Mouse Phosphatidylserine/prothrombin complex (PS/PT) IgM Antibody ELISA Kit

Sample

Serum, plasma

Species Reactivity

Mouse

Detection Method

iELISA

Intended Use

The Mouse Phosphatidylserine/prothrombin complex (PS/PT) IgM Antibody ELISA Kit is designed for the Semi-quantitative analysis of Mouse Serum, plasma samples.

Contents of Kit

1. Polystyrene microwell ELISA plate coated with a purified antigen, PS/PT (12-1 x 8 wells), with holder in foil package containing desiccants
2. ELISA Negative Control, 1 vial of buffer containing preservative and mouse serum with no mouse antibodies to PS/PT prediluted, 1.2 mL
3. aPS/PT IgM ELISA Control, 1 vial of buffer containing preservative and mouse serum IgM antibodies to PS/PT prediluted, 1.2 mL
4. aPS/PT IgM ELISA Calibrator A, 1 vial of buffer containing preservative and mouse serum IgM antibodies to PS/PT prediluted, 1.2 mL
5. aPS/PT IgM ELISA Calibrator B, 1 vial of buffer containing preservative and mouse serum IgM antibodies to PS/PT prediluted, 1.2 mL
6. aPS/PT IgM ELISA Calibrator C, 1 vial of buffer containing preservative and mouse serum IgM antibodies to PS/PT prediluted, 1.2 mL
7. aPS/PT IgM ELISA Calibrator D, 1 vial of buffer containing preservative and mouse serum IgM antibodies to PS/PT prediluted, 1.2 mL
8. aPS/PT IgM ELISA Calibrator E, 1 vial of buffer containing preservative and mouse serum IgM antibodies to PS/PT prediluted, 1.2 mL
9. HRP aPS/PT IgM Conjugate, (goat), anti-mouse, 1 vial – colored green containing buffer, protein stabilizers and preservative, 10 mL
10. HRP Sample Diluent Plus, 1 vial – colored pink containing Tris-buffered saline, Tween 20, Calcium protein stabilizers and preservative, 50 mL
11. HRP Wash Buffer Plus (40x), 1 vial of 40x concentrate - colored red containing Tris-buffered saline, Tween 20 and Calcium, 25 mL, Refer to the Methods Section for dilution instructions.
12. TMB Chromogen, 1 vial containing stabilizers, 10 mL
13. HRP Stop Solution, 0.344 M Sulfuric Acid, 1 vial – colorless

Storage

1. Store all the kit reagents at 2-8°C. Do not freeze. Reagents are stable until the expiration date when stored and handled as directed.
2. Unused antigen coated microwell strips should be resealed securely in the foil pouch containing desiccants and stored at 2-8°C.
3. Diluted wash buffer is stable for 1 week at 2-8°C.

Detection Range

aPS/PT IgG: 5.1-150 units
aPS/PT IgM: 3.6-150 units
For samples with values above 150 units, report the result as >150 units or positive. For samples with values less than 5.1 units for IgG or 3.6 units for IgM report the result as <5.1 or <3.6 or negative.

General Description

Antiphospholipid antibodies (aPL) represent a large group of immunoglobulins of considerable clinical importance due to their association with arterial and/or venous thrombosis, recurrent pregnancy loss, neurological disorders, pulmonary hypertension and thrombocytopenia. Anticardiolipin antibodies (ACA) detected by ELISA and lupus anticoagulant (LA) detected by clotting assays have been the most established and standardized tests for the diagnosis of antiphospholipid syndrome (APS). The family of aPL has recently expanded to include a heterogeneous group of auto antibodies whose specificity is directed against phospholipid binding proteins or their complex with phospholipids.

Among the phospholipid-binding proteins, the best studied is β2-Glycoprotein I (β2 GPI). β2 GPI bears the cryptic epitope(s) for ACA binding which are exposed when β2 GPI binds to negatively charged phospholipids such as cardiolipin, or immobilized on irradiated plastic ELISA plates. A number of studies have highlighted the significance of anti-β2 GPI antibody by ELISA as an alternative assay to conventional ACA ELISA with higher specificity and this method has been embraced by the clinical community.

Prothrombin (Factor II) is another phospholipid binding protein with a molecular weight of 72,000, present in normal plasma in an approximate concentration of 2.5 μmol/L. Prothrombin exerts a procoagulant activity via a prothrombinase complex, triggering fibrinogen conversion to fibrin. LA has been shown to be directed against multiple plasma proteins, including β2 GPI and prothrombin.

In 1995, Arvieux et al showed that aPT could be detected by ELISA in lupus anticoagulant (LA) positive samples using irradiated plates in a similar manner to the anti-β2 GPI assay. They hypothesized an analogy with anti-β2 GPI: aPT could recognize cryptic or neoepitopes formed when prothrombin interacts with the anionic surface.

There are some reports regarding the prevalence of aPT in patients with antiphospholipid antibodies (aPL). Arvieux et al16 reported a prevalence of 55.4% in LA positive patients. Galli et al found aPT in 58% of APS patients. Petri et al preliminarily reported that aPT have potential predictive value for thrombotic events in 100 SLE patients. Horbach et al investigated the clinical significance of aPT in 175 SLE patients and found that both IgG and IgM aPT were more frequent in patients with a history of thrombosis and were associated with venous, but not arterial, thrombosis. The titers of IgM aPT were slightly higher in patients with thrombosis than without, whereas there were no differences in titers of IgG aPT between the 2 groups. Vaarala et al showed a predictive value of 2.5-fold increase in the risk of myocardial infarction or cardiac death in middle-aged men with high levels of aPT.

The association between aPT and LA has been studied widely. In 1988, Fleck et al showed that aPT had LA activity. It has been also shown that antibodies responsible for LA activity bind to prothrombin in certain experimental conditions. Additionally, Bevers et al showed that prothrombin was required for expressing LA activity in 11/16 ACA depleted LA positive plasma samples implying that, at least, 69% of LA activity depends on the prothrombin binding antibodies. The mechanism by which these antibodies act remains unclear. It has been speculated that aPT inhibit thrombin-mediated endothelial cell prostacyclin release and that aPT inhibit protein C activation.

Recently it has been hypothesized that prothrombin binds to anionic phospholipids exposed on the endothelial cells and that aPT activate endothelial cells and induce procoagulant substances via prothrombin. Bertolaccini et al examined the presence of IgG and IgM aPT by ELISA in SLE patients. These antibodies were found in 28% of SLE patients with 18% positive for IgG and 14% positive for IgM, with 4% of patients positive for both IgG and IgM. This group reported an association between presence of aPT and the occurrence of vascular events as 53% of SLE patients with aPT had a history of a thrombotic event. This study also reported an association of aPT with the presence of ACA and anti-β2 GPI, although the authors state that the aPT ELISA should not be considered as a substitute for conventional LA, ACA or β2 GPI assays.

Over the last several years a cleaner picture has emerged concerning the clinical relevance of aPT antibodies. We now know that the antibodies most closely associated with the antiphospholipid syndrome and the LA are actually directed to a complex of anionic phospholipids such as phosphatidylserine and prothrombin (PS/PT) rather to PT alone. These data are clearly outlined in a recent review article.

A number of studies have shown that anti-PS/PT assays have a significant clinical relevance and more closely correlate with clinical features of the antiphospholipid syndrome and the lupus anticoagulant.