DESCRIPTION

Thrombomodulin (THBD, also known as CD141) is an endothelial-specific type I membrane glycoprotein of 105 kDa that binds thrombin, which plays an important role as a natural anticoagulant. It is expressed mostly on the surface of vascular endothelial cells, where it probably be involved in cell differentiation or inflammation. Soluble forms of CD141 have been found in plasma and urine. It has been reported that plasma CD141 is increased in patients with diabetes mellitus, particularly those with diabetic nephropathy.

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	4,059.3	221.3	5.5
2	8	1,041.6	60.1	5.8
3	8	306.5	8.8	2.9

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	4,014.1	168.0	4.2
2	16	1,005.6	59.2	5.9
3	16	302.2	19.3	6.4

RECOVERY

The recovery of human CD141/Thrombomodulin spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:8	104	101-106
	1:16	114	103-131
Urine	1:16	94	84-99
	1:32	96	90-100
Cell lysate	1:16	96	93-98

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 urine was initially diluted 1:4)

		Human plasma(Sample Diluent PT 3-ef)	Urine(Sample Diluent PT 4B1)	Cell lysate(Sample Diluent PT 3-ef)
1:2	Average% of Expected	100	100	100
	Range (%)	-	-	-
1:4	Average% of Expected	119	87	103
	Range (%)	116-122	81-93	102-103
1:8	Average% of Expected	101	89	110
	Range (%)	98-104	85-93	110-111
1:16	Average% of Expected	96	90	111
	Range (%)	93-100	85-94	108-114

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human CD141/Thrombomodulin One-Step ELISA Kit produced at Sunlong Medical™.