Anti-Phospholipid Screen ELISA Kit

$350.00

The Anti-Phospholipid Screen ELISA Assay Kit is used for the separate quantitative determination of IgG and / or IgM antibodies to phospholipides (cardiolipin, phosphatidyl-serine, -inositol, -ethanolamine, phosphatidic acid and 2 glycoprotein I) in human serum for the diagnosis of anti-phospholipid antibody syndrome (APAS). The Eagle Biosciences Anti-Phospholipid Screen ELISA Assay kit is for research use only and not for diagnostic procedures.

SKU: PHS31-K01 Categories: , ,

Anti-Phospholipid Screen ELISA Kit

Anti-Phospholipid Screen ELISA 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-Phospholipid Screen ELISA, Human Phospholipid Antibody Screen ELISA
For Research Use Only


Assay Principle


The Anti-Phospholipid Screen ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of IgG and / or IgM antibodies to negatively charged phospholipids.

The antibodies of the standards, controls and diluted patient samples react with an antigen complex consisting of cardiolipin, phosphatidyl-serine, -inositol, -ethanolamine, phosphatidic acid and the cofactor beta-2 GP-I immobilized on the solid phase of microtiter plates. The use of highly purified human beta-2 GP-I guarantees the specific binding of autoimmune related phospholipid antibodies of the specimen under investigation. Following an incubation period of 60 min 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 min 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.


Related Products

DotDiver Anti‐Phospholipid IgM
DotDiver Anti‐Phospholipid IgG
Anti-Beta-2 GP-I Screen ELISA

Additional Information

Assay Background


APAS is an autoimmune disorder comprising such clinical symptoms like arterial or venous thrombosis, 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) (2,3). 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 such as Cardiolipin. 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 (18…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 (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 or E) solution 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 (F) to each well.
  9. Incubate 15 min protected from light at room temperature (18…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


Anti-Phospholipid Screen ELISA Assay Standard Curve

Manual

Product Manual