DESCRIPTION

Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. TFPI is produced as two major isoforms in humans, TFPIa and TFPIb that result from alternative splicing. The TFPIb isoform localizes to the endothelium surface where it is a potent inhibitor of tissue factor-factor VIIa complexes that initiate blood coagulation. The TFPIa isoform is present in platelets. TFPI is a Kunitz-type serine protease inhibitor that exerts anticoagulant activity by blocking early procoagulant stimulia. TFPI can enhance anti-thrombotic treatment in sepsis, inflammatory diseases, and cardiovascular diseases. In plasma, 80% of TFPI is carboxy-terminal truncated and circulates bound primarily to low-density lipoproteins (LDL), with the levels of the latter having an impact on plasma TFPI levels. The other 20% is unbound free-form TFPI characterised by the K3 domain.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with biotin) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with biotin also binds to the analyte. Streptavidin-HRP binds to the biotin. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns solution yellow. The color intensity is proportional to the quantity of bound protein which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	20	23.3	2.2	9.4
2	20	75.7	5.4	7.1
3	20	312.9	15.6	5.0

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	22.9	1.5	6.6
2	24	66.8	5.6	8.5
3	24	314.2	18.1	5.8

RECOVERY

The recovery of human TFPI spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:40	113	104-122
	1:80	101	75-117
Cell culture supernatant	1:40	96	72-116
	1:80	82	72-120

LINEARITY

To assess the linearity of the assay, human serum, plasma and cell culture supernatant samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

Sample Type		Range (%)	Average% of Expected
Human plasma(Sample Diluent PT 3-eg )	1:10	-	100
	1:20	90-95	92
	1:40	89-93	91
	1:80	84-101	92
Human serum(Sample Diluent PT 3-eg )	1:10	-	100
	1:20	91-102	97
	1:40	93-102	97
	1:80	96-101	98
Cell culture supernatant(Sample Diluent PT 4)	1:10	-	100
	1:20	101-103	102
	1:40	95-99	97
	1:80	84-106	95

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human TFPI produced at Sunlong Medical™.