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QIA56 MMP-9 ELISA Kit

MMP-9 ELISA Kit: Ficha de dados de segurança do material (FDSM) ou ficha de segurança do material (FSM), certificado de análise (CA) e certificado de qualidade (CQ), dossiês e outros documentos disponíveis.

Sinônimos: Gelatinase B ELISA Kit, 92 kDa Gelatinase ELISA Kit, Matrix Metalloproteinase 9 ELISA Kit

Detection Methods: Colorimetric
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Species ReactivityDetection Methods
HColorimetric

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      A non-isotopic, sandwich ELISA for the in vitro measurement of human MMP-9 in tissue culture supernatant, serum, or plasma.
      Catalogue NumberQIA56
      Brand Family Calbiochem®
      SynonymsGelatinase B ELISA Kit, 92 kDa Gelatinase ELISA Kit, Matrix Metalloproteinase 9 ELISA Kit
      Application Data
      The mean signal of each standard run in replicates of four in eight assays using two different lots of plates and two different lots of detector antibody.


      The pooled coefficients of variation (according to the formula of Henry et. al., 1974) and between assay coefficients of variation are plotted against MMP-9 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 four on two separate occasions.

      The study tested dilution-recovery of ten positive samples. The dilutions were run in the MMP-9 ELISA Kit and the "found" doses were plotted against the "expected" doses to determine linearity of dilution. The slope is not significantly different from 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; NHP = Normal Human Plasma; PMA = Phorbol 12-myristate 13-acetate; EGF = Epidermal Growth Factor; AR = Amphiregulin; Both = EGF and AR; A = 2 x 106 cells/ml; B = 1.0 x 106 cells/ml.

      Levels of MMP-9 detected by the ELISA after immunoaffinity extraction of MMP-9 positive samples (PMA treated HT1080 and HL-60 cells) with a MMP-9 antibody that is not used in the ELISA, a TIMP-1 antibody, a TIMP-2 antibody and a TIMP-3 antibody.

      APMA promotes the autocatalytic cleavage of the N-terminal propeptide of the latent 92-kDa enzyme to yield the active form of the enzyme. 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-9 ELISA. Several methods of MMP-9 induction were used to generate positive samples such as treatment with 50 ng/ml amphiregulin (AR), 50 ng/ml epidermal growth factor (EGF), 1 ng/ml transforming growth factor β (TGFβ) and 25 ng/ml phorbol 12-myristate 13-acetate (PMA). Normal human sera (NHS) containing moderate levels of MMP-9 were treated with APMA and analyzed.
      Materials Required but Not Delivered• 2-20 µl, 20-200 µl and 200-1000 µl precision pipettors with disposable tips
      • Wash bottle or multichannel dispenser for washing
      • 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 450/540nm
      References
      ReferencesMassova, I., et al. 1998. FASEB J. 12, 1075.
      Xie, B.,et al. 1998. J. Biol. Chem. 273, 11576.
      Borden, P., and Heller, R. A. 1997. Crit. Rev. Eukaryot. Gen. Expression 7, 159.
      Chambers, A. F., and Matrisian, L. M. 1997. J. Natl. Cancer Inst. 89, 1260.
      DeClerck, Y.A., et al. 1997. Adv. Exp. Med. Biol. 425, 89.
      Gomis-Ruth, F. X., et al. 1997. Nature 389, 77.
      Murphy, G., and Knauper, V. 1997. Matrix Biol. 15, 511.
      Olson, M. W., et al. 1997. J. Biol. Chem. 272, 29975.
      Woodhouse, F. C., et al. 1997. Cancer 80, 1529.
      Yu, A. E., et al. 1997. Drugs Aging 11, 229.
      Coussens, L. M., and Werb, Z. 1996. Chem. Biol. 3, 895.
      Lim Y.T., et al. 1996. J. Cell. Physiol. 167, 333.
      Kohn, E. C., and Liotta, L. A. 1995. Cancer Res. 55, 1856.
      Shapiro, S.D., et al. 1995. J. Biol. Chem. 270, 6351.
      Murphy, G., et al. 1994. J. Biol. Chem. 269, 6632.
      Fridman, R., et al. 1993. Biochem J. 289, 411.
      Goldberg, G. L., et al. 1992. J. Biol. Chem. 267, 4583.
      Morodomi T.,et al. 1992. Biochem. J. 285, 603.
      Product Information
      Detection methodColorimetric
      DeclarationNot available for sale in Japan.
      Form34 or 41 Tests
      Format96-well plate
      Kit contains96-Well Coated Plate, MMP-9 Standard, Biotinylated Detector Antibody, 400X Streptavidin Peroxidase Conjugate, Diluent, Substrate, Assay Buffers, Stop Solution, Plate Sealer, and a user protocol.
      Applications
      Biological Information
      Assay range0.625 - 20 ng/ml
      Assay time3.5 h
      Sample TypeTissue culture media, serum, plasma
      Species Reactivity
      • Human
      Physicochemical Information
      Sensitivity0.1 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 23/24/25-35-36-40-43

      Toxic by inhalation, in contact with skin and if swallowed.
      Causes severe burns.
      Irritating to eyes.
      Limited evidence of a carcinogenic effect.
      May cause sensitization by skin contact.
      S PhraseS: 26-36/37/39-45

      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 useThe Calbiochem® MMP-9 ELISA Kit is a non-isotopic immunoassay for the in vitro quantitation of human MMP-9 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 ConditionsUpon 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 contains96-Well Coated Plate, MMP-9 Standard, Biotinylated Detector Antibody, 400X Streptavidin Peroxidase Conjugate, Diluent, Substrate, Assay Buffers, Stop Solution, Plate Sealer, and a user protocol.
      Specifications
      Global Trade Item Number
      Número de catálogo GTIN
      QIA56-1EA 04055977209112

      Documentation

      MMP-9 ELISA Kit Certificados de análise

      TítuloNúmero do lote
      QIA56

      Referências

      Visão geral de referência
      Massova, I., et al. 1998. FASEB J. 12, 1075.
      Xie, B.,et al. 1998. J. Biol. Chem. 273, 11576.
      Borden, P., and Heller, R. A. 1997. Crit. Rev. Eukaryot. Gen. Expression 7, 159.
      Chambers, A. F., and Matrisian, L. M. 1997. J. Natl. Cancer Inst. 89, 1260.
      DeClerck, Y.A., et al. 1997. Adv. Exp. Med. Biol. 425, 89.
      Gomis-Ruth, F. X., et al. 1997. Nature 389, 77.
      Murphy, G., and Knauper, V. 1997. Matrix Biol. 15, 511.
      Olson, M. W., et al. 1997. J. Biol. Chem. 272, 29975.
      Woodhouse, F. C., et al. 1997. Cancer 80, 1529.
      Yu, A. E., et al. 1997. Drugs Aging 11, 229.
      Coussens, L. M., and Werb, Z. 1996. Chem. Biol. 3, 895.
      Lim Y.T., et al. 1996. J. Cell. Physiol. 167, 333.
      Kohn, E. C., and Liotta, L. A. 1995. Cancer Res. 55, 1856.
      Shapiro, S.D., et al. 1995. J. Biol. Chem. 270, 6351.
      Murphy, G., et al. 1994. J. Biol. Chem. 269, 6632.
      Fridman, R., et al. 1993. Biochem J. 289, 411.
      Goldberg, G. L., et al. 1992. J. Biol. Chem. 267, 4583.
      Morodomi T.,et al. 1992. Biochem. J. 285, 603.
      Protocolo do usuário

      Revision09-January-2009 JSW
      SynonymsGelatinase B ELISA Kit, 92 kDa Gelatinase ELISA Kit, Matrix Metalloproteinase 9 ELISA Kit
      Form34 or 41 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival, store the entire contents at -20°C. Avoid multiple freeze/thaw cycles. Do not expose reagents to excessive light.
      Intended useThe Calbiochem® MMP-9 ELISA Kit is a non-isotopic immunoassay for the in vitro quantitation of human MMP-9 protein in tissue culture medium, serum, and plasma.
      BackgroundMatrix metalloproteinase-9 (MMP-9; 92-kDa gelatinase B) is an important member of the MMP family of neutral endopeptidases responsible for the degradation of the extracellular matrix (ECM) and is involved in tumor invasion and metastasis. MMP-9 is a collagenase with specificity for type IV collagen, which makes up the backbone of the basement membrane. The proteolytic degradation of the ECM is an important aspect of many physiological and pathological conditions associated with alterations in connective tissue proteins such as embryo implantation, morphogenesis, wound healing, ovulation, cell migration, tissue involution, angiogenesis, and tumor invasion. MMP-9 is secreted from stimulated macrophages, neutrophils and transformed cells in a latent form. In fact all MMPs, except membrane-type metalloproteinases, are secreted as inactive zymogens into the extracellular matrix, where subsequent activation results in cleavage of the proenzymes into the active species. The presence or absence of activators and the binding of tissue inhibitors of metalloproteinases (TIMPs) maintains strict control on the activation of such enzymes in the extracellular space. The TIMP family has four members: TIMP-1, TIMP-2, TIMP-3, and TIMP-4. Binding of the TIMPs to their specific MMPs results in efficient inhibition of enzymatic activity of MMPs. In the case of gelatinases (MMP-9 and MMP-2), 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-9 protein has four structural domains: an amino-terminal propeptide, a catalytic domain, a fibronectin type-II like domain within the catalytic domain and a hemopexin-like domain at the carboxyl-terminal. Cleavage of the propeptide results in zymogen activation. The catalytic domain contains two zinc ions and a calcium ion. MMP-9 incorporates three repeats homologous to the type-II module of fibronectin into the catalytic domain. This region is also known as the gelatin-binding domain and is involved in binding to denatured collagen or gelatin. The hemopexin-like domain is highly conserved among MMPs and shows sequence similarity to the plasma protein, hemopexin. The hemopexin like domain has been shown to play a functional role in substrate binding and/or in interactions with the tissue inhibitors of metalloproteinases (TIMPs). There is a high degree of specificity in the binding of TIMPs to the latent forms of these enzymes. TIMP-1 binds exclusively to the zymogen form of MMP-9 (Kd~35 nM), whereas TIMP-2 binds to the zymogen form of MMP-2 (Kd ~5 nM). There is a biphasic binding of TIMP-1 to the zymogen form of MMP-9, with the hemopexin-like domain representing the high affinity binding site.
      Principles of the assayThe Calbiochem® MMP-9 ELISA Kit is a "sandwich" enzyme immunoassay employing a mouse monoclonal antibody and a sheep polyclonal antibody. An antibody, specific for the human MMP-9 protein, has been immobilized onto the surface of the 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 2 h, during which time any MMP-9 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-9 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-9 (provided lyophilized). By comparing the absorbance obtained from a sample containing an unknown amount of MMP-9 with that obtained from the standards, the concentration of MMP-9 in the sample can be determined.
      Materials providedStandards 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-9 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-9 Coated 96-Well Plate (Kit Component No. JA1592-1EA): 1 plate, 96 removable wells coated with MMP-9 polyclonal antibody

      • MMP-9 Standard (Kit Component No. JA1597-1EA): 2 vials containing lyophilized MMP-9 protein; reconstituted standards should be discarded after one use

      • Detector Antibody (Kit Component No. JA1594-6ML): 1 vial, 6 ml, biotinylated monoclonal anti-human MMP-9 antibody

      • 400X Conjugate (Kit Component No. JA1596-.1ML): 1 vial, 100 µl, streptavidin-peroxidase conjugate supplied as a 400X solution

      • Conjugate Diluent (Kit Component No. JA1615-12ML): 1 vial, 12 ml

      • TMB, Soluble (Kit Component No. JA1608-12ML): 1 vial, 12 ml

      • Sample Diluent (Kit Component No. JA1593-25ML): 1 bottle, 25 ml

      • 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 µl precision pipettors with disposable tips
      • Wash bottle or multichannel dispenser for washing
      • 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 450/540nm
      Precautions and recommendations• Wear disposable gloves and eye protection.
      • Use only the 96-well plate 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 suspension cells by centrifugation at 1000 x g for 10 min, 4°C. Remove supernatant for testing. Samples may be stored at -20°C. • Adherent Cells: Remove tissue culture medium and centrifuge at 1000 x g for 10 min. Remove supernatant for testing. Samples may be store at -20°C. • Serum and Plasma: Serum and plasma samples should be diluted with Sample Diluent 1:10 (normal samples) or 1:40 (most cancer samples) or greater as needed. Samples found to contain greater than 20 ng/ml MMP-9 (i.e., outside the range of the standard curve) must be diluted with Sample Diluent (provided), so that the MMP-9 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 20X Wash Buffer Concentrate to 475 ml of dH2O. Mix well. • MMP-9 Standard and Standard Curve: Each time an assay is performed, reconstitute a Lyophilized Standard by carefully and accurately pipetting dH2O and sample diluent, as described on the lyophilized MMP-9 Standard vial label to give a concentration of 20 ng/ml. Let the reconstituted standard sit for 15 min at room temperature, with occasional swirling. Avoid excessive agitation of the standard. After reconstituting the MMP-9 Standard it should be diluted with Sample Diluent. Obtain seven tubes and label them 20, 10, 5, 2.5, 1.25, 0.625 and 0 ng/ml. Add 250 µl of Sample Diluent into each tube except the 20 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 (20 ng/ml) and add it to the second tube (10 ng/ml) and mix gently. Repeat this procedure until you reach the sixth tube (0.625 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 volume of 400X Conjugate 1:400 with Conjugate Diluent to provide 100 µl 1X Conjugate for each sample and standard well. For example add 30 µl to 12 ml of Conjugate Diluent, mix gently. Filter with a 0.2 µm syringe filter prior to use. Discard any unused 1X Conjugate.
      Detailed protocolThe MMP-9 ELISA Kit is provided with removable strips of wells so the assay can be carried out on two separate occasions. Since conditions may vary, a standard curve MUST be determined each time the assay is performed. 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 strips 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.
      2. Prepare all samples and standards for assay.
      3. Add 50 µl Detector Antibody to each well designated for samples and standards.
      4. Add 50 µl each sample and diluted MMP-9 standards (in duplicate) to designated wells using clean pipette tips for each sample.
      5. Cover the wells with a plate sealer and incubate at room temperature for 2 h.
      6. Wash the wells with 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 removed 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.
      9. FLOOD ENTIRE PLATE WITH dH2O. Remove contents of wells by inverting over sink and tapping on paper towels.
      10. Add 100 µl of TMB, Soluble to each well and incubate IN THE DARK at room temperature for 30 min.
      11. Add 100 µl Stop Solution to each well in the same order as the previously added TMB Solution.
      12. 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.
      Calculations1. 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 data, which simplifies this process. 4. For samples that have been diluted, the MMP-9 concentration must be multiplied by the dilution factor (ie., if the sample was diluted five-fold, then the MMP-9 value obtained from the standard curve must be multiplied by five).
      Standard curve

      Figure 1: Example Standard Curve

      The mean signal of each standard run in replicates of four in eight assays using two different lots of plates and two different lots of detector antibody.
      Example data

      Figure 2: Phorbol ester induced up-regulation of MMP-9 protein in HL-60 cells

      It has been shown that the induction of MMP-9 gene expression is associated with macrophage differentiation. Phorbol 12-myristate 13-acetate (PMA) at all concentrations studied induced the synthesis of MMP-9 protein. This up regulation closely parallels the timing of PMA induced cell adhesion and spreading, a hallmark of macrophage differentiation.

      Figure 3: MMP-9 Detection in PMA-Treated HT1080 Cells

      PMA has been shown to increase the expression of MMP-9 by HT1080 cells. The MMP-9 ELISA detects this increase in synthesis in a time dependent matter.

      Figure 4: Detection of MMP-9 in Human Serum and Plasma

      Levels of MMP-9 detected by the assay are significantly elevated in cancer sera samples compared to normal sera and plasma samples (left figure is the data on a logarithmic scale and the right is a linear scale). The following samples were assayed: normal human sera (NHS), normal human plasma (NHP), sera from cervical cancer (CV), breast cancer (BR) and prostate cancer (PT) patients.

      Figure 5: Clinical Utility of the MMP-9 Immunoassay

      The assay has a 90% percent clinical sensitivity (ability to recognize affected individuals) at a 95% clinical specificity (ability to recognize unaffected individuals).
      Sensitivity0.1 ng/ml
      Sensitivity NotesThe assay can easily distinguish 0.1 ng/ml of MMP-9 from zero.

      Figure 6: Sensitivity
      Assay Range0.625 - 20 ng/ml
      Assay Range NotesThese results demonstrate that the assay specifically recognizes free and TIMP bound zymogen form of MMP-9.
      Precision

      Figure 7: 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-9 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 four on two separate occasions.
      Parallelism

      Figure 8: Parallelism

      The study tested dilution-recovery of ten positive samples. The dilutions were run in the MMP-9 ELISA Kit and the "found" doses were plotted against the "expected" doses to determine linearity of dilution. The slope is not significantly different from 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; NHP = Normal Human Plasma; PMA = Phorbol 12-myristate 13-acetate; EGF = Epidermal Growth Factor; AR = Amphiregulin; Both = EGF and AR; A = 2 x 106 cells/ml; B = 1.0 x 106 cells/ml.
      SpecificityThe MMP-9 ELISA Kit detects free and TIMP-1 bound MMP-9 in human serum and plasma samples and cell culture supernatants. Specificity was demonstrated by immunoaffinity extraction (inhibition of assay signal) of MMP-9 positive samples by a specific MMP-9 antibody, a specific TIMP-1 antibody, a specific TIMP-2 antibody and a specific TIMP-3 antibody. The MMP-9 antibody, which is not a component of the ELISA, extracted almost all MMP-9 (almost the entire signal was lost), while the control antibody (non MMP-9 antibody) did not affect the signal of the MMP-9 positive samples. The TIMP-1 antibody extracted most of the pro-MMP-9 activity detected by this immunoassay. Addition of TIMP-1 and TIMP-2 recombinant proteins to both positive and negative samples does not change the level of MMP-9 detected demonstrating that the assay has the same specificity for the free and the TIMP bound form of MMP-9.

      It is known that 2-aminophenylmercuric acetate (AMPA) treatment promotes the autocatalytic cleavage of the N terminal propeptide of the latent 92-kDa enzyme to yield an 84-kDa enzyme. Further treatment causes the autocatalytic cleavage of the carboxyl terminus to generate the 68-kDa form of MMP-9. Both tissue culture supernatants and sera samples were tested in the MMP-9 ELISA after APMA treatment. In every case almost all the signal disappeared after APMA treatment. 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-9 ELISA. In addition the assay recognizes pure recombinant pro-MMP-9 protein, but not active MMP-9 recombinant protein.

      Figure 9: Specificity

      Levels of MMP-9 detected by the ELISA after immunoaffinity extraction of MMP-9 positive samples (PMA treated HT1080 and HL-60 cells) with a MMP-9 antibody that is not used in the ELISA, a TIMP-1 antibody, a TIMP-2 antibody and a TIMP-3 antibody.


      Figure 10: Levels of MMP-9 Following APMA Treatment

      APMA promotes the autocatalytic cleavage of the N-terminal propeptide of the latent 92-kDa enzyme to yield the active form of the enzyme. 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-9 ELISA. Several methods of MMP-9 induction were used to generate positive samples such as treatment with 50 ng/ml amphiregulin (AR), 50 ng/ml epidermal growth factor (EGF), 1 ng/ml transforming growth factor β (TGFβ) and 25 ng/ml phorbol 12-myristate 13-acetate (PMA). Normal human sera (NHS) containing moderate levels of MMP-9 were treated with APMA and analyzed.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.

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