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48-602MAG
Buffer Detection Kit for Magnetic Beads
1 Kit
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The assay has a 30% percent clinical sensitivity (ability to recognize affected individuals) at a 95% clinical specificity (ability to recognize unaffected individuals). Astrocytoma and HS578T cells were plated out at various cell densities into 96 plate wells. Cells were incubated at 37°C for 24 h. Supernatant was measured in the MMP-2 ELISA. MMP-2 levels were detectable at cell concentrations of 4 X 103 Astrocytoma cells/well and 0.6 X 104 HS578T cells/well and above. The lower limit of detection (LLD), commonly used to define sensitivity, was measured by assaying four replicates of zero eight times using two different lots of plates and two different lots of detector antibody. The grand mean signal and pooled standard deviation of zero was calculated. The grand mean of each standard (run in replicates of four in the eight assays) was used for the standard curve, and the response, mean signal of zero plus two standard deviations, read in dose from the standard curve is the LLD; that is, the smallest dose that is not zero with 95% confidence. The pooled coefficients of variation (according to the formula of Henry et. al., 1974) and between assay coefficients of variation are plotted against MMP-2 concentrations. The pooled data were collected from samples run eight times using two different lots of plates and two different lots of detector antibody in replicates of three on two separate occasions. The study tested dilution-recovery of 28 positive samples. Dilutions were run in the MMP-2 ELISA and the "found" doses were plotted against the "expected" doses to determine linearity of dilution. The slope is very close to one and the intercept is not significantly different from zero. These studies are consistent with the absence of cross-reacting and matrix effects such as pH, salts, and endogenous binders that interfere with the reagents used in the assay. Note: Ca = Cancer; NHS = Normal Human Sera; TC= Tissue Culture. Levels of MMP-2 detected by the ELISA after immunoaffinity extraction of MMP-2 positive samples with a MMP-2 antibody that is not used in the ELISA and a control antibody. Levels of a highly purified 72 kDa MMP-2 that exists in a stable, but non-covalent 1:1complex with TIMP-2 detected after neutralization with a MMP-2 antibody that is not used in the ELISA. Levels of MMP-2 detected by the MMP-2 ELISA after 2-aminophenylmercuric acetate (APMA) treatment. APMA promotes the autocatalytic cleavage of the N-terminal prosequence of the latent 72-kDa enzyme to yield the active form of the enzyme. HT1080 and astrocytoma tissue culture supernatants, recombinant proMMP-2 and normal human serum samples were either untreated (-APMA) or incubated for four hours at 37°C with 2 mM APMA (+APMA) or a volume of DMSO to control for the DMSO base of APMA (Vehicle Ctrl). Levels of MMP-2 detected by the ELISA after immunoaffinity extraction of MMP-2 positive samples with a MMP-2 antibody that is not used in the ELISA and a control antibody.
Materials Required but Not Delivered
• 2-20 µl, 20-200 µl and 200-1000 • l precision pipettors with disposable tips • Wash bottle or multichannel dispenser for • 500 ml graduated cylinder • Deionized or distilled H2O • Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/595 nm or 450/540 nm
References
Product Information
Detection method
Colorimetric
Declaration
Not available for sale in Japan.
Form
34 or 41 Tests
Format
96-well plate
Kit contains
Anti-MMP-2 Coated 96-Well Plate, MMP-2 Standard, Detector Antibody, 400X Conjugate, Conjugate Diluent, Substrate, Sample Diluent, Plate Wash Concentrate, Stop Solution, Plate Sealers, and a user protocol.
Toxic by inhalation, in contact with skin and if swallowed. Causes severe burns. Irritating to eyes, respiratory system and skin. Limited evidence of a carcinogenic effect. May cause sensitization by skin contact.
S Phrase
S: 23-26-36/37/39-45
Do not breathe fumes. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing, gloves and eye/face protection. In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Product Usage Statements
Intended use
The Calbiochem® MMP-2 ELISA Kit is a non-isotopic immunoassay for the in vitro quantitation of human MMP-2 protein in tissue culture medium, serum, and plasma.
Storage and Shipping Information
Ship Code
Dry Ice Only
Toxicity
Multiple Toxicity Values, refer to MSDS
Hazardous Materials Attention:
Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
Storage
-20°C
Storage Conditions
Upon arrival, store the entire contents at -20°C. Avoid multiple freeze/thaw cycles. Do not expose reagents to excessive light.
Avoid freeze/thaw
Avoid freeze/thaw
Do not freeze
Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit contains
Anti-MMP-2 Coated 96-Well Plate, MMP-2 Standard, Detector Antibody, 400X Conjugate, Conjugate Diluent, Substrate, Sample Diluent, Plate Wash Concentrate, Stop Solution, Plate Sealers, and a user protocol.
Upon arrival, store the entire contents at -20°C. Avoid multiple freeze/thaw cycles. Do not expose reagents to excessive light.
Intended use
The Calbiochem® MMP-2 ELISA Kit is a non-isotopic immunoassay for the in vitro quantitation of human MMP-2 protein in tissue culture medium, serum, and plasma.
Background
Matrix metalloproteinase-2 (MMP-2; 72-kDa gelatinase A) is usually found only in mesenchymal cells (mainly fibroblasts) during development and tissue regeneration. MMP-2 and MMP-9 degrade type IV collagen, which makes up the backbone of the basement membrane and denatured collagen (gelatin). MMP-2 is not active in benign tumors; it only becomes activated once the tumors become invasive. The enzyme apparently helps tumor cells spread by degrading the collagen in basement membranes, a protein mesh around blood vessels and other organs that is usually the first barrier that roaming tumor cells have to cross. MMP-2 is secreted as an inactive proenzyme with one end of the molecule, the propeptide, serving as a built-in inhibitor that has to be cleaved before the enzyme can act. The inhibitory propeptide folds into several staircase-like helices, one of which completely shields the catalytic center. To activate the molecule, other proteins cleave a loop connecting the helices. This disrupts the structure of the propeptide leading to its release from the active site. Increasing evidence is accumulating that the newly discovered membrane associated MMPs (MT-MMPs) act as cell surface activator(s) of proMMP-2 under physiological or pathophysiological conditions.
MMP activity is controlled by transcriptional regulation, zymogen activation, specific tissue inhibitors of metalloproteinases (TIMPs) and general proteinase inhibitors. The TIMP family has four members: TIMP-1, TIMP-2, TIMP-3, and TIMP-4. Binding of the TIMPs to their specific MMP results in efficient inhibition of proteinase activity. In the case of gelatinases (MMP-2 and MMP-9), the TIMPs have been shown to bind to the zymogen forms of the enzymes. This interaction provides an extra level of regulation by inhibiting activation. The MMP-2 proenzyme cannot be activated by most of the suggested physiological activators of other MMPs including serine proteinases. In serum, α2-macroglobulins very rapidly sequester and clear active MMP-2 molecules. They bind MMP-2 through an exposed peptide stretch, known as the "bait" region, which leads to the cleavage of MMP-2 and large conformational changes in the macroglobulin molecule. This results in entrapment of MMP-2 and exposes binding sites for high affinity cell surface receptors that target the whole complex for degradation.
Principles of the assay
The MMP-2 ELISA Kit is a "sandwich" enzyme immunoassay employing two mouse monoclonal antibodies. An antibody, specific for the human MMP-2 protein, has been immobilized onto the surface of wells provided in the kit. The sample to be assayed and biotinylated detector monoclonal antibody are pipetted into the wells and allowed to incubate for 4 h, during which time any MMP-2 present binds to the capture and detecting antibodies. Unbound material is washed away and horseradish peroxidase-conjugated streptavidin is added, which binds to the detector antibody. The horseradish peroxidase catalyzes the conversion of the chromogenic substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stopping reagent), the intensity of which is proportional to the amount of MMP-2 protein in the sample. The colored reaction product is quantified using a spectrophotometer. Quantitation is achieved by the construction of a standard curve using known concentrations of MMP-2 (provided lyophilized). By comparing the absorbance obtained from a sample containing an unknown amount of MMP-2 with that obtained from the standards, the concentration of MMP-2 in the sample can be determined.
Materials provided
Standards should be assayed in duplicate. A standard curve must be performed on the same plate and at the same time as the samples. The MMP-2 ELISA provides sufficient reagents to run two sets of standard curves, and 41 samples (if assayed in duplicate all at once using one standard curve), or 34 samples (if assayed on two separate occasions using two standard curves).
• Anti-MMP-2 Coated 96-Well Plate (Kit Component No. JA1868-1EA): 1 plate, 96 removable wells coated with MMP-2 antibody • MMP-2 Standard (Kit Component No. JA1869-1EA): 2 vials containing lyophilized recombinant MMP-2 protein; reconstituted standards should be discarded after one use • Detector Antibody (Kit Component No. JA2034-6ML): 1 vial, 6 ml, biotinylated monoclonal anti-human MMP-2 antibody • 400X Conjugate (Kit Component No. JA2032-.1ML): 1 vial, 100 µl streptavidin-peroxidase conjugate, supplied as a 400X solution • Conjugate Diluent (Kit Component No. JA1615-12ML): 1 vial, 12 ml • Substrate (Kit Component No. JA1608-12ML): 1 vial, 12 ml • Sample Diluent (Kit Component No. JA2033-25ML): 1 bottle, 25 ml • ELISA 20X Plate Wash Concentrate (Kit Component No. JA1617-100ML): 1 bottle, 100 ml, supplied as a 20X solution of PBS and surfactant; contains 2% chloroacetamide • Stop Solution (Kit Component No. JA1616-12ML): 1 vial, 12 ml, 2.5N sulfuric acid • Plate Sealers: (Kit Component No. JB155-EA):
Materials Required but not provided
• 2-20 µl, 20-200 µl and 200-1000 • l precision pipettors with disposable tips • Wash bottle or multichannel dispenser for • 500 ml graduated cylinder • Deionized or distilled H2O • Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/595 nm or 450/540 nm
Precautions and recommendations
• Wear disposable gloves and eye protection. • Use only the wells provided with the kit. • Do not mix reagents from different kits. • Do not mouth pipette or ingest any of the reagents. • The buffers and reagents used in this kit contain anti-microbial and anti-fungal reagents. Care should be taken to prevent direct contact with these products. • Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled. • Human samples may be contaminated with infectious agents. Do not ingest, expose to open wounds, or breathe aerosols. Dispose of samples properly.
Preparation
Suspension Cells: Pellet cells by centrifugation at 1000 x g for 10 min, 4°C and remove supernatant for testing. Samples may be stored at -80 or -20°C.
Adherent Cells: Remove tissue culture medium and centrifuge the medium at 1000 x g for 10 min. Remove supernatant for testing. Samples may be stored at -80 or -20°C.
Cells in 96-Well Plates: Grow cells in desired medium and plate cells at desired concentration at a volume of 100 µl/well in a sterile 96-well tissue culture plate. The MMP-2 ELISA is sensitive enough to detect MMP-2 levels from 2.5 x 104 cells/well. Treat cells with drug(s) of choice at the appropriate dose and time courses. Centrifuge at 1000 x g for 10 min, 4°C and remove supernatant for testing. Samples may be stored at -80 or -20°C.
• Serum and Plasma: Serum and plasma samples should be diluted with Sample Diluent 1:20.
Note: Samples found to contain greater than 50 ng/ml MMP-2 (i.e., outside the range of the standard curve) must be diluted with Sample Diluent (provided), so that the MMP-2 concentration falls within the range spanned by the standard curve, and assayed again.
Reagent preparation
• 1X Wash Buffer: Prepare 1X Wash Buffer by adding 25 ml ELISA 20X Plate Wash Concentrate to 475 ml of deionized water. Mix well.
• MMP-2 Standard and Standard Curve: Each time an assay is performed, reconstitute a Lyophilized Standard as described on the lyophilized MMP-2 Standard vial label to give a concentration of 50 ng/ml by carefully and accurately pipetting the stated amount of dH2O and if required sample diluent. Let the reconstituted standard sit for 15 min at room temperature, with occasional swirling. Avoid excessive agitation of the standard. After reconstituting the MMP-2 Standard it should be diluted with Sample Diluent. Obtain seven tubes and label them 50, 25, 12.5, 6.25, 3.13, 1.56 and 0 ng/ml. Add 250 µl of Sample Diluent into each tube except the 50 ng/ml tube (first tube) that gets "undiluted" reconstituted standard. Remove 500 µl from the original vial of lyophilized material and add it to the first tube. Remove 250 µl from the first tube (50 ng/ml) and add it to the second tube (25 ng/ml) and mix gently. Repeat this procedure until you reach the sixth tube (1.56 ng/ml). The last tube (0 ng/ml) should just be Sample Diluent. Reconstituted standards should be discarded after one use.
• 1X Conjugate: Just prior to use, dilute a sufficient amount of the 400X Conjugate 1:400 with Conjugate Diluent to provide 100 µl of 1X Conjugate for each sample and standard well. For example: add 30 µl 400X Conjugate to 12 ml Conjugate Diluent and mix gently. Filter with a 0.2 µm syringe filter prior to use. Discard any unused 1X Conjugate.
Detailed protocol
A standard curve MUST be performed each time the assay is carried out. Standards should be assayed in duplicate. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross-contamination of reagents or samples.
1. Remove the appropriate number of wells from the foil pouch and place them into the empty well holder. Return any unused wells to the foil pouch, reseal and store at 4°C. 3. Add 50 µl Detector Antibody to each well. 4. Add 50 µl samples and standards to designated wells using clean pipette tips for each sample. 5. Cover wells with a plate sealer and incubate at 37°C for 4 h. 6. Wash the wells 1X Wash Buffer making sure each well is filled completely. Shake the contents into the sink; repeat for a total of 3 washes. Following the final wash, gently tap the inverted plate on paper towels to remove excess liquid. 7. Add 100 µl 1X Conjugate to each well, cover with a plate sealer, and incubate at room temperature for 30 min. 8. Wash the wells 3 times as outlined in step 6 above. Following the final wash FLOOD ENTIRE PLATE WITH dH2O. Shake the contents into the sink and gently tap the inverted plate on paper towels to remove excess liquid. 9. Add 100 µl of Substrate to each well and incubate IN THE DARK at room temperature for 30 min. 10. Add 100 µl Stop Solution to each well in the same order as the previously added Substrate. 11. Measure absorbance in each well using a spectrophotometric plate reader set at dual wavelengths of 450/595 nm (or 450/540nm). Wells must be read within 30 min of adding the Stop Solution.
Calculations
1. Average the duplicate absorbance values for each standard, including the zero and all sample values.
2. On graph paper, plot the mean absorbance values for each of the standards on the Y axis, versus the concentration of each standard (ng/ml) on the X axis.
3. Determine the concentration of unknowns by interpolation from the standard curve. There are a variety of plate reader software packages available (Softmax, Molecular Devices Corporation, Menlo Park, CA; KinetiCalc, BioTek Instruments, Inc. Winooski, VT) for analysis of plate data, which simplifies this process. All concentrations of MMP-2 reported in this booklet used point to point curve fitting.
4. For samples that have been diluted, the MMP-2 concentration must be multiplied by the dilution factor (i.e., if the sample was diluted five-fold, then the MMP-2 value obtained from the standard curve must be multiplied by five).
Standard curve
Figure 1: Standard Curve
The mean signal of each standard run in replicates of three in eight assays using two different lots of plates and two different lots of detector antibody.
Example data
Figure 2: Clinical Utility of the MMP-2 Immunoassay
The assay has a 30% percent clinical sensitivity (ability to recognize affected individuals) at a 95% clinical specificity (ability to recognize unaffected individuals).
Sensitivity
0.1 ng/ml
Sensitivity Notes
Figure 3: Sensitivity Using 96-Well Format
Astrocytoma and HS578T cells were plated out at various cell densities into 96 plate wells. Cells were incubated at 37°C for 24 h. Supernatant was measured in the MMP-2 ELISA. MMP-2 levels were detectable at cell concentrations of 4 X 103 Astrocytoma cells/well and 0.6 X 104 HS578T cells/well and above.
Figure 4: Sensitivity
The lower limit of detection (LLD), commonly used to define sensitivity, was measured by assaying four replicates of zero eight times using two different lots of plates and two different lots of detector antibody. The grand mean signal and pooled standard deviation of zero was calculated. The grand mean of each standard (run in replicates of four in the eight assays) was used for the standard curve, and the response, mean signal of zero plus two standard deviations, read in dose from the standard curve is the LLD; that is, the smallest dose that is not zero with 95% confidence.
Assay Range
1.56 - 50 ng/ml
Precision
Figure 5: Precision
The pooled coefficients of variation (according to the formula of Henry et. al., 1974) and between assay coefficients of variation are plotted against MMP-2 concentrations. The pooled data were collected from samples run eight times using two different lots of plates and two different lots of detector antibody in replicates of three on two separate occasions.
Parallelism
Figure 6: Parallelism
The study tested dilution-recovery of 28 positive samples. Dilutions were run in the MMP-2 ELISA and the "found" doses were plotted against the "expected" doses to determine linearity of dilution. The slope is very close to one and the intercept is not significantly different from zero. These studies are consistent with the absence of cross-reacting and matrix effects such as pH, salts, and endogenous binders that interfere with the reagents used in the assay. Note: Ca = Cancer; NHS = Normal Human Sera; TC= Tissue Culture.
Specificity
The MMP-2 ELISA Kit detects proMMP-2, active MMP-2 and the MMP-2:TIMP-2 complex. Specificity was demonstrated by neutralization (inhibition of assay signal) or extraction of active MMP-2 protein samples by a specific MMP-2 antibody. The MMP-2 antibody, which is not a component of the ELISA, neutralized or extracted almost all of the active MMP-2, proMMP-2 and MMP-2:TIMP-2 complexes in samples. (almost the entire signal was lost), while the control antibody (non MMP-2 antibody) did not affect the signal.
It is known that 2-aminophenylmercuric acetate (APMA) treatment promotes the autocatalytic cleavage of the N terminal prosequence of the latent 72-kDa enzyme to yield a 65-kDa enzyme. Recombinant proMMP-2, tissue culture supernatants and sera samples were tested in the MMP-2 ELISA after APMA treatment. There was no lost of signal after activation of recombinant proMMP-2 indicating that the ELISA detects both forms of MMP-2 to the same extent. In supernatants and sera samples there was a significant lost of signal after activation which could be do to degradation of MMP-2 protein by other activated MMPs in the sample or the lost of active MMP-2 to the general serum proteinase inhibitor α2 macroglobulin. Analysis of the samples by zymography showed both cleavage of the proenzyme by APMA and a good correlation with the levels of the proenzyme determined with the MMP-2 ELISA.
Figure 7: Specificity for Active MMP-2
Levels of MMP-2 detected by the ELISA after immunoaffinity extraction of MMP-2 positive samples with a MMP-2 antibody that is not used in the ELISA and a control antibody.
Figure 8: Specificity for the MMP-2:TIMP-2 Complex
Levels of a highly purified 72 kDa MMP-2 that exists in a stable, but non-covalent 1:1complex with TIMP-2 detected after neutralization with a MMP-2 antibody that is not used in the ELISA.
Figure 9: Detection of MMP-2 Following APMA Treatment
Levels of MMP-2 detected by the MMP-2 ELISA after 2-aminophenylmercuric acetate (APMA) treatment. APMA promotes the autocatalytic cleavage of the N-terminal prosequence of the latent 72-kDa enzyme to yield the active form of the enzyme. HT1080 and astrocytoma tissue culture supernatants, recombinant proMMP-2 and normal human serum samples were either untreated (-APMA) or incubated for four hours at 37°C with 2 mM APMA (+APMA) or a volume of DMSO to control for the DMSO base of APMA (Vehicle Ctrl).
Figure 10: Specificity for Active MMP-2
Levels of MMP-2 detected by the ELISA after immunoaffinity extraction of MMP-2 positive samples with a MMP-2 antibody that is not used in the ELISA and a control antibody.
Registered Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
