DiaPharma Group, Inc.

For research use only

Two-site ELISA method for the measurement of human PF4 (platelet factor 4), on platelet poor human plasma, or any biological sample where PF4 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 PF4. It is insensitive to the presence of heparin in the tested sample and PF4 is measured homogeneously whether its presentation is. The assay calibrator is standardized with the NIBSC international standard.

Structure & Mechanism of Action

Platelet Factor 4 is known as an "anti-heparin". It is a platelet-released protein that promotes blood clotting by neutralizing heparin. PF4 is a 70 amino acid platelet specific protein with a MW of about 32 kD for the tetramer. It is released from platelet a granules upon activation in the tetrameric form as a complex with a platelet proteoglycan. The half-life in plasma is short (<5 minutes), as PF4 binds to endothelial cell GAG's (glycosaminoglycans) where it is stored. PF4 has a strong anti-heparin activity. It forms stoichiometric macromolecular complexes with heparin with an optimal ratio of 27 IU of heparin per mg of PF4.

Significance

Elevated PF4 concentrations (>10 IU/ml) are observed in various clinical situations associated with the presence of platelet activation such as CVD, surgery, extracorporeal circulation, diabetes, malignancies, etc. Heparin therapy releases PF4 from the endothelial cell storage pool and its concentration is then strongly increased in plasma. Evidence has shown a connection between PF4 levels and an increased thrombotic tendency.

Assay Principle

Plasma is collected in an anticoagulant containing platelet inhibitors, such as CTAD tubes or ETP. DiaPharma PF4 is a sandwich ELISA. The diluted sample is added to the microwell, coated with affinity purified rabbit polyclonal antibodies (F(ab')2 fragments) specific for human PF4, which allows to avoid the interference from rheumatoid factor. When present, this protein is captured onto the solid phase. Following a washing step, the immunoconjugate, an affinity purified rabbit polyclonal antibody coupled to HRP is introduced, and binds to the free epitopes of immobilized PF4. Following a new washing step, the substrate OPD in the presence of H2O2 is added and color develops. The amount of color developed is directly proportional to the concentration of human PF4 in the tested sample.

Assay Features

· Complete, ready to use, & optimised -- kit contains calibrators and controls
· No interference of rheumatoid factor
· Homogeneous reactivity whatever the PF4 presentation
· Insensitive to heparin
· Standardised against the 1st International Standard (NIBSC)
· High sensitivity and specificity -- measuring range of 0.1-10 ng/ml