DESCRIPTION

Endogenous inhibitors of matrix metalloproteinases (MMPs) called tissue inhibitors of matrix metalloproteinases (TIMPs) form a family of four members, TIMP1-4. Although possessing many similarities, TIMPs show several structural and biochemical differences, and their expression patterns differ. TIMP4 is characterized by being primarily restricted to cardiovascular structures. Experimental studies have suggested a role for TIMP4 in cardiovascular disorders exhibiting an inflammatory component. Overexpression of TIMP4 has been observed within the arterial wall in rats after a balloon injury. TIMP4 has been shown to suppress vascular smooth muscle cell migration and induce cell apoptosis and to inhibit capillary endothelial cell migration.

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	480.5	24.5	5.1
2	20	893.5	58.5	6.5
3	20	3,844.1	208.5	5.4

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	515.1	51.2	9.9
2	24	1,058.1	67.8	6.4
3	24	4,065.2	271.9	6.7

RECOVERY

The recovery of human TIMP4 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human Serum	1:15	105	85-116
	1:30	98	77-117
Cell culture supernatant	1:2	118	107-129
	1:4	109	88-125
Human milk	1:40	105	90-127
	1:80	99	88-113

LINEARITY

To assess the linearity of the assay, cell culture supernatant was spiked with high concentrations of human TIMP4 in various matrices and diluted with the Sample Diluent to produce samples with values within the dynamic range of the assay. Human serum and human milk samples were diluted with the Sample Diluent to produce samples with values within the dynamic range of the assay.

Sample Type		Range (%)	Average% of Expected
Human serum	1:5	-	100
	1:10	94-113	103
	1:20	75-109	96
	1:40	96-106	101
Cell culture supernatant	1:2	88-117	103
	1:4	90-97	93
	1:8	89-93	91
	1:16	88-95	91
Human milk	1:20	-	100
	1:40	96-99	97
	1:80	101-107	104
	1:160	104-110	107

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human TIMP4 produced at Sunlong Medical™.