AMA-M2 Ab ELISA Kit

Sample

Serum, plasma

Species Reactivity

Human

Detection Method

iELISA

Intended Use

AMA-M2 Ab ELISA Kit is an indirect solid phase ELISA for the quantitative measurement of IgG class autoantibodies against mitochondrial M2 subtype antigen in human serum or plasma.

Contents of Kit

1. Divisible microplate consisting of 12 modules of 8 wells each. Ready to use. -- 96 wells
2. Calibrators (A-F) (0,12.5, 25, 50,100, 200 IU/mL), containing serum/buffer matrix (PBS, BSA, detergent, NaN₃ 0.09%), yellow. Ready to use. -- 1.5 mL × 6
3. Control positive (1) and negative (2), containing AMA-M2 antibodies in a serum/buffer matrix (PBS, BSA, detergent, NaN₃ 0.09%), yellow. Ready to use. The concentration is specified on the certificate of analysis. -- 1.5 mL × 2
4. Sample buffer P (5×),containing PBS, BSA detergent, preservative NaN₃ 0.09%, yellow, 5× conc. -- 20 mL
5. Enzyme conjugate containing anti-human IgG; HRP labelled; PBS, BSA, detergent, preservative ProClin 300 0.05%, light red. Ready to use. -- 15 mL
6. TMB substrate; containing 3, 3', 5, 5'-Tetramethylbenzidin, colorless. Ready to use. -- 15 mL
7. Stop solution; contains acid. Ready to use. -- 15 mL
8. Wash solution (50×), containing Tris, detergent, presearvative NaN₃ 0.09%; 50× conc. -- 20 mL

Storage

Store test kit at 2-8°C in the dark. Do not expose test reagents to heat, sun or strong light during storage and usage. Store microplate sealed and desiccated in the clip bag provided. Unopened reagents are stable until expiration of the kit. See labels for individual batch. Diluted Wash Solution and Sample Buffer are stable for at least 30 days when stored at 2-8°C. We recommend consumption on the same day.

Performance Characteristics

Calibration
The assay system is calibrated against the international reference preparation WHO67/183 for AMA-M2
as 100 IU/mL.

Detection Range

The calculation range of this ELISA assay is 0-200 IU/mL.

Detection Limit

Functional sensitivity was determined to be: 1IU/mL

General Description

Anti-mitochondrial antibodies (AMA) are a heterogeneous group of autoantibodies directed against various proteins that are located in the outer and inner membrane of mitochondria. Specific anti-mitochondrial antibodies have been described for the primary biliary cirrhosis (PBC) as subtypes M2, M4, M8 and M9. Other AMA subtypes are related to other diseases, like collagenosis (AMA-M5) and drug induced LE and Hepatitis (AMA-M3 and AMA-M6).
The heterogeneously reacting specific anti-mitochondrial antibodies of the M2 subtype are directed against three related proteins of the α-keto acid dehydrogenase complex which is located at the inside of the mitochondrial membrane. The recognized major epitope is located on the E2 subunit and the protein X of the pyruvate dehydrogenase complex (PDC). Additionally AMA-M2 autoantibodies recognise the (E1a und E1b) subunits of the same complex and the E2 subunit of several other multi enzyme complexes, such as the 2-oxo-glutarate dehydrogenase complex (OGDC) and the branched chain 2-oxo acid dehydrogenase complex (BCOADC).
Using HEp 2 Cell monolayers for indirect immune fluorescence AMA-M2 autoantibodies are characterised as a fine-speckled cytoplasmatic, perinuclear condensed fluorescence pattern. For differential diagnosis of the primary biliary cirrhosis (PBC) determination of AMA-M2 by ELISA is recommended because of its high sensitivity and specificity.
In patients with other autoimmune diseases determination of AMA antibodies allows an early screening for the occurence of subtype M2 and M9 antibodies which may be related with the development and / or association of PBC.
Profiling the AMA subtypes allows an immunological and prognostic classification of the primary biliary cirrhosis. Beginning cases of symptomatic PBC often exhibit only AMA-M2 sub-type antibodies (sometimes in combination with AMA-M9), whereas progressive cases and mixed syndromes with chronic acute hepatitis (CAH) are related with the occurrence of AMA-M2, -M4 and -M8 antibody subtypes.