (7 positive (IMN+) and 3 negative (IMN-)), as well as 5 normal healthy controls (NHC). The positive control rabbit antibody to PLA₂R reacted with the expected peptide used as the immunogen. Consensus epitopes and their respective PLA₂R domains derived from this analysis are illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061669#pone-0061669-g003" target="_blank">Figure 3</a> and summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061669#pone-0061669-t001" target="_blank">Table 1</a>.</p

Clinical and Serological Features of Patients Referred through a Rheumatology Triage System because of Positive Antinuclear Antibodies

<div><p>Background</p><p>The referral of patients with positive anti-nuclear antibody (ANA) tests has been criticized as an inappropriate use of medical resources. The utility of a positive ANA test in a central triage (CT) system was studied by determining the autoantibody profiles and clinical diagnoses of patients referred to rheumatologists through a CT system because of a positive ANA test.</p><p>Methods</p><p>Patients that met three criteria were included: (1) referred to Rheumatology CT over a three year interval; (2) reason for referral was a “positive ANA”; (3) were evaluated by a certified rheumatologist. The CT clinical database was used to obtain demographic and clinical information and a serological database was used to retrieve specific ANA and/or extractable nuclear antigen (ENA) test results. Clinical information was extracted from the consulting rheumatologist's report.</p><p>Results</p><p>15,357 patients were referred through the CT system; 643 (4.1%) of these because of a positive ANA and of these 263 (40.9%) were evaluated by a certified rheumatologist. In 63/263 (24%) of ANA positive patients, the specialist provided a diagnosis of an ANA associated rheumatic disease (AARD) while 69 (26.2%) had no evidence of any disease; 102 (38.8%) had other rheumatologic diagnoses and 29 (11%) had conditions that did not meet AARD classification criteria. Of ANA positive archived sera, 15.1% were anti-DFS70 positive and 91.2% of these did not have an AARD.</p><p>Conclusions</p><p>This is the first study to evaluate the serological and clinical features of patients referred through a CT system because of a positive ANA. The spectrum of autoantibody specificities was wide with anti-Ro52/TRIM21 being the most common autoantibody detected. Approximately 15% of referrals had only antibodies to DFS70, the vast majority of which did not have clinical evidence for an AARD. These findings provide insight into the utility of autoantibody testing in a CT system.</p></div