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

Abstract

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.

Figure 1

Anti-Rpp25 antibodies in Th/To immunoprecipitation (IP)-positive and IP-negative samples measured by enzyme-linked immunosorbent assay (ELISA). Comparative descriptive analysis is shown in a). In b) the receiver operating characteristic (ROC) analysis shows good discrimination between anti-Th/To IP-positive patients (n = 14) and controls (anti-Th/To-negative SSc and healthy individuals, n = 156). AUC, area under the curve.

Figure 2

Correlation between enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA). The results of 53 samples tested by anti-Rpp25 ELISA and CLIA (QUANTA Flash) showed good agreement (rho = 0.71; P < 0.0001, Spearman correlation test).

Figure 3

Anti-Rpp25 antibodies in samples from anti-Th/To immunoprecipitation (IP)-positive patients, an independent cohort of patients with systemic sclerosis and controls measured by QUANTA Flash anti-Rpp25. Comparative descriptive analysis is shown of samples from anti-Th/To IP-positive patients (n = 8), patients with SSc (n = 70) and controls (n = 367) including patients with rheumatoid arthritis (RA, n = 141), systemic lupus erythematosus (SLE, n = 67), undifferentiated connective tissue disease (UCTD, n = 17), osteoarthritis (OA, n = 47), ankylosing spondylitis (AS, n = 13), polymyalgia rheumatica (PR, n = 20), degenerative spine disease (n = 6), fibromyalgia (n = 5), psoriasis arthritis (n = 13), other pathologies (n = 28) and healthy individuals (HI, n = 10). SE, standard error.

Figure 4

Receiver-operating characteristics (ROC) analysis. a) ROC analysis shows good discrimination between Th/To immunoprecipitation (IP)-positive patients (n = 8) and controls (n = 367) including rheumatoid arthritis (RA, n = 141), systemic lupus erythematosus (SLE, n = 67), undifferentiated connective tissue disease (UCTD, n = 17), osteoarthritis (OA, n = 47), ankylosing spondylitis (AS, n = 13), polymyalgia rheumatica (PMR, n = 20), degenerative spine disease (n = 6), fibromyalgia (n = 5), psoriasis arthritis (n = 13), other pathologies (n = 28) and healthy individuals (HI, n = 10). b) ROC analysis shows discrimination between SSc (n = 70) and controls (n = 367). AUC, area under the curve; SSc, systemic sclerosis.