Anti-Phosphatidylserine ELISA Assay Kit

$525.00

The Anti-Phosphatidylserine ELISA Assay Kit is used for the separate quantitative determination of IgG and / or IgM antibodies to phosphatidylserine in human serum for the diagnosis of anti-phospholipid antibody syndrome (APAS). The Anti-Phosphatidylserine ELISA Assay is for research use only and not for diagnostic procedures.

SKU: PDL31-K01 Categories: , ,

Anti-Phosphatidylserine ELISA Assay Kit

Anti-Phosphatidylserine ELISA Assay Kit Developed and Manufactured by Medipan

Size: 1×96 wells
Sensitivity: 1 U/ml
Dynamic Range: 1 – 300 U/ml
Incubation Time: 2.5 hours
Sample Type: Serum
Sample Size: 10 µL
Alternative Names: Human Anti-Phosphatidylserine ELISA, Human Phosphatidylserine Antibody ELISA, APAS
For Research Use Only


Linearity
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.
Specificity
Measuring a group of blood donors (n=62) in both Anti-Phosphatidyl-Serin IgG and IgM assays a specificity of 100% was found. All samples were tested negative.


Assay Principle

The Anti-Phosphatidylserine ELISA Assay Kit is used for the highly sensitive determination of autoantibodies to phosphatidyl-serine in human serum.  The antibodies of calibrators, control and diluted patient samples react with an antigen complex consisting of phosphatidyl-serine and the cofactor ß2-GP I immobilized on the solid phase of microtiter plates. Following an incubation period of 60 minutes at room temperature, unbound sample components are removed by a wash step. The bound antibodies react specifically with anti-human-IgG or anti-human-IgM conjugated to horseradish peroxidase (HRP) within the incubation period of 30 minutes at room temperature (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 into the wells after 15 min at room temperature 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 calibrators (x-axis) and their corresponding OD values (y-axis) measured. The concentration of antibodies of the specimen is directly read off the standard curve. Evaluating the test by a semi-quantitative method using a cut-off calibrator is also possible.


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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 antibodies are not completely revealed yet, the detection of such autoantibodies is widely established and plays an important role in the diagnostics of systemic autoimmune diseases.  Unlike phospholipid antibodies which appear in some infectious disease patients autoimmune patients exhibit phospholipid antibodies that seem to recognize phospholipids in association with plasma protein cofactors such as ß2-glycoprotein I (ß2-GP I, apolipoprotein H) (2). 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 such as phosphatidyl-serine. This interaction results in conformational changes of the protein and the creation of new epitopes apparently recognized by autoimmune phospholipid autoantibodies.

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 Controls P, N (N optionally), 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) 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 (E) to each well.
  9. Incubate 15 min protected from light at room temperature (20…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-Phosphatidylserine ELISA Assay Kit Standard Curve

Documents


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
  • 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

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