Intended use

The DIASTAT^? anti-mitochondrial antibody test is a qualitative enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM autoantibodies specific for the M2 antigen of the mitochondrial membrane in human serum or plasma. Detection of these autoantibodies is a useful aid in the diagnosis of Primary Biliary Cirrhosis and is not definitive in isolation. Autoantibody levels represent one parameter in a multicriterion diagnostic process.

Background

Primary Biliary Cirrhosis (PBC) is a chronic and often fatal cholestatic autoimmune liver disease, primarily affecting young to middle-aged women. It is characterised by slow, progressive destruction of intrahepatic bile ducts, portal inflammation and scarring, eventually leading to cirrhosis and liver failure.

Clinical manifestations, biochemical tests and the detection of circulating anti-mitochondrial antibodies (AMAs) are used for diagnosis of PBC. Histological examination provides confirmatory information and is helpful in assessing prognosis. AMAs are reportedly present in over 95% of biopsy-proven PBCs, they have been observed at low levels in patients with other diseases and in apparently healthy individuals. Disease-specific subtypes of AMAs have been identified and are regarded as specific and sensitive markers for PBC. To date, several autoantigens have been identified; of these, the M2 antigen is believed to be specific for PBC. It is composed of two immunoreactive polypeptides of approximately 70kD and 50kD and has been identified as part of the pyruvate dehydrogenase complex, a major mitochondrial enzyme.

Current techniques available for the detection of AMAs include immunofluorescence, radio-immunoassay, enzyme?immunoassay and immunoblotting. The most commonly used method is immunofluorescence on the HEp 2 substrate, however immunofluorescent staining patterns can be non-specific, making identification of the antigen subtype difficult. ELISAs offer increased sensitivity, simplicity and more rapid results.
 

Technical information

The wells of the microtitre strips are coated with M2 antigen. During the first incubation, specific autoantibodies in diluted serum or plasma bind to the antigen-coated surface. The wells are then washed to remove unbound components. In the second incubation, the Conjugate, enzyme-labelled antibodies to human IgG and IgM, binds any surface-bound autoantibodies. After further washing, specific autoantibodies are traced by incubation with the Substrate. Addition of Stop Solution terminates the reaction, resulting in a coloured end-product. The amount of Conjugate bound is measured in absorbance units and compared with that bound by the Reference Control.