Anti-Beta 2 GP-1 (Glycoprotein 1) ELISA Assay Kit

$340.00

The Eagle Biosciences Anti-ß2 GP-I ELISA Assay Kit is used for the quantitative determination of IgG or IgM antibodies to ß2 glycoprotein-I (ß2 GP-I) in human serum or plasma for the diagnosis of anti-phospholipid antibody syndrome (APAS). The Anti-ß2 GP-I ELISA is for research use only and not for diagnostic procedures.

SKU: B2G31-K01 Categories: , ,

Anti-Beta 2 GP-1 (Glycoprotein 1) ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 1 U/ml
Dynamic Range: 1 – 300 U/ml
Incubation Time: 2.5 hours
Sample Type: Serum, Plasma
Sample Size: 10 µL

Additional Information

Assay Background

APAS is an autoimmune disorder comprising such clinical symptoms like arterial or venous thrombosis, thrombocytopenia and recurrent fetal loss. Primary APAS as well as systemic lupus erythematosus (SLE) are characterized by the appearance of autoantibodies to negatively charged phospholipids (1). Although significance and pathological relevance of phospholipid anti-bodies are not completely revealed yet, the detection of several autoantibody specificities is usually applied to the differential diagnosis and follow-up of systemic rheumatic inflammatory diseases.
Unlike phospholipid antibodies which occur in some patients having infectious disease, phospholipid antibodies of autoimmune disease patients seem to recognize the relevant phospholipids in association with a plasma protein cofactor.
One of these cofactors has been identified as ß2 glycoprotein-I (ß2 GP-I) (apolipoprotein H) (2,3). ß2 GP-I, a serum protein with a molecular weight of 50 kDa affects platelet aggregation and coagulation.

The positively charged fifth domain of ß2 GP-I interacts with negatively charged phospholipids or activated polystyrol surfaces of ELISA wells. This interaction results in conformational changes of ß2 GP-I and the creation of new epitopes apparently recognized by autoimmune phospholipid autoantibodies.

Assay Principle

The Eagle Biosciences Anti- ß2 GP-I ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of IgG or IgM antibodies to ß2 glycoprotein-I in human serum or plasma. The antibodies of the standards, the positive control and diluted patient samples react with the human ß2 GP-I, immobilized on the solid phase of microtiter plates. The use of highly purified ß2 GP-I guarantees the specific binding of antibodies to ß2 glycoprotein-I of the specimen under investigation. Following an incubation period of 60 min at room temperature (RT), unbound serum components are removed by a wash step.

The bound antibodies react specifically with anti-human-IgG or IgM conjugated to horseradish peroxidase (HRP) within the next incubation period of 30 min at RT. Excessive conjugate is separated from the solid-phase immune complexes by an additional 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 added acidic solution (H2SO4) into the wells, after 15 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 specifically bound antibodies.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.

Assay Procedure

  1. Bring all reagents to room temperature (20…25°C) before use. Mix gently without causing foam.
  2. Dispense 100 µl calibrators (0 optional) 1 – 4 (quantitative) or 100 µl calibrator 1 (semi-quantitative)100 µl control P (N optional) 100 µl diluted patient samples into the respective wells.
  3. Incubate 60 min at room temperature (20…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 or E) solution to each well.
  6. Incubate 30 min at room temperature (20…25°C).
  7. Decant, then wash each well three times using 300 µl wash solution (made of B).
  8. Add 100 µl of substrate (F) to each well.
  9. Incubate 15 min protected from light at room temperature (20…25°C).
  10. Add 100 µl of stop solution (G) 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

Manual

Product Manual


Publications

Literature

  • Harris EN, Gharavi AE, Boey ML, Patel BM, Mackworth-Young GG, Loizou S and Hughes GRV: Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983 11:1211
  • Galli M, Comfurius P, Maassen C, Hemker HC, DeBaets MHVan Breda-Vriesman PJC, Barbui T, Zwaal RFA, Bevers EM: Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein factor. Lancet 1990 335:1544-1547
  • McNeil HP, Simpson RJ, Chesterman CN, Krilis SA: Anti-phospho¬lipid antibodies are directed against a complex antigen that includes a lipid-binding factor of coagulation: beta 2-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 1990 87:4120-4124