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

Abstract

The antimitochondrial response in primary biliary cirrhosis (PBC) is the most highly directed and specific self-reacting antibody in human immunopathology. Originally, antimitochondrial antibodies (AMAs) were detected by indirect immunofluorescence (IIF) and found in approximately 90% of well-documented patients with PBC. The introduction of recombinant autoantigens and the use of immunoblotting have increased the sensitivity and specificity of AMAs, and they are now considered positive in approximately 95% of patients with PBC. Clearly, accurate autoantibody detection represents one of the fundamental requirements for reliable diagnostics in autoimmunity. To address the 5% of AMA-negative patients with PBC, we have generated and validated a bead assay for the detection of AMA. We enrolled 120 patients with PBC, including a non-random group of 30 rigorously proven AMA-negative patients, 50 healthy subjects, and 74 controls with autoimmune diseases ( T he presence of circulating anti-mitochondrial antibodies (AMAs) represents a key diagnostic feature of primary biliary cirrhosis (PBC). Using highly sensitive assays with recombinant antigens, up to 95% of PBC patients have sera autoantibodies directed against the ubiquitous 2-oxo-acid-dehydrogenase complexes involved in mitochondrial energy metabolism. AMA positivity with its unique prevalence rate among autoimmune diseases 1 represents one of the three diagnostic criteria for PBC, the others being liver histology compatible with the disease and a persistent elevation of alkaline phosphatase. 2 Approximately 5% of well-documented PBC patients do not react with any of the mitochondrial antigens using currently available assays. Over the past decade, the "AMA-negative" population has been progressively reduced in our laboratory by the development of more sensitive detection techniques. 3 Nonetheless, the persistence of some AMA-negative subjects raises the question of whether these patients represent a subpopulation with distinct clinical features or simply have antibody titers and specificities undetectable with available technology. 4 Recent technological advances have led to the development of increasingly automated detection methods associated with more rapid, accurate, and reliable assaying, 5 which could address the question. The From the

Antimitochondrial antibodies in acute liver failure: Implications for primary biliary cirrhosis

“…In studies of AMA reactivity in large numbers of sera, we have demonstrated that when recombinant antigens are used for immunodiagnosis, they are strikingly specific for PBC; we have also shown that such specificity and sensitivity is dramatically increased with the use of ELISA and/or immunoblot assays compared with immunofluorescence. 21,24,25 In our initial analysis and our observations that AMAs were indeed found in ALF, we decided to replicate the data in a second site-hence the duplicate analysis at both UC Davis and Teikyo University (both sites had identical results). Recent work has suggested that posttranslational modification of the mitochondrial 2-OADC lipoyl moiety is a possible mechanism in the initiation of AMAs in PBC.…”

Autoimmune liver serology: Current diagnostic and clinical challenges

“…This preparation contains all three immunodominant mitochondrial antigenic epitopes, namely PDC-E2, BCOADC-E2 and OGDC-E2 [88] . Although assays based on MIT3 are reported to give positive results for PBC sera that test negative for AMA by conventional IFL techniques [89,90] , IFL testing for AMA should remain the screening procedure.…”

“…They may be of diagnostic assistance, especially in those cases where it is difficult to interpret the IFL staining patterns due to concurrent autoantibody reactivities or in true AMAnegative PBC cases [ of ACA reactivity in the combined PBC/CREST disease. Assays to detect multiple reactivities (multiplex) and to provide a full autoimmune serological profile of relevance to PBC are being developed [89] . At present, a lack of guidelines for the detection of PBC-specific autoantibodies by scientific bodies responsible for the standardization of autoimmune serological tests is a significant handicap and perpetuates uncertainties on which are the clinically relevant tests (see below).…”

Experimental evidence on the immunopathogenesis of primary biliary cirrhosis

“…64,65 Autoantibodies Circulating AMAs are highly specific for PBC and are detected in nearly 100% of patients when tested for using techniques based on recombinant mitochondrial antigens (immunoblotting or enzymelinked immunosorbent assay). The high sensitivity and specificity of AMAs make them one of the most specific diagnostic tests of human immunopathology, 66 different from other autoimmune conditions, 67,68 including autoimmune hepatitis. 69 In most clinical settings for initial screening, AMAs are usually detected by indirect immunofluorescence using rodent liver, kidney and stomach sections as a substrate, leading in some cases to false positive or negative results.…”