Human CXCL4/PF4 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human CXCL4/PF4 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human PF4 in cell culture supernates, serum, and platelet-poor plasma. It contains E. coli-expressed recombinant human PF4 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human PF4 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human PF4.

Preparation and Storage

Background: CXCL4/PF4

CXCL4/PF4 is a secreted chemokine that is expressed by megakaryocytes and restrains the coagulation cascade. It is released by activated platelets and regulates Thrombin/Thrombomodulin complexes, activated Protein C (APC), Coagulation Factor Xa, and Fibrin. The anticoagulant heparin neutralizes CXCL4 procoagulant effects. Complexes of heparin and CXCL4 trigger antibody formation that causes the pathological syndrome HITT. Immunogenic complexes of CXCL4 with Apolipoprotein H contribute to antiphospholipid syndrome (APS). CXCL4 interferes with the proliferative and angiogenic functions of FGF-2 and VEGF. However, it can also exert proinflammatory and pro-atherogenic effects on monocytes, macrophages, and endothelial cells. CXCL4 may signal through CXCR3B in humans.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.