DESCRIPTION

Factor IX, also known as PTC, circulates as an inactive zymogen until the proteolytic release of its activation peptide allows it to assume the conformation of an active serine protease. Upon activation by Factor XIa or Factor VIIa/tissue factor complex, Factor IXa forms a complex with Factor VIIIa, calcium ions, and phospholipids on the surface of activated platelets. This complex, known as the tenase complex, efficiently activates Factor X to Factor Xa, leading to thrombin generation and fibrin clot formation. Deficiencies or mutations in Factor IX result in Hemophilia B, an X-linked recessive bleeding disorder.

PRINCIPLE OF THE ASSAY

An anti-tag antibody is pre-coated onto the bottom of wells. After adding antigen or samples, Tagged-Capture antibody and HRP-conjugated detection antibody, a sandwich complex is formed in the solution. TMB acts as a HRP substrate, and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns the 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 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	3,564.2	104.1	2.9
2	8	819.6	14.8	1.8
3	8	425.1	7.7	1.8

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	3,663.4	147.2	4.0
2	16	838.7	26.4	3.1
3	16	441.8	19.8	4.5

RECOVERY

The recovery of human Factor IX/PTC spiked to three different levels throughout the range of the assay in human serum was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:1,600	103	84-119
	1:3,200	108	103-114

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The human serum was initially diluted 1:400.)

		Human serum
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	100
	Range (%)	99-101
1:8	Average% of Expected	96
	Range (%)	84-109
1:16	Average% of Expected	90
	Range (%)	82-98

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human Factor IX/PTC One-Step ELISA Kit produced at Sunlong Medical™.