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

Mahler M, Gascon C, Patel S, Ceribelli A, Fritzler MJ, Swart A, Chan EK, Satoh M - Arthritis Res. Ther. (2013)

Bottom Line: 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).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).

View Article: PubMed Central - HTML - PubMed

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.

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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).
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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).

Mentions: To verify the results using a second method, a total of 53 samples that were tested by ELISA and QUANTA Flash CLIA for anti-Rpp25 reactivity showed high correlation (rho = 0.71, 95% CI 0.56, 0.81; P < 0.0001; see Figure 2). Next we analyzed the precision of the QUANTA Flash Rpp25 CLIA, which demonstrated good precision with a total variation of 6.6% (Table 1). To analyze the prevalence of anti-Rpp25 antibodies in different cohorts, anti-Th/To-positive sera (n = 8 identified by IP), unselected SSc samples (n = 70) and disease and healthy controls (n = 367) were tested using the anti-Rpp25 assay on the BIO-FLASH instrument (Figure 3). The anti-Th/To-positive sera were from four SSc patients, two with RP, one with SS and one with ILD. There was no significant difference between the Th/To-positive SSc and non-SSc patients (P = 0.4857). When comparing anti-Th/To IP-positive samples and controls by ROC analysis, showing an AUC value of 0.919 (95% CI 0.919, 1.000), a preliminary cutoff was defined (10,000 RLU, see Figure 4). At this cutoff 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 by QUANTA Flash Rpp25 (P < 0.0001). Thus a positive percent agreement of 100.0% (95% CI 63.1, 100.0%) and a negative percent agreement of 99.5% (95% CI 98.0, 99.9%) were found. At this cutoff, 2/70 (2.9%) of the second cohort of SSc patients were positive for anti-Rpp25 antibodies compared to 2/367 (0.5%) of the controls (P = 0.032). Positive (LR+) and negative (LR-) likelihood ratios were 5.24 and 0.98, respectively (Table 2). The two control patients with a positive result had low levels of anti-Rpp25 antibodies and one had RA (RLU = 25,259) and the other patient had a diagnosis of PR (RLU = 19,561). ROC analysis showed discrimination between SSc patients and controls with AUC values of 0.732 (95% CI 0.655, 0.809).


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

Mahler M, Gascon C, Patel S, Ceribelli A, Fritzler MJ, Swart A, Chan EK, Satoh M - Arthritis Res. Ther. (2013)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672760&req=5

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).
Mentions: To verify the results using a second method, a total of 53 samples that were tested by ELISA and QUANTA Flash CLIA for anti-Rpp25 reactivity showed high correlation (rho = 0.71, 95% CI 0.56, 0.81; P < 0.0001; see Figure 2). Next we analyzed the precision of the QUANTA Flash Rpp25 CLIA, which demonstrated good precision with a total variation of 6.6% (Table 1). To analyze the prevalence of anti-Rpp25 antibodies in different cohorts, anti-Th/To-positive sera (n = 8 identified by IP), unselected SSc samples (n = 70) and disease and healthy controls (n = 367) were tested using the anti-Rpp25 assay on the BIO-FLASH instrument (Figure 3). The anti-Th/To-positive sera were from four SSc patients, two with RP, one with SS and one with ILD. There was no significant difference between the Th/To-positive SSc and non-SSc patients (P = 0.4857). When comparing anti-Th/To IP-positive samples and controls by ROC analysis, showing an AUC value of 0.919 (95% CI 0.919, 1.000), a preliminary cutoff was defined (10,000 RLU, see Figure 4). At this cutoff 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 by QUANTA Flash Rpp25 (P < 0.0001). Thus a positive percent agreement of 100.0% (95% CI 63.1, 100.0%) and a negative percent agreement of 99.5% (95% CI 98.0, 99.9%) were found. At this cutoff, 2/70 (2.9%) of the second cohort of SSc patients were positive for anti-Rpp25 antibodies compared to 2/367 (0.5%) of the controls (P = 0.032). Positive (LR+) and negative (LR-) likelihood ratios were 5.24 and 0.98, respectively (Table 2). The two control patients with a positive result had low levels of anti-Rpp25 antibodies and one had RA (RLU = 25,259) and the other patient had a diagnosis of PR (RLU = 19,561). ROC analysis showed discrimination between SSc patients and controls with AUC values of 0.732 (95% CI 0.655, 0.809).

Bottom Line: 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).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).

View Article: PubMed Central - HTML - PubMed

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.

Show MeSH
Related in: MedlinePlus