DiaPharma Group, Inc.

For research use only. Not for use in diagnostic procedures.

Two-site ELISA method for the measurement of annexin V on human plasma (citrated or EDTA) or any biological sample where annexin V must be measured. This assay is designed with affinity purified rabbit antibodies as well for coating the microELISA plate (immunocapture) as for the horseradish peroxidase (HRP) immunoconjugate used for revelation. This assay measures native or recombinant annexin V. In addition to annexin V measurements in plasma, this assay is very useful for the quality control of platelet and blood cell concentrates (annexin V released correlates with platelet and blood cell surface lesion).

Structure & Mechanism of Action

The normal range of Annexin V in human plasma is < 10 ng/ml. Annexin V is a calcium-dependent protein with anticoagulant properties. It has a MW of 35 kD and is present in many tissues and mainly in endothelial cells and in placenta. It is present at low concentrations in platelets and at higher concentrations in red blood cells and leukocytes. Annexin V binds to anionic phospholipids and to activated cell membranes in a calcium-dependent manner. It inhibits blood coagulation and phospholipase A2 (an enzyme that hydorlyses ester bonds in phospholipids and release fatty acids), regulates exocytosis (release of material from a cell), and interacts with cytoskeletal proteins. It inhibits pro-coagulant and pro-inflammatory activities of dying cells during apoptosis.

Significance

Annexin V concentrations can be elevated in autoimmune diseases and SLE (Systemic Lupus Erythematosus)

It is reported to be a marker for early stage of myocardial infarction. Measurement of Annexin V can also be useful for studying hepatic disorders. The assay is also useful for studies of subjects infused with recombinant Annexin V, where pharmacokinetic and pharmacodynamic data is needed.

Assay Principle

DiaPharma Annexin V is a sandwich ELISA designed with affinity purified rabbit polyclonal antibodies specific for human Annexin V. The diluted test plasma or biological fluid is introduced in a microwell coated with affinity purified rabbit polyclonal antibodies (F(ab?) 2 fragments to avoid the interference of Rheumatoid Factor) specific for human Annexin V. When present, this protein is captured onto the solid phase. Following a washing step, the immunoconjugate, a rabbit polyclonal antibody coupled to HRP, is introduced and binds to the free epitopes of immobilized Annexin V. Following a new washing step, the peroxidase substrate, OPD in the presence of H2O2, is added and a color develops, which is read at 492 nm. The amount of color developed is directly proportional to the concentration of human Annexin V in the tested sample. See the package insert for details.

Assay Features

· Complete, ready to use, & optimized -- kit contains calibrators and controls
· No interference of rheumatoid factor
· Total assay time of only 2 hours, 15 minutes
· Fast, easy to perform, reliable and accurate
· High sensitivity and specificity -- measuring range of 0.2 - 20 ng/ml
· Can be used for measurement of Annexin V in platelet and blood cell concentrates or plasma
· Measures recombinant Annexin V as well as native Annexin V in plasma

Kit Contents

· Micro ELISA plate coated with F(ab)'2 fragments from rabbit polyclonal antibodies specific for Annexin V1, disposable (6x16 well strips).
· Anti-Annexin V-peroxidase immunoconjugate (3 vials).
· Ready to use sample diluent.
· Ready to use conjugate diluent.
· Substrate tablets (H1O1/OPD) (3x2 tablets).

Assay Characteristics

· Two site ELISA
· Total assay time: <2h 15 min
· Detection threshold: 0.2 ng/ml
· Dynamic range: 0.2 to 20 ng/ml
· Intra-assay CV: 1 to 5%
· Inter-assay CV: 2.5 to 8%
· No interference of rheumatoid factor.
· Tested specimen: platelet or blood cell supernatants (tested diluted at 1:2 or 1:5, ...)
· Trisodium citrated or NA1EDTA anticoagulated human plasma.

What is Annexin V?

· 35 KDa, Ca++ dependent, protein
· Present in many tissues (endothelial cells, placenta, ...)
· Present in platelets (5.9 10 g/cell and, at higher concentration, in red blood cells and leukocytes
· Binds to anionic phospholipids via Ca++
· Inhibits blood coagulation and phopholipase A
· Regulates exocytosis and interacts with cytoskeletal proteins
· Inhibits pro-coagulant and pro-inflammatory activities of dying cells during apoptosis

Diagnostic Value of Annexin V Assay

· Autoimmune disease and Systemic Lupus Erythematosus (SLE) (10 to > 250 ng/ml)
· Early stage of myocardial infarction (10 to > 50 ng/ml)
· Hepatic disorders (10 to > 100 ng/ml)
· Follow-up of patients treated with Annexin V
· Normal range in human plasma: < 10 ng/ml

Selected References

Released Annexin V during platelet storage correlates with EDTA induced shape change. (P. Krailadsiri et Al.: Transf. Scien. 1997)

Annexin V increases from 16.0 ng/ml (± 13.31) to 33.68 ng/ml (± 20.22) during a 5-6 day platelet concentrate storage. (P. Krailadsiri et Al.: Transf. Scien. 1997)

Note: a minimum order of 20 kits is required for this item.