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  • 產(chǎn)品名稱：HumanMMP-9/CLG4B/MMP9ELISAPairSet

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HumanMMP-9/CLG4B/MMP9ELISAPairSet
詳情介紹：
Purpose The ELISA Pair Set is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utlizes monoclonal antibody specific for MMP-9 / CLG4B coated on a 96-well plate. Standards and samples are added to thewells, and any MMP-9 / CLG4B present binds to the immobilized antibody. The wells are washed and a biotinylatedrabbit anti-MMP-9 monoclonal antibody is then added, producing an antibody-antigen-antibody "sandwich". To producescolor in proportion to the amount of MMP-9 / CLG4B present in the sample strepavidin-HRP and TMB substrate solutionare loaded. The absorbances of the microwell are read at 450 nm.
Sensitivity The minimum detectable dose of human MMP-9 / CLG4B was determined to be approximately 31.25 pg/mL. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
Characteristics Pair Set
Components Capture Antibody - 0.5 mg/mL of mouse anti-MMP9 monoclonal antibody. Dilute to a workingconcentration of 2.0 μg/mL in CBS before coating.
Detection Antibody - 0.5 mg/mL of biotinylated rabbit anti-MMP9 monoclonal antibody. Dilute to aworking concentration of 0.25 μg/mL in detection antibody dilution buffer before use.
Standard - Each vial contains 70 ng of recombinant MMP9. Reconstitute standard powder with1mL detection antibody dilution buffer. A seven-point standard curve using 2-fold serial dilutions insample dilution buffer, and a high standard of 2000 pg/mL is recommended.
Streptavidin-HRP - 50 μL of streptavidin conjugated to horseradish-peroxidase. 1:2000 Dilution indetection antibody dilution buffer before use.
Material not included CBS - 0.05 M
Na2CO3 - 0.05 M
NaHCO3, pH 9.6, 0.2 μm filtered
TBS - 25 mM
Tris, adjust pH to 7.4 by HCl
Wash Buffer - 0.05 % Tween20 in TBS, pH 7.2 - 7.4
Blocking Buffer - 5 % BSA in Wash Buffer
Sample dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Detection antibody dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Substrate Solution: To achieve best assay results, fresh substrate solution is recommended Substrate stock solution - 10 mg/mL TMB (Tetramethylbenzidine ) in DMSO Substrate dilution buffer - 0.05 M Na2HPO4 and 0.025 M citric acid , adjust pH to 5.5
Substrate working solution - For each plate dilute 250 μL substrate stock solution in 25 mL substrate dilutionbuffer and then add 80 μL 0.75 % H2O2 , mix it well
Stop Solution - 2 N H2SO4
Alternative Name MMP9
Background Matrix metalloproteinase-9, also known as 92 kDa type IV collagenase, MMP-9 and CLG4B is a secretedprotein which belongs to the peptidase M10A family. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade components of the extracellular matrix (ECM) and play essentialroles in various physiological processes such as morphogenesis, differentiation, angiogenesis and tissueremodeling, as well as pathological processes including inflammation, arthritis, cardiovascular diseases, pulmonary diseases and tumor invasion. MMP-9 contains three fibronectin type-II domains and fourhemopexin-like domains. It is secreted from neutrophils, macrophages, and a number of transformed cells. Itis the most complex family member in terms of domain structure and regulation of its activity. MMP-9 isinvolved in a variety of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis, and be regarded as a potential therapeutic target. MMP-9 may play an essential role in local proteolysis of the extracellular matrix and in leukocyte migration. MMP-9 could play a role in bone osteoclastic resorption. It cleaves type IV and type V collagen into large C-terminal three quarter fragments and shorter N-terminal one quarter fragments. MMP-9 also degradesfibronectin but not laminin or Pz-peptide. MMP-9 and MMP-13 catalyze the degradation of extracellularmatrix (ECM) components in the growth plate and at the same time cleave and release biologically activemolecules stored in the ECM, such as VEGFA. In mice, ablation of MMP-9, MMP-13, or both MMP-9 andMMP-13 causes severe distortion of the metaphyseal growth plate. MMP-9 has also been implicated innumerous somatic illnesses, including cardiovascular disorders and cancer. MMP-9 has been shown to beincreasingly important in several aspects of central nervous system activity. Defects in MMP-9 may be acause of susceptibility to intervertebral disc disease (IDD) , also known as lumbar disk herniation (LDH) . IDDis one of the most common musculo-skeletal disorders and the predominant cause of low-back pain andunilateral leg pain.
Research Area Cardiovascular, Atherosclerosis, Proteolysis / Ubiquitin, Metalloprotease, Angiogenesis, Extracellular Matrix, Matrix Metalloproteinases
Application Notes Optimal working dilution should be determined by the investigator.
Comment

The human MMP-9 / CLG4B ELISA Pair Set is for the quantitative determination of human MMP-9.This ELISA Pair Set contains the basic components required for the development of sandwich ELISAs.
Reagent Preparation
  1. Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100 μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4 °C.
    2. Aspirate each well and wash with at least 300 μL wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
    3. Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.
    4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
  1. Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at roomtemperature.
    2. Repeat the aspiration/wash as in step 2 of plate preparation.
    3. Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1hour at room temperature.
    4. Repeat the aspiration/wash as in step 2 of plate preparation.
    5. Add 100 μL of Streptavidin-HRP to each well. Incubate for 1 hour at room temperature.
    6. Repeat the aspiration/wash as in step 2 of plate preparation.
    7. Add 200 μL of substrate solution to each well. Incubate for 20?minutes at room temperature (if substrate solutionis not as requested, the incubation time should be optimized). Avoid placing the plate in direct light.
    8. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.
Calculation of Results

Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each. 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. To determine the concentration of the unknowns, find the unknowns' mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.
Restrictions For Research Use only
Format Lyophilized
Precaution of Use The Stop Solution suggested for use with this Pair Set is an acid solution. Wear eye, hand, face, andclothing protection when using this material.
Handling Advice Avoid repeated freeze-thaw cycles.
Storage 4 °C/-20 °C/-80 °C
Storage Comment Capture Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Detection Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Standard: Store lyophilized standard at -20°C to -80°C for up to 6 months from date of receipt. Aliquot and store the reconstituted Standard at -80°C for up to 1 month. Streptavidin-HRP: Store at 4°C and protect it from prolonged exposure to light. DO NOT FREEZE! It is stable for up to 6 months from date of receipt.
Expiry Date 6 months
Supplier Images
image for Human MMP-9 / CLG4B / MMP9 ELISA Pair Set (ABIN2010457) Human MMP-9 / CLG4B / MMP9 ELISA Pair Set
Product cited in: Kragstrup, Jalilian, Hvid, Kj?rgaard, ?stg?rd, Schi?ttz-Christensen, Jurik, Robinson, Vorup-Jensen, Deleuran: "Decreased plasma levels of soluble CD18 link leukocyte infiltration with disease activity in spondyloarthritis." in: Arthritis research & therapy, Vol. 16, Issue 1, pp. R42, 2014 (PubMed).