MMP 9 ELISA Kit

Catalog No. ABIN578882

Product Details MMP 9 ELISA Kit

Target: MMP 9 (MMP9) (Matrix Metallopeptidase 9 (Gelatinase B, 92kDa Gelatinase, 92kDa Type IV Collagenase) (MMP9))

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.78-50 ng/mL

Minimum Detection Limit: 0.78 ng/mL

Application: ELISA

Purpose: This immunoassay kit allows for the in vitro quantitative determination of rat MMP-9 concentrations in serum, plasma and other biological fluids.

Sample Type: Plasma, Serum

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural rat MMP-9.

Cross-Reactivity (Details): No significant cross-reactivity or interference was observed.

Sensitivity: The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.

Characteristics: Rattus norvegicus,Rat,Matrix metalloproteinase-9,MMP-9,92 kDa gelatinase,92 kDa type IV collagenase,Gelatinase B,GELB,Mmp9,3.4.24.35

Components: Reagent (Quantity ): Assay plate (1), Standard (2), Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1 × 120μl), Detection Reagent B (1 × 120μl), Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1x10ml), Stop Solution (1x10ml), Plate sealer for 96 wells (5), Instruction (1)

Material not included: Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water.

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: The microtiter plate provided in this kit has been pre-coated with an antibody specific to MMP-9. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for MMP-9. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain MMP-9, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm 2 nm. The concentration of MMP-9 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reagent Preparation:

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution 3 produces a stock solution of 10 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (10 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.

Sample Collection: Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.

Assay Procedure:

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample* per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C . Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. 4
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

Calculation of Results:

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the MMP-9 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Restrictions: For Research Use only

Handling

Handling Advice: 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Storage: 4 °C/-20 °C

Storage Comment: The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

References for MMP 9 ELISA Kit (ABIN578882)

Wu, Dong, Liu, Xu, Li, Jing: "Erythropoietin attenuates ischemia-reperfusion induced lung injury by inhibiting tumor necrosis factor-alpha and matrix metalloproteinase-9 expression." in: European journal of pharmacology, Vol. 602, Issue 2-3, pp. 406-12, (2009) (PubMed).

Target Details for MMP 9

Target: MMP 9 (MMP9) (Matrix Metallopeptidase 9 (Gelatinase B, 92kDa Gelatinase, 92kDa Type IV Collagenase) (MMP9))

Alternative Name: Mmp9 (MMP9 Products)

Synonyms: CLG4B ELISA Kit, GELB ELISA Kit, MANDP2 ELISA Kit, MMP-9 ELISA Kit, AW743869 ELISA Kit, B/MMP9 ELISA Kit, Clg4b ELISA Kit, pro-MMP-9 ELISA Kit, mmp-9 ELISA Kit, fgMMP-9 ELISA Kit, mmp9 ELISA Kit, ZFMMP-9 ELISA Kit, wu:fb02g06 ELISA Kit, wu:fb07b05 ELISA Kit, wu:fi98c09 ELISA Kit, wu:fj05a08 ELISA Kit, zgc:64165 ELISA Kit, clg4b ELISA Kit, gelb ELISA Kit, mandp2 ELISA Kit, matrix metallopeptidase 9 ELISA Kit, collagenase ELISA Kit, matrix metalloproteinase 9 ELISA Kit, matrix metallopeptidase 9 S homeolog ELISA Kit, MMP9 ELISA Kit, RB3913 ELISA Kit, BPSS0666 ELISA Kit, Sbal_3732 ELISA Kit, Bcer98_0486 ELISA Kit, Shew185_0630 ELISA Kit, Shal_3056 ELISA Kit, Sbal195_0657 ELISA Kit, Lbys_3550 ELISA Kit, Palpr_2084 ELISA Kit, Mmp9 ELISA Kit, mmp9 ELISA Kit, mmp9.S ELISA Kit

Background: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. They are secreted as zymogens (pro-MMPs) that are activated by a variety of proteinases or by reaction with organic mercurials. They are inhibited by specific tissue inhibitors of metalloproteinases (TIMPs) and by alpha 2-macroglobulin. The regulation of MMP activity is important in tissue remodeling, inflammation, tumor growth and metastasis. Human MMP-9 (also known as gelatinase B) is secreted as a 92 kDa zymogen. Cleavage of pro-MMP-9 at or near residue 87 results in the active enzyme with a mass of approximately 82 kDa. MMP-9 has three fibronectin type II domains, a hemopexin-like domain and a proline-rich type V collagen-homologous domain. Pro-MMP-9 can be activated by MMP-3 or by certain bacterial proteinases. MMP-9 is inhibited by alpha 2-macroglobulin or by TIMP-1, which binds to pro-MMP-9 as well as to active MMP-9. Pro-MMP-9 is secreted by monocytes, macrophages, neutrophils, keratinocytes, fibroblasts, osteoclasts, chondrocytes, skeletal muscle satellite cells, endothelial cells, and various tumor cells. Pro-MMP-9 expression is upregulated by TGF- beta 1, IL-1 beta, TGF- alpha, PDGF-AB, TNF- alpha and IL-1 alpha . Substrates for MMP-9 include denatured collagen type I (gelatin), native collagens type IV, V, VII, X and XI, fibrinogen, vitronectin, IL-1 beta and entactin, a molecule that bridges laminin and type IV collagen. MMP-9 is involved in inflammation, tissue remodeling, wound healing, mobilization of matrix-bound growth factors and processing of cytokines. Its expression correlates with the desmoplasia (abnormal collagen deposition) that accompanies pancreatic cancer, with the metastasis to lymph nodes by humanbreast carcinoma cells and with the invasion of regional vessels in giant cell tumors of bones. MMP-9 may be elevated in gingival crevicular fluid and saliva in patients with gingivitis and periodontal diseases. 2

Gene ID: 3131

Pathways: Cellular Response to Molecule of Bacterial Origin, Positive Regulation of Immune Effector Process, CXCR4-mediated Signaling Events