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Abstract Number: 713

Rpp25 Is a Major Target of Autoantibodies to the Th/to Complex As Measured by ELISA and a New Chemilumiscence Assay

Michael Mahler1, Cristina Gascon2, Sima Patel2, Angela Ceribelli3, Edward K.L. Chan3 and Minoru Satoh4, 1Research, INOVA Diagnostics, San Diego, CA, 2Research, INOVA Diagnostics, Inc., San Diego, CA, 3Oral Biology, University of Florida, Gainesville, FL, 4Medicine, University of Florida, Gainesville, FL

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords: Antinuclear antibodies (ANA), autoantibodies, autoantigens, diagnosis and systemic sclerosis

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Session Information

Title: Systemic Sclerosis, Fibrosing Syndromes, and Raynaud’s – Clinical Aspects and Therapeutics

Session Type: Abstract Submissions (ACR)

Background/Purpose:

Anti-nuclear antibodies (ANA) play an important role in the diagnosis of systemic autoimmune diseases including systemic sclerosis (SSc). A significant portion of ANA in SSc are directed against nucleolar antigens including the Th/To antigen. Several proteins of the Th/To complex have been reported to react with anti-Th/To antibodies. Although known for over 20 years, anti-Th/To antibodies are rarely used in routine testing algorithms to aid in the diagnosis of SSc. Furthermore, little is known about the clinical association of autoantibodies targeting the individual components of the Th/To antigen. The objective of the present study is to evaluate the newly developed ELISA and chemiluminescence immunoassay (CIA, QUANTA Flash, INOVA) to measure autoantibodies to Rpp25 using immunoprecipitation (IP) as reference method.

Methods:

A component of Th/To antigen (Rpp-25) was expressed as histidine-tagged recombinant protein in E.coliand purified by a standard method. Anti-Rpp25 antibodies in human sera were tested by ELISA and QUANTA Flash (fully automated on the BIO-FLASH System), and compared with results by immunoprecipitation as a standard.  Anti-Th/To by IP was based on detection of 7-2 and 8-2 RNA by immunoprecipitation and silver staining of RNAs. The first cohort consisted of 121 SSc patients including 8 anti-Th/To positive samples confirmed by IP, enrolled in the University of Florida Center for Autoimmune Diseases (UFCAD) registry from 2000-2012. Previously described eight anti-Th/To positive Italian SSc sera from Spedali Civili di Brescia (Brescia, Italy) were also included in some analysis. For evaluation of the QUANTA Flash Rpp25 assay, 10 anti-Th/To positive sera were randomly selected based on the available amount of sera. As controls, sera were collected from ANA positive asymptomatic healthy individuals (n=42), from rheumatoid arthritis patients (n=20) and from random healthy individuals (n=10).

Results:

The reactivity to Rpp25 by ELISA was significantly higher in anti-Th/To IP positive than in negative samples (p<0.0001). A sensitivity of 64.7% (95% CI 38.3-85.8%) and a specificity of 99.1% (95% CI 95.3-100.0%) was determined. To verify the results using a second method, anti-Th/To IP positive sera and negative controls were tested using the Rpp25 assay on the BIO-FLASH. At cut-off selected by receiver operating characteristic analysis 9/10 (90.0%) of the anti-Th/To positive sera but none of the controls were positive for anti-Rpp25 antibodies by BIO-FLASH (p<0.0001). Thus a sensitivity of 90.0% (95% CI 55.5-99.7%) and a specificity of 100.0% (95% CI 95.0-100.0%) were found. Ten samples were tested by ELISA and BIO-FLASH for anti-Rpp25 reactivity and the results were highly correlated (rho=0. 96, 95% CI 0.85-0.99; p<0.0001

Conclusion:

Rpp25 is a major target of autoantibodies to the Th/To autoantigen complex. Autoantibodies to Rpp25 detected by ELISA and especially CIA show excellent agreement with IP for anti-Th/To antibodies. The new assays may help to make this long-known antibody specificity widely available to clinicians. Further studies are needed to evaluate the clinical utility of the new assays.


Disclosure:

M. Mahler,

Inova Diagnostics, Inc.,

3;

C. Gascon,

Inova Diagnostics, Inc.,

3;

S. Patel,

Inova Diagnostics, Inc.,

3;

A. Ceribelli,
None;

E. K. L. Chan,
None;

M. Satoh,
None.

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