Anti-M2 ELISA Assay Kit


The Anti-M2 (Mitochondrial Anitbodies) ELISA Assay Kit is used for the quantitative determination of IgG antibodies to the mitochondrial antigen M2 in human serum or plasma for the diagnosis of primary biliary cirrhosis (PBC). The Eagle Biosciences Anti-M2 (Mitochondrial Anitbodies) ELISA is for research use only and not for diagnostic procedures.
This product was previously known as AM231-K01.

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Anti-M2 ELISA Assay Kit

Anti-M2 ELISA Assay Kit Developed and Manufactured by Medipan

Size: 1×96 wells
Sensitivity: 3 U/ml
Dynamic Range: 3 – 300 U/ml
Incubation Time: 2 hours
Sample Type: Serum, Plasma
Sample Size: 10 µL
Alternative Names: Human Anti-M2 ELISA, Mitochondrial Antibody ELISA, Human Mitochondrial Antibody ELISA
For Research Use Only

Selected positive serum samples have been tested by this assay and found to dilute linearly. However, due to the heterogeneous nature of human autoantibodies there might be sera that do not follow this rule.
No cross reactivity to other autoantigens have been found.

Assay Principle

The Eagle Biosciences Anti-M2 ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of IgG antibodies to the mitochondrial antigen M2.  The antibodies of the standards, controls, and diluted patient samples react with to the native mitochondrial antigen M2 immobilized on the solid phase of microtiter plates. The use of highly purified native mitochondrial antigen M2 guarantees the specific binding of M2 antibodies of the specimen under investigation. Following an incubation period of 30 min at room temperature (RT), unbound serum components are removed by a wash step. The bound IgG antibodies react specifically with anti-human-IgG conjugated to horseradish peroxidase (HRP) within the incubation period of 30 min at RT. Excessive conjugate is separated from the solid-phase immune complexes by the following wash step. HRP converts the colorless substrate solution of 3,3’,5,5’-tetramethyl¬benzidine (TMB) added into a blue product. The enzyme reaction is stopped by dispensing an acidic solution (HCl) into the wells after 30 min at RT turning the solution from blue to yellow.

The optical density (OD) of the solution at 450 nm is directly proportional to the amount of specific antibodies bound. The standard curve is established by plotting the antibody concentrations of the standards (x-axis) and their corresponding OD values (y-axis) measured. The concentration of antibodies of the specimen is directly read off the standard curve.

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Additional Information

Assay Background

The group of primary autoimmune liver disease (PAL) comprises autoimmune hepatitis (AIH), PBC and primary sclerosing cholangitis (PSC).

The clinical picture of PAL is in most cases not different from other chronic liver diseases. About 15% of all cases with chronic liver diseases show an autoimmune pathogenesis. Therefore, after exclusion of infectious etiology especially by viruses, the determination of different autoantibodies is recommended.

PBC is a chronic inflammatory disorder of the small and medium bile ducts and serologically characterized by the occurrence of circulating M2 autoantibodies. M2 autoantibodies belong to the group of mitochondrial antibodies (AMA) and recognize three related proteins of the alpha-keto acid dehydrogenase complex. This complex is located at the inner mitochondrial membrane. The major epitopes of this protein complex is found on the E2 subunit and the protein X of the pyruvate dehydrogenase complex (PDC). Furthermore, M2 autoantibodies bind the E1alpha and E1beta 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).

M2 autoantibodies have been found in up to 96% of patients suffering from PBC thus representing a powerful serological tool for the diagnosis of PBC.

Assay Procedure

  1. Bring all reagents to room temperature (18-25°C) before use. Mix gently without causing foam.
  2. Dispense 100 µl standards (1 – 6), 100 µl positive (P) and negative (N) control, 100 µl diluted patient samples into the respective wells.
  3. Incubate 30 min at room temperature (18-25°C).
  4. Decant, then wash each well three times using 300 µl  wash solution (made of B).
  5. Add 100 µl of conjugate (D) to each well.
  6. Incubate 30 min at room temperature (18-25°C).
  7. Decant, then wash each well three times using 300 µl  wash solution (made of B).
  8. Add 100 µl of substrate (E) to each well.
  9. Incubate 30 min protected from light at room temperature (18-25°C).
  10. Add 100 µl of stop solution (F) to each well and mix gently.
  11. Read the OD at 450 nm versus 620 or 690 nm within 30 min after adding the stop solution.

Typical Standard Curve

Anti-M2 ELISA Assay Standard Curve

Package Inserts

Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.

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