Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	0.16-10 ng/mL
Sensitivity:	0.09 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human MMP-2 in samples. No significant cross-reactivity or interference between Human MMP-2 and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human MMP-2. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human MMP-2 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human MMP-2, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human MMP-2. The concentration of Human MMP-2 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	MMP2: Ubiquitinous metalloproteinase that is involved in diverse functions such as remodeling of the vasculature, angiogenesis, tissue repair, tumor invasion, inflammation, and atherosclerotic plaque rupture. As well as degrading extracellular matrix proteins, can also act on several nonmatrix proteins such as big endothelial 1 and beta-type CGRP promoting vasoconstriction. Also cleaves KISS at a Gly-\|-Leu bond. Appears to have a role in myocardial cell death pathways. Contributes to myocardial oxidative stress by regulating the activity of GSK3beta. Cleaves GSK3beta in vitro. Interacts (via the C-terminal hemopexin-like domains- containing region) with the integrin alpha-V/beta-3; the interaction promotes vascular invasion in angiogenic vessels and melamoma cells. Interacts (via the C-terminal PEX domain) with TIMP2 (via the C-terminal); the interaction inhibits the degradation activity. Interacts with GSK3B. Aspirin appears to inhibit expression. Produced by normal skin fibroblasts. PEX is expressed in a number of tumors including gliomas, breast and prostate. Inhibited by histatin-3 1/24 (histatin-5). Belongs to the peptidase M10A family.
UniProt Protein Details:	Protein type:Secreted; Motility/polarity/chemotaxis; Cell development/differentiation; Protease; Apoptosis; EC 3. 4. 24. 24; Secreted, signal peptide Chromosomal Location of Human Ortholog: 16q12. 2 Cellular Component: extracellular space; proteinaceous extracellular matrix; sarcomere; mitochondrion; extracellular region; plasma membrane; nucleus Molecular Function:protein binding; zinc ion binding; serine-type endopeptidase activity; metalloendopeptidase activity Biological Process: extracellular matrix disassembly; collagen catabolic process; axon guidance; extracellular matrix organization and biogenesis; intramembranous ossification; cellular protein metabolic process; positive regulation of innate immune response; ephrin receptor signaling pathway; response to hypoxia; angiogenesis; proteolysis; blood vessel maturation; embryo implantation Disease: Multicentric Osteolysis, Nodulosis, And Arthropathy
NCBI Summary:	This gene is a member of the matrix metalloproteinase (MMP) gene family, that are zinc-dependent enzymes capable of cleaving components of the extracellular matrix and molecules involved in signal transduction. The protein encoded by this gene is a gelatinase A, type IV collagenase, that contains three fibronectin type II repeats in its catalytic site that allow binding of denatured type IV and V collagen and elastin. Unlike most MMP family members, activation of this protein can occur on the cell membrane. This enzyme can be activated extracellularly by proteases, or, intracellulary by its S-glutathiolation with no requirement for proteolytical removal of the pro-domain. This protein is thought to be involved in multiple pathways including roles in the nervous system, endometrial menstrual breakdown, regulation of vascularization, and metastasis. Mutations in this gene have been associated with Winchester syndrome and Nodulosis-Arthropathy-Osteolysis (NAO) syndrome. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Oct 2014]
UniProt Code:	P08253
NCBI GenInfo Identifier:	116856
NCBI Gene ID:	4313
NCBI Accession:	P08253. 2
UniProt Secondary Accession:	P08253,Q9UCJ8, B2R6U1, B4DWH3, E9PE45,
UniProt Related Accession:	P08253
Molecular Weight:	68,831 Da
NCBI Full Name:	72 kDa type IV collagenase
NCBI Synonym Full Names:	matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase)
NCBI Official Symbol:	MMP2
NCBI Official Synonym Symbols:	CLG4; MONA; CLG4A; MMP-2; TBE-1; MMP-II
NCBI Protein Information:	72 kDa type IV collagenase; gelatinase A; 72 kDa gelatinase; collagenase type IV-A; neutrophil gelatinase; matrix metalloproteinase-2; matrix metalloproteinase-II
UniProt Protein Name:	72 kDa type IV collagenase
UniProt Synonym Protein Names:	72 kDa gelatinase; Gelatinase A; Matrix metalloproteinase-2; MMP-2; TBE-1Cleaved into the following chain:PEX
Protein Family:	Matrix metalloproteinase
UniProt Gene Name:	MMP2
UniProt Entry Name:	MMP2_HUMAN

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (ng/mL)	RLU	Average	Corrected
10	30781 34161	32471	32447
5	14732 16208	15470	15446
2.5	7759 7247	7503	7479
1.25	3395 3911	3653	3629
0.63	1805 1717	1761	1737
0.31	862 786	824	800
0.16	357 357	357	333
0	23 25	24	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human MMP-2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human MMP-2 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	0.52	1.55	3.62	0.55	1.69	3.74
Standard deviation	0.05	0.13	0.29	0.06	0.19	0.24
C V (%)	9.62	8.39	8.01	10.91	11.24	6.42

Recovery

The recovery of Human MMP-2 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	98-114	104
EDTA plasma (n=5)	88-101	93
Cell culture media (n=5)	98-115	105

Linearity

Samples were spiked with high concentrations of Human MMP-2 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	88-101	84-97	90-104
1:2	Average (%)	94	90	98
1:4	Range (%)	103-120	102-114	91-107
1:4	Average (%)	110	108	98
1:8	Range (%)	100-112	89-104	98-115
1:8	Average (%)	106	96	106
1:16	Range (%)	91-102	99-111	85-100
1:16	Average (%)	96	104	92

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.