PBC Screen: An IgG/IgA dual isotype ELISA detecting multiple mitochondrial and nuclear autoantibodies specific for primary biliary cirrhosis
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)
- et al.
Definition of human autoimmunity–autoantibodies versus autoimmune disease
Autoimmun Rev
(2010) - et al.
Use of a designer triple expression hybrid clone for three different lipoyl domain for the detection of antimitochondrial autoantibodies
Hepatology
(1996) - et al.
Detection of Gp210 autoantibodies in primary biliary cirrhosis using a recombinant protein containing the predominant autoepitope
Hepatology
(1995) - et al.
Specificity and sensitivity of gp210 autoantibodies detected using an enzyme-linked immunosorbent assay and a synthetic polypeptide in the diagnosis of primary biliary cirrhosis
Hepatology
(1996) - et al.
Management of primary sclerosing cholangitis
Am J Gastroenterol
(2002) - et al.
Penetration and co-localization in MDCK cell mitochondria of IgA derived from patients with primary biliary cirrhosis
J Autoimmun
(1998) - et al.
International autoimmune hepatitis group report: review of criteria for diagnosis of autoimmune hepatitis
J Hepatol
(1999) - et al.
Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group
J Hepatol
(2004) - et al.
Evaluation of newly developed ELISA using "MESACUP-2 test mitochondrial M2" kit for the diagnosis of primary biliary cirrhosis
Clin Biochem
(2003) - et al.
Detection of antimitochondrial autoantibodies in immunofluorescent AMA-negative patients with primary biliary cirrhosis using recombinant autoantigens
Hepatology
(2001)
Autoantibodies against nuclear pore complexes are associated with more active and severe liver disease in primary biliary cirrhosis
J Hepatol
Primary biliary cirrhosis
N Engl J Med
Antinuclear antibodies in primary biliary cirrhosis
Semin Liver Dis
Interpreting serological tests in diagnosing autoimmune liver diseases
Semin Liver Dis
Autoimmune liver serology: current diagnostic and clinical challenges
World J Gastroenterol
EASL clinical practice guidelines: management of cholestatic liver diseases
J Hepatol
Primary biliary cirrhosis
Hepatology
A sensitive bead assay for antimitochondrial antibodies: chipping away at AMA-negative primary biliary cirrhosis
Hepatology
New ELISA for detecting primary biliary cirrhosis-specific antimitochondrial antibodies
Clin Chem
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
Cited by (121)
Prevalence and significance of antimitochondrial antibodies in autoimmune hepatitis (AIH): Results from a large multicentre study of the International AIH Group
2023, European Journal of Internal MedicineAutoantibodies testing in autoimmunity: Diagnostic, prognostic and classification value
2023, Autoimmunity ReviewsAutoantibodies in Primary Biliary Cholangitis
2022, Clinics in Liver DiseaseLaboratory skills for immunologists: utility and limitations with emphasis on allergy research
2022, Allergic and Immunologic Diseases: A Practical Guide to the Evaluation, Diagnosis and Management of Allergic and Immunologic DiseasesChallenges for diagnosis and treatment of primary biliary cholangitis
2022, Translational Autoimmunity: Challenges for Autoimmune Diseases: Volume 5
- 1
These authors contributed equally to this study.