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QIA130 MMP-13 ELISA Kit

MMP-13 ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: Collagenase-3 ELISA Kit, Matrix Metalloproteinase ELISA Kit

Detection Methods: Colorimetric
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QIA130
  
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      Overview

      Replacement Information

      Key Spec Table

      Species ReactivityDetection Methods
      HColorimetric
      Description
      Overview

      This product has been discontinued.



      A highly sensitive and specific kit for detection of MMP-13 from human samples, including serum, synovial fluid, and cell culture supernatant. Recognizes both the latent and active forms of MMP-13, however exhibits 10-fold higher sensitivity towards the active than the latent form of MMP-13. Does not recognize MMP-1, MMP-2, MMP-3, MMP-8, or MMP-9.
      Catalogue NumberQIA130
      Brand Family Calbiochem®
      SynonymsCollagenase-3 ELISA Kit, Matrix Metalloproteinase ELISA Kit
      Materials Required but Not Delivered• Pipettes with disposable tips (100 µl, 500 µl and 1 ml), a multi-channel pipette (100-200 µl)
      • Distilled or deionized water
      • Horizontal orbital microplate shaker
      • Microplate reader capable of measuring absorbance at 450 nm
      • Squirt bottle or automated microplate washer
      • 500 ml graduated cylinder
      References
      ReferencesBluteau G., et al. 2001. Biochim. Biophys. Acta 1526, 147.
      Brew, K., et al. 2000. Biochim Biophys. Acta 1477, 267.
      Pendas A.M., et al. 2000. Clin. Chim. Acta 291, 137.
      Nagase, H., and Woessner, F. Jr. 1999. J. Biol. Chem. 274, 21491.
      Westhoff C.S., et al. 1999. Arthritis Rheum. 42, 1517.
      Knauper V., et al. 1996. J. Biol. Chem. 271, 17124.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsCoated 96-Well Plate, Assay Buffer, Wash Buffer Concentrate, Standard, Serum Standard Diluent, Detection Buffer, Biotinylated Antibody, Conjugate Solution, TMB Substrate, Stop Solution, and a user protocol.
      Applications
      Biological Information
      Assay range32 - 2000 pg/ml
      Assay time~5 hours
      Sample TypeSerum, synovial fluid, tissue culture supernatant
      Species Reactivity
      • Human
      Physicochemical Information
      Sensitivity7 pg/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe MMP-13 ELISA Kit is a non-isotopic immunoassay for the in vitro quantitation of latent and active human MMP-13 protein in body fluids such as serum and synovial fluids. Note: When analyzing pro-MMP-13 and active MMP-13 in parallel, the level of pro-MMP-13 must be ~10 times higher than the level of active MMP-13 in order to obtain a comparable signal.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsAll components of the kit can be stored in the refrigerator (4°C). Once reconstituted, the standard solution should be used immediately or stored at -20°C. The diluted Biotinylated Detection Antibody and diluted Conjugate Solution should be prepared freshly directly before use. When running a partial plate, only a suitable aliquot should be activated.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCoated 96-Well Plate, Assay Buffer, Wash Buffer Concentrate, Standard, Serum Standard Diluent, Detection Buffer, Biotinylated Antibody, Conjugate Solution, TMB Substrate, Stop Solution, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      QIA130 0

      Documentation

      MMP-13 ELISA Kit SDS

      Title

      Safety Data Sheet (SDS) 

      MMP-13 ELISA Kit Certificates of Analysis

      TitleLot Number
      QIA130

      References

      Reference overview
      Bluteau G., et al. 2001. Biochim. Biophys. Acta 1526, 147.
      Brew, K., et al. 2000. Biochim Biophys. Acta 1477, 267.
      Pendas A.M., et al. 2000. Clin. Chim. Acta 291, 137.
      Nagase, H., and Woessner, F. Jr. 1999. J. Biol. Chem. 274, 21491.
      Westhoff C.S., et al. 1999. Arthritis Rheum. 42, 1517.
      Knauper V., et al. 1996. J. Biol. Chem. 271, 17124.

      Brochure

      Title
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      User Protocol

      Revision09-November-2016 JSW
      SynonymsCollagenase-3 ELISA Kit, Matrix Metalloproteinase ELISA Kit
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageAll components of the kit can be stored in the refrigerator (4°C). Once reconstituted, the standard solution should be used immediately or stored at -20°C. The diluted Biotinylated Detection Antibody and diluted Conjugate Solution should be prepared freshly directly before use. When running a partial plate, only a suitable aliquot should be activated.
      Intended useThe MMP-13 ELISA Kit is a non-isotopic immunoassay for the in vitro quantitation of latent and active human MMP-13 protein in body fluids such as serum and synovial fluids. Note: When analyzing pro-MMP-13 and active MMP-13 in parallel, the level of pro-MMP-13 must be ~10 times higher than the level of active MMP-13 in order to obtain a comparable signal.
      BackgroundMatrix metalloproteinases (MMPs) comprise a family of secreted and membrane-bound endopeptidases that hydrolyze extracellular matrix proteins. Based on their preferred substrates and on structural features MMPs can be divided into collagenases, gelatinases, stromelysins, and membrane-type matrix metalloproteinases. The activity of these enzymes is tightly regulated by tissue inhibitors of metalloproteinases (TIMP). Collagenase 3 (MMP-13) is a secreted 452 amino acid protein that is released from cells as an inactive zymogen and activated extracellularly by removal of its propeptide. Cleavage of the 84 amino acid propeptide can be catalyzed by other MMPs such as MMP-2 and MMP-14, or by factors like plasmin. Under normal conditions, MMP-13 is expressed during embryogenesis (fetal bone development) and is present only at very low levels in adult tissue. However, this enzyme is reported to be involved in the development and metastasis of breast and lung carcinomas, chondrosarcomas and osteosarcomas, head and neck carcinomas, and some forms of skin cancer. Additionally, this enzyme plays an important role in degenerative bone diseases including osteoarthritis and rheumatoid arthritis.
      Principles of the assayThe MMP-13 ELISA Kit provides a highly sensitive, specific, and quantitative determination of latent and active human MMP-13 in serum, synovial fluid, and cell culture supernatants.

      This quantitative assay is based on a two-site sandwich format. A highly specific monoclonal antibody recognizing the MMP-13 is immobilized on the plate. MMP-13 is bound to the wells and other components of the sample are removed by aspiration and washing of the plate. Two steps using a secondary biotinylated antibody and a highly polymerized streptavidin-peroxidase conjugate detect the analyte. Excess antibody is removed by aspiration and washing after each detection step. The amount of peroxidase bound to each well is determined by the addition of TMB Substrate. The reaction is stopped by addition of Stop Solution and the resultant color is read in a microtiter plate reader at 450 nm. The concentration of MMP-13 in samples is determined by interpolation from the standard curve.
      Materials provided• Coated 96-Well Plate (Kit Compnent No. JA7710): The plate contains 6 x 16 strips coated with anti-MMP-13. The strips are ready to use.
      • Assay Buffer (Kit Component No. JA7711): Bottle contains a phosphate buffer with additives. Ready to use.
      • Wash Buffer Concentrate (Kit Compnent No. JA7712): Each bottle (total of 2 bottles) contains a buffer concentrate that has to be diluted 20 fold to get 500 ml Wash Buffer.
      • MMP-13 Standard (Kit Compnent No. JA7713): The vial contains lyophilized activated MMP-13 in Assay Buffer. It has to be reconstituted with 1 ml of distilled water prior to use to get a 20 ng/ml stock solution.
      • Serum Standard Diluent (Kit Compnent No. JA7714): This solution contains certified human serum in a buffer with additives. Ready to use. Warning: Component contains human serum! Wear eye, hand, face, and clothing protection when using this material!
      • Detection Buffer (Kit Compnent No. JA9210): This solution consists of phosphate buffer containing additives.
      • Biotinylated Antibody (Kit Compnent No. JA9211): This solution contains biotinylated monoclonal anti-procollagenase 3 antibody in a buffer with additives. Antibody has to be diluted 100-fold with Detection Buffer before use.
      • Conjugate Solution (Kit Compnent No. JA7717): This solution contains a highly polymeric streptavidin-peroxidase conjugate in stabilizer with preservatives. It has to be diluted 40-fold with Detection Buffer prior to use.
      • TMB Substrate (Kit Compnent No. JA7718): Bottle contains a TMB solution ready to use.
      • Stop Solution (Kit Compnent No. JA7719): Bottle contains 0.25 M H₂SO₄. Ready to use. See warning under Safety Warning and Precautions.
      Materials Required but not provided• Pipettes with disposable tips (100 µl, 500 µl and 1 ml), a multi-channel pipette (100-200 µl)
      • Distilled or deionized water
      • Horizontal orbital microplate shaker
      • Microplate reader capable of measuring absorbance at 450 nm
      • Squirt bottle or automated microplate washer
      • 500 ml graduated cylinder
      Precautions and recommendations• Stop solution contains 0.25 M sulphuric acid. Wear eye, hand, face, and clothing protection when using this material.
      • The Serum Standard Diluent contains human serum that has been tested negatively for HAV, HBV, HCV and HIV. Wear suitable protective clothing, such as laboratory overalls and gloves and observe caution when working with this material.
      • All chemicals should be considered as potentially hazardous. We therefore recommend that only those persons who have been trained in laboratory techniques handle this product and that it is used only in accordance with the principles of good laboratory practice. Wear suitable protective clothing, such as laboratory overalls, safety glasses, and gloves. Avoid contact with skin and eyes. In case of contact, wash immediately with water.
      • Allow samples and all reagents to equilibrate to room temperature (20-30°C) prior to performing the assay. This is an essential prerequisite for the TMB Substrate.
      • For the highly sensitive determination of collagenase-3 in serum or synovial fluid samples, use the Serum Standard Diluent to prepare the standard curve.
      • It is absolutely essential that all wells be washed thoroughly and uniformly. When washing is done by hand, use a squeeze bottle and ensure that all wells are completely filled and emptied at each step.
      • Use only reagents from the same lot for each assay. This is especially important when running more than one plate per sample group.
      • A separate standard curve must be run on each plate.
      • Mix all reagents thoroughly prior to use, avoiding foaming.
      • Keep the wells sealed with the foil except when adding reagents and during reading.
      • Any variation in the protocol can cause variation in binding.
      • The values obtained by the samples should be within the standard range. If this is not the case, dilute the sample and repeat the assay.
      • We take great care to ensure that this product is suitable for all validated sample types, as designated in this manual. However, it is possible that in some cases, high levels of interfering substances may cause unusual results.
      • Samples should not contain EDTA or other chelating reagents as they inhibit the assay.
      PreparationSerum: • Serum samples may be stored at -80°C. • Avoid freeze/thaw cycles. • When the serum is stored at -80°C, it is absolutely necessary to mix the samples thoroughly prior to measuring. • Dilute the serum samples 1:10 with Assay Buffer, depending on the possible concentration of the analyte. For measuring these dilutions use the standards prepared with Serum Standard Diluent. • If you need samples with a dilution of more than 1:10, initially prepare a 1:10 dilution with Assay Buffer and do further dilution steps using Serum Standard Diluent. This is necessary to maintain a serum concentration of 10% in the sample. Synovial Fluid: • Centrifuge the collected synovial fluid for 15 min at 4000 x g or higher. • The supernatant can be stored at -80°C. • Avoid freeze/thaw cycles. • When the synovial fluid was stored at -80°C, it is absolutely necessary to mix the samples thoroughly prior to measuring. • Dilute the samples 1:10 or more with Assay Buffer, depending on the possible concentration of the analyte. For measuring these samples, use the standards prepared with Serum Standard Diluent. • If you need samples with a dilution of more than 1:10, initially prepare a 1:10 dilution with Assay Buffer and do further dilution steps using Serum Standard Diluent. • It is important to note that the recovery of MMP-13 in synovial fluid is only 50% or less (see table 5). Tissue culture supernatant • Centrifuge the samples to remove any particles. • The supernatants can be stored at -80°C. • Avoid freeze/thaw cycles. • Dilute the samples 1:3 or more with Assay Buffer, depending on the possible concentration of the analyte. For measuring diluted samples use the standards prepared with Assay Buffer Note: Fetal or neonatal calf serum may contain Collagenase 3. Always measure your culture media as background controls!
      Reagent preparationIt is absolutely necessary to equilibrate all reagents to room temperature (1 h incubation on the bench) prior to use in order to prevent margin effects. Use either distilled or deionized water for the reconstitution of the standard and for the dilution of the wash buffer concentrate. Always seal the plates with the provided foil during incubation. • Assay Buffer: After equilibration to room temperature, the buffer is ready to use. • Wash Buffer: Dilute the contents of the bottle (25 ml) to 500 ml with distilled or deionized water. Thoroughly wash the stock bottle to ensure that the entire contents are used. • Standard: Add 1 ml distilled or deionized water to the standard tube (yellow lid) and allow the contents to dissolve for 30 min. Gently mix, but avoid foaming of the reagent! • Serum Standard Diluent: After equilibration to room temperature, the reagent is ready to use. • Detection Buffer: After equilibration to room temperature, the reagent is ready to use. • Biotinylated Antibody: Dilute Biotinylated Antibody 100-fold with Detection Buffer. For the whole plate, add 120 µl from the antibody solution tube (red lid) to 12 ml Detection Buffer. When running half a plate, add 60 ml antibody solution to 6 ml Detection Buffer. • Conjugate Solution: Dilute the provided Conjugate Solution 40-fold with Detection Buffer. For the whole plate, add 300 µl of the conjugate tube (blue lid) to 12 ml Detection Buffer. When running half a plate, add 150 µl of the conjugate to 6 ml of Detection Buffer. • Substrate and Stop Solution: After equilibration to room temperature, the reagents are ready to use. Preparation of Standards with Assay Buffer (For tissue culture supernatants) 1. Label 7 tubes with 32, 63, 125, 250, 500, 1000, and 2000 pg/ml. 2. Pipette 900 µl of Assay Buffer into the 2000 pg/ml tube, the remaining tubes pipette 500 µl of Assay Buffer. 3. Pipette 100 µl of the stock standard (20 ng/ml) into the 2000 pg/ml tube and mix thoroughly. 4. Pipette 500 µl of the 2000 pg/ml standard into the tube labeled with 1000 pg/ml and mix thoroughly. 5. Repeat this dilution procedure with the other standard tubes. 6. The blank value (0 pg/ml) is obtained by using simple Assay Buffer. 7. The stock solution is not part of the standard curve and can be stored at -20°C. Preparation of standards with Serum Standard Diluent (For the highly sensitive measurement of serum and synovial fluid samples) 1. Label 7 tubes with 32, 63, 125, 250, 500, 1000, and 2000 pg/ml. 2. Pipette 900 µl of Serum Standard Diluent into the 2000 pg/ml tube, in the remaining tubes pipette 500 µl of Serum Standard Diluent. 3. Pipette 100 µl of the stock standard (20 ng/ml) into the 2000 pg/ml tube and mix thoroughly. 4. Pipette 500 µl of the 2000 pg/ml standard into the tube labeled with 1000 pg/ml and mix thoroughly. 5. Repeat this dilution procedure with the other standard tubes. 6. The blank value (0 pg/ml) is obtained by using Serum Standard Diluent. 7. The stock solution is not part of the standard curve and can be stored at -20°C.
      Detailed protocol1. Prepare reagents and standards as described in the sections above. For assessing serum samples, prepare the standard using the Serum Standard Diluent. NOTE: It is necessary to equilibrate the reagents to room temperature before use.
      2. Prepare the unknown samples as described above by appropriate dilution with Assay Buffer.
      3. Prepare the 96-well plate by inserting the required amount of wells into the frame. Note that you need 16 wells for the standard curve.
      4. Pipette 100 µl of the reconstituted standards in duplicate in the wells using a clean pipette tip for each standard. Assay Buffer serves as zero blank.
      5. Pipette 100 µl of the prepared unknown samples in duplicate into the wells.
      6. Seal the plate with the provided plate sealer and incubate on a shaker at room temperature for exactly 120 min.
      7. Aspirate and wash the wells 4 times with Wash Buffer. Take care that all wells are completely filled and emptied at each wash.
      8. Blot the plate on paper towels to remove residual fluid from the plate.
      9. Add 100 µl of diluted Biotinylated Antibody into each well.
      10. Seal the plate and incubate on a shaker at room temperature for exactly 90 min.
      11. Aspirate and wash the wells 4 times with Wash Buffer. Take care that all wells are completely filled and emptied at each wash.
      12. Blot the plate on paper towels to remove residual fluid from the plate
      13. Add 100 µl of diluted Conjugate Solution into each well.
      14. Seal the plate with the provided foil and incubate on a shaker at room temperature for exactly 30 min.
      15. Aspirate and wash the wells 6 times with Wash Buffer. Take care that all wells are completely filled and emptied at each wash.
      16. Blot the plate on paper towels to remove residual fluid from the plate.
      17. Add 100 µl of Substrate Solution to each well.
      18. Seal the plate with foil provided and incubate in the dark at room temperature for exactly 15 min.
      19. Stop the reaction by adding 100 µl of Stop Solution to each well.
      20. Read the plate at 450 nm (540, 570 or 620 nm reference filter) within 30 min. Note: Reading of the plate without reference may yield higher absorbances and thus may be less accurate.
      CalculationsCalculations and Evaluation of Results The calculation is illustrated using representative data: the assay data should be similar to that shown in Table 1. 1. Calculate the average absorbance for each set of standard wells. 2. A standard curve is generated by plotting the mean absorbance (y axis) against pg/ml standard (x-axis, Figure 1). 3. The pg/ml values of the samples can be read directly from the graph or calculated by the regression coefficients. 4. Multiply the calculated pg/ml values by the dilution factor of the samples.
      Assay characteristics and examples

      Table 1: Typical Assay Data


      Figure 1: Standard Curve

      Prepared with Assay Buffer.
      Sensitivity7 pg/ml
      Sensitivity NotesThe sensitivity, defined as two standard deviations above the concentration mean of the calculated concentrations of 48 blank replicates was determined. The sensitivity was determined as 7 pg/ml.
      Assay Range32 - 2000 pg/ml
      Precision

      Table 2: Intra-assay precision in Assay Buffer (Precision in Serum Standard Diluent is si

      Intra-assay precision was measured by assaying four control samples twenty times on one plate.


      Table 3: Inter-assay precision in Assay Buffer (Precision in Serum Standard Diluent is si

      The inter-assay variation was measured by assaying four control samples 16 times on different plates.


      Table 4: Precision Profile

      The precision profile was calculated as % CV from the mean and standard deviation of absorbance for each standard. The results for the determination in Assay Buffer are shown in Table 4.
      Recovery

      Table 5: Recovery

      The recovery of MMP-13 standard spiked to levels throughout the range of the assay in various matrix types diluted down to 1:10 is shown in Table 5.
      Linearity

      Table 6: Linearity

      The following samples were measured after dilution with Assay Buffer to assess linearity of the assay.
      SpecificityThe assay recognizes the activated form of MMP-13. It does not recognize the latent or activated forms of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, or the catalytic domain of MT-1 MMP, MT-2 MMP, MT-3 MMP, MT-4 MMP, or MT-5 MMP. High concentrations of latent MMP-13 may contribute to absorbance values measured in the assay. When present at equal concentrations, latent MMP-13 yields about 1/10 the absorbance value of active MMP-13.
      Protocol Summary

      Figure 2: Protocol Summary
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.