DESCRIPTION

MMP9 (matrix metallopeptidase 9), also named as Gelatinase B, is a member of matrix metalloproteinase (MMP) family. The MMP family of enzymes is comprised of critically important extracellular matrix remodeling proteases whose activity has been implicated in normal embryogenesis, tissue remodelling and many diseases such as arthritis, cancer, periodontitis, glomerulonephritis, encephalomyelitis, atherosclerosis and tissue ulceration. These proteases have come to represent important therapeutic and diagnostic targets for the treatment and detection of human cancers. MMP9 is produced by a variety of normal and transformed cells including neutrophils, monocytes, macrophages, astrocytes, fbroblasts, osteoclasts and so on. In tumors, MMP-9 destroys collagen (type IV) in the vascular basal membrane in the vicinity of tumor cells which invade the surrounding tissues and contributes to metastasis. Circulating levels of MMP-9 are increased in many inflammatory disorders such as atherosclerosis, hepatitis C virus infection and colorectal cancer.

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	1,328.9	34.7	2.6
2	8	339.0	7.9	2.3
3	8	164.4	4.4	2.7

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,331.5	34.2	2.6
2	16	333.4	8.3	2.5
3	16	160.2	7.5	4.7

RECOVERY

The recovery of human MMP-9 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:200	98	86-119
Cell culture supernatant	1:3,200	95	86-101
Urine	1:4	86	80-92
Saliva	1:160	102	90-110

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 plasma was initially diluted 1:25. The cell culture supernatant was initially diluted 1:200. The saliva was initially diluted 1:25.)

		Human plasma	Cell culture supernatant	Urine	Saliva
1:2	Average% of Expected	100	100	100	100
	Range (%)	-	-	-	-
1:4	Average% of Expected	113	114	112	104
	Range (%)	100-126	107-120	111-113	98-111
1:8	Average% of Expected	93	117	123	93
	Range (%)	81-106	108-126	122-124	91-95
1:16	Average% of Expected	90	114	123	98
	Range (%)	89-91	109-120	119-127	85-111

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human MMP-9 produced at Sunlong Medical™.