Elsevier

Journal of Autoimmunity

Volume 35, Issue 4, December 2010, Pages 436-442
Journal of Autoimmunity

PBC Screen: An IgG/IgA dual isotype ELISA detecting multiple mitochondrial and nuclear autoantibodies specific for primary biliary cirrhosis

https://doi.org/10.1016/j.jaut.2010.09.005Get rights and content

Abstract

A dual isotype (IgG, IgA) enzyme-linked immunosorbent assay (ELISA) designed to provide enhanced detection of primary biliary cirrhosis (PBC)-specific autoantibodies against both major mitochondrial and nuclear antigens has been developed and recently become commercially available. The assay (PBC Screen) simultaneously detects IgG and IgA autoantibodies to the immunodominant portions of the 3 major mitochondrial (MIT3) and nuclear (gp210, and sp100) antigens. The aim of this study was to compare the performance of the PBC Screen to the combined performance obtained with individual IgG ELISAs to MIT3, gp210, and sp100 on a large group of selected patients from multiple centers. A total of 1175 patients with PBC and 1232 subjects without PBC were evaluated. Non-PBC groups included healthy controls (624) as well as individuals with autoimmune hepatitis (281), primary sclerosing cholangitis (77), viral hepatitis (91 hepatitis B and 98 hepatitis C), other liver diseases (31), and other infectious or autoimmune diseases (30). The PBC Screen at the receiver operator characteristic optimized cutoff of 27.8 units, had an overall sensitivity of 83.8%, specificity of 94.7% and area under curve of 0.9212. This was similar to the specificity of 96.1% obtained by the combined results of individual MIT3, sp100, and gp210 IgG ELISAs (kappa index at 0.898). Of the 253 PBC patients without AMA detectable by immunofluorescence, 113 (44.7%) were interpreted as positive for PBC-specific autoantibodies. In conclusion, the PBC Screen is an appropriate first-line test for the diagnosis of PBC, including for patients negative for markers assessed using conventional methods.

Introduction

Primary biliary cirrhosis (PBC) is a chronic liver disease characterized by the progressive destruction of the small intra-hepatic bile ducts, increasing functional impairment of the liver and, over time, to liver failure and the necessity of liver transplantation [1]. Serological assays are important for the diagnosis of PBC [2], [3], [4]. Anti-mitochondrial antibodies (AMA), detected by indirect immunofluorescence microscopy (IIF) on rodent kidney/stomach/liver sections or HEp-2 cell line substrates, are the classic serological markers of PBC and are found in up to 90–95% of PBC patients [5], [6]. IIF, however, is labor-intensive and highly dependent on skill and experience of the observer for interpretation of the diagnostic labeling patterns. Moreover, patients with liver or extra-hepatic autoimmune diseases often have antibodies to multiple targets and this can make interpretation of the resulting complex patterns difficult, leading to both false positive and negative results [7]. ELISAs developed using a purified mitochondrial antigen fraction originally designated “M2” have permitted more sensitive and objective detection of AMA antibodies. More recently, Gershwin, Leung, and colleagues created a recombinant fusion protein (MIT3), which includes the immunodominant portions of the 3 primary targets of AMA, namely the E2 subunits of the pyruvate dehydrogenase complex (PDC-E2), the branched-chain 2-oxo-acid dehydrogenase complex (BCOADC-E2) and the 2-oxo-glutarate dehydrogenase complex (OGDC-E2) [8]. This antigen has demonstrated a higher diagnostic sensitivity than conventional ELISAs for AMA [8]. Lastly, it has been suggested that Luminex and an ELISA using a mixture of purified PDC and MIT3 as antigenic targets may increase sensitivity of the AMA detection systems [9], [10].

AMA are considered to be the hallmark of PBC, but they are not the only disease-specific autoantibodies. Antinuclear antibodies (ANA) from patients with PBC recognize 2 highly specific nuclear antigens. ANA in patients with PBC that label the nuclear periphery in a punctate rim-like pattern most often recognize gp210, an integral membrane protein associated with the nuclear pore complex [11]. ANA that give a multiple nuclear dot pattern recognize the nuclear body-associated protein sp100 [12]. Autoantibodies against gp210 and sp100 are detected in approximately 25% of patients with PBC using anti-total IgG antiserum but up to 65% when anti-IgG isotype specific antisera was used [13], some of whom do not have detectable AMA, and are highly specific for the disease [3], [4]. Hence, tests to detect gp210 and sp100 antibodies increase the specificity and sensitivity in diagnosing PBC, especially in cases where the clinical presentation and/or serological picture may be unclear. ELISAs utilizing recombinant fusion proteins or synthetic polypeptides have been developed to detect gp210 and sp100 antibodies in patients with PBC [12], [14], [15]. In addition, we and others showed that these nuclear antibodies are associated with more severe disease and have prognostic as well as diagnostic value in PBC (in contrast to AMA alone) [16], [17].

Optimal detection of antibodies against the immunodominant portions of the major mitochondrial as well as the nuclear antigens would require performing 3 separate assays. Practically, however, the costs and logistics involved in running 3 tests may discourage this approach and, as a result, some individuals with PBC may remain undiagnosed or present clinicians with diagnostic dilemmas. To address this problem, a new multi-analyte, dual isotype ELISA, which simultaneously screens for IgG and IgA antibodies to the 3 immunodominant epitopes of AMA, gp210, and sp100 (PBC Screen) in each test serum has been developed. The present multi-center study was performed to assess this newly developed ELISA in a large cohort of well-characterized PBC patients and patients with other diseases assembled from centers in North America, Europe, and Asia, and to test the concordance of the method with results obtained using the individual MIT3, gp210, and sp100 ELISAs.

Section snippets

Subjects

The study was performed in 2 phases. The first phase included subjects from the United States, Canada, United Kingdom, and Greece from 2005–2006, while the second phase was performed from 2007 to 2009 on an independent collection of sera from subjects in the United States, Italy, and Japan. We enrolled a total of 1175 well-defined PBC patients, including 922 with detectable AMA and 253 without detectable AMA as determined by IIF, and 1232 controls (Table 1). A specific effort was made to enroll

Sensitivity and specificity of the PBC Screen for diagnosis of PBC

ROC analysis resulted in a sensitivity of 83.8% (985/1175) for the complete cohort of PBC patients, including those with and without AMA detectable by IIF, with a specificity of 94.7% at an optimal cutoff value of 27.8 units for diagnosis of PBC (Fig. 1). The area under curve value for this assay was 0.9212. At the optimal cutoff, PLR, NLR, and diagnostic accuracy were 15.81, 0.17, and 0.894, respectively. The diagnostic characteristics of the new cutoff (27.8 units) determined from the data in

Discussion

IIF microscopy is the standard for detection of AMA in many clinical laboratories. However, ELISAs, especially those using recombinant proteins such as MIT3, are significantly more sensitive [8]. In addition, ELISAs make detection of antibodies against nuclear pore protein gp210 and the nuclear body protein sp100 more accurate and objective [2], [3], [4]. Close to half of the specimens in our study that were originally classified as “AMA-negative” PBC by experienced hepatologists were shown to

Acknowledgements

DPB was supported by a CSL award from the Higher Education Funding Council for England.

References (40)

  • P. Invernizzi et al.

    Autoantibodies against nuclear pore complexes are associated with more active and severe liver disease in primary biliary cirrhosis

    J Hepatol

    (2001)
  • M.M. Kaplan et al.

    Primary biliary cirrhosis

    N Engl J Med

    (2005)
  • P. Invernizzi et al.

    Antinuclear antibodies in primary biliary cirrhosis

    Semin Liver Dis

    (2005)
  • P. Invernizzi et al.

    Interpreting serological tests in diagnosing autoimmune liver diseases

    Semin Liver Dis

    (2007)
  • D.P. Bogdanos et al.

    Autoimmune liver serology: current diagnostic and clinical challenges

    World J Gastroenterol

    (2008)
  • EASL clinical practice guidelines: management of cholestatic liver diseases

    J Hepatol

    (2009)
  • K.D. Lindor et al.

    Primary biliary cirrhosis

    Hepatology

    (2009)
  • S. Oertelt et al.

    A sensitive bead assay for antimitochondrial antibodies: chipping away at AMA-negative primary biliary cirrhosis

    Hepatology

    (2007)
  • C. Dahnrich et al.

    New ELISA for detecting primary biliary cirrhosis-specific antimitochondrial antibodies

    Clin Chem

    (2009)
  • J.C. Courvalin et al.

    The 210-kD nuclear envelope polypeptide recognized by human autoantibodies in primary biliary cirrhosis is the major glycoprotein of the nuclear pore

    J Clin Invest

    (1990)
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    These authors contributed equally to this study.

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