IMUBIND Total TFPI ELISA

INTENDED USE
The IMUBIND^® Total Tissue Factor Pathway Inhibitor (TFPI) ELISA is an enzyme-linked sandwich immunoassay for the measurement of TFPI (aliases: EPI, LACI)⁽¹⁾ in human plasma and cell culture supernatants. This assay is intended for research use only.  It is not intended for diagnostic or therapeutic use.

EXPLANATION OF THE TEST
TFPI circulates in human plasma as complexes with LDL, HDL and VLDL⁽²⁾ and can be found in several forms: a 36,000 D molecule, a 43,000 D molecule and as truncated moieties. This heterogeneity of size appears, in part, to be the result of the formation of mixed disulfide complexes between TFPI and apolipoprotein AII.⁽³⁾ In humans, approximately 10% of total TFPI is carried by platelets which release TFPI once they are activated by thrombin.⁽⁴⁾ Thus, at the site of a wound, where platelets aggregate, elevated levels of TFPI are present. Based on the initial isolation of the inhibitor it was found that TFPI inhibits Tissue Factor (TF) procoagulant activity; i.e., the TF/FVIIa complex, and directly inhibits factor Xa by binding at or near its serine active site.^(5,6)

The C-terminal region of TFPI is required for a high affinity binding between TFPI and factor Xa and the subsequent factor X a inhibition.⁽⁷⁾ It has been found that TFPI is released into blood following administration of heparin and that heparin enhances TFPI inhibition of factor Xa, and that the C-terminal region is the major heparin binding site.⁽⁸⁾

PRINCIPLE OF THE METHOD
The IMUBIND Total TFPI ELISA is a "sandwich" ELISA employing a rabbit anti-human TFPI polyclonal antibody as the capture antibody. Specificity of the capture antibody for native, complexed and truncated TFPI was confirmed by Western blot analysis, visualizing a single band at 34 kDa, corresponding to the mobility of intact native TFPI and visualizing a single band at 21 kDa, corresponding to the mobility of a truncated form of TFPI.  Diluted plasma samples or cell culture supernatants incubate in microwells precoated with this capture antibody. TFPI is detected using a biotinylated monoclonal antibody specific for the Kunitz domain 1 of TFPI. The subsequent binding of the streptavidin conjugated horseradish peroxidase (HRP) completes the formation of the antibody enzyme detection complex.  The addition of TMB substrate and its reaction with the HRP creates a blue colored solution. The reaction is stopped by the addition of a citrate stop solution, changing the color of the reaction solution to yellow. TFPI levels are determined by measuring sample solution absorbance at 450 nm and comparing against those of a standard curve.

Category: Research use only

Type: ELISA

Product Availability: Worldwide

Manufacturer: ImmBioMed GmbH & Co KG, Germany

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