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QIA40 TIMP-2 ELISA Kit

TIMP-2 ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Detection Methods: Colorimetric
QIA40
  
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      Overview

      Replacement Information

      Key Spec Table

      Species ReactivityDetection Methods
      B, Gp, H, M, Rb, RColorimetric
      Description
      Overview

      This product has been discontinued.



      A sandwich ELISA based kit specific for human TIMP-2.
      A non-isotopic immunoassay for thein vitromeasurement of human TIMP-2 protein in tissue culture medium, serum, plasma and tissue lysates.
      Catalogue NumberQIA40
      Brand Family Calbiochem®
      Application Data
      Materials Required but Not Delivered• 2-20 µl, 20-200 µl and 200-1000 µl precision pipettors with disposable tips; 1 ml, 5 ml, and 10 ml pipettes for reagent preparation.
      • Wash bottle or multi-channel dispenser for washing.
      • Deionized or distilled H2O.
      • Graduated cylinder (100 ml and 500 ml)
      • Disposable polypropylene test tubes.
      • Spectrophotometer capable of measuring absorbance at 630 nm or 450 nm.
      • 1.0 M sulfuric acid
      • Automatic plate washer or wash bottle
      References
      ReferencesFujimoto, N., et al. 1995. J. Immunol. Methods 187, 33.
      Baker, T., et al. 1994. Br. J. Cancer 70, 506.
      Martel-Pelletier, J., et al. 1994. Lab. Invest. 70, 807.
      Hayakawa, T., et al. 1994. J. Cell Sci. 107, 2373.
      Hayakawa, T., et al. 1994. Cell Struct. Funct. 19, 109.
      Wilde, C.G., et al. 1994. DNA Cell Biol. 13, 711.
      Birkedal-Hansen, H., et al. 1993. Oral Biol. Med. 4, 250.
      Fujimoto, N., et al. 1993. Clin. Chim. Acta 220, 31.
      Stetler-Stevenson, W. G., et al. 1993. FASEB J. 7, 1434.
      Stetler-Stevenson, W. G., et al. 1992. FEBS J. 296, 231.
      Kolkenbrock, H., et al. 1991. Eur. J. Biochem. 198, 775.
      Stetler-Stevenson, W. G., et al. 1989. J. Biol. Chem. 264, 17374.
      Cawston, T.E., et al. 1981. Biochem. J. 195, 159.
      Product Information
      Detection methodColorimetric
      DeclarationNot available for sale in Japan.
      Form96 Tests
      Format96-well plate
      Kit contains96-Well Coated Plate, TIMP-2 Standard, HRP Conjugate, Assay Buffers, Color Reagent, Plate Sealers, and a user protocol.
      Applications
      Biological Information
      Assay range8 - 128 ng/ml
      Assay time4.5 h
      Sample TypeTissue culture medium, serum, plasma, and tissue lysates
      Species Reactivity
      • Bovine
      • Guinea Pig
      • Human
      • Mouse
      • Rabbit
      • Rat
      Physicochemical Information
      Sensitivity3 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 20/21-36-61

      Harmful by inhalation and in contact with skin.
      Irritating to eyes.
      May cause harm to the unborn child.
      S PhraseS: 45-53

      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Avoid exposure - obtain special instructions before use.
      Product Usage Statements
      Intended useThe Calbiochemreg; TIMP-2 ELISA is a non-isotopic immunoassay for the in vitro quantitation of TIMP-2 protein in serum, plasma, tissue samples, and cell culture supernatant. The ELISA system has been developed to measure the free form of TIMP-2 and the complexed form of TIMP-2 with active MMPs, but does not detect the complexed form with proMMP2. This assay will detect TIMP-2 from human, mouse, rat, guinea pig, rabbit, and bovine.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage -20°C
      Storage ConditionsUpon receipt store unopened kit at -20°C. Avoid freeze/thaw cycles of solutions. Protect from light.
      Protect from Light Protect from light
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit contains96-Well Coated Plate, TIMP-2 Standard, HRP Conjugate, Assay Buffers, Color Reagent, Plate Sealers, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      QIA40 0

      Documentation

      TIMP-2 ELISA Kit Certificates of Analysis

      TitleLot Number
      QIA40

      References

      Reference overview
      Fujimoto, N., et al. 1995. J. Immunol. Methods 187, 33.
      Baker, T., et al. 1994. Br. J. Cancer 70, 506.
      Martel-Pelletier, J., et al. 1994. Lab. Invest. 70, 807.
      Hayakawa, T., et al. 1994. J. Cell Sci. 107, 2373.
      Hayakawa, T., et al. 1994. Cell Struct. Funct. 19, 109.
      Wilde, C.G., et al. 1994. DNA Cell Biol. 13, 711.
      Birkedal-Hansen, H., et al. 1993. Oral Biol. Med. 4, 250.
      Fujimoto, N., et al. 1993. Clin. Chim. Acta 220, 31.
      Stetler-Stevenson, W. G., et al. 1993. FASEB J. 7, 1434.
      Stetler-Stevenson, W. G., et al. 1992. FEBS J. 296, 231.
      Kolkenbrock, H., et al. 1991. Eur. J. Biochem. 198, 775.
      Stetler-Stevenson, W. G., et al. 1989. J. Biol. Chem. 264, 17374.
      Cawston, T.E., et al. 1981. Biochem. J. 195, 159.
      User Protocol

      Revision15-August-2008 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Speciesbovine, guinea pig, human, mouse, rabbit, rat
      StorageUpon receipt store unopened kit at -20°C. Avoid freeze/thaw cycles of solutions. Protect from light.
      Intended useThe Calbiochemreg; TIMP-2 ELISA is a non-isotopic immunoassay for the in vitro quantitation of TIMP-2 protein in serum, plasma, tissue samples, and cell culture supernatant. The ELISA system has been developed to measure the free form of TIMP-2 and the complexed form of TIMP-2 with active MMPs, but does not detect the complexed form with proMMP2. This assay will detect TIMP-2 from human, mouse, rat, guinea pig, rabbit, and bovine.
      BackgroundMatrix metalloproteinases (MMPs) are a family of enzymes that are responsible for the degradation of extracellular matrix components such as collagen, laminin and proteoglycans. In addition to sequence homology, all MMPs share the following characteristics: the catalytic mechanism is dependent upon a zinc ion at the active center, they cleave one or more extracellular matrix components, they are secreted as zymogens which are activated by removal of an ~10 kDa segment from the N-terminus and their activity is regulated by endogenous inhibitors. These enzymes are involved in normal physiological processes such as embryogenesis and tissue remodeling and may play an important role in arthritis, periodontitis, and metastasis. 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. TIMP-2, a 194 amino acid unglycosylated protein of 21 kDa, exhibits a 43% and 44% amino acid sequence homology with TIMP-1 and TIMP-3, respectively. TIMP-2 inhibits the activity of all active MMPs and regulates the activation of proMMP-2by binding to the C-terminal region of proMMP-2. In addition to its inhibitory function, TIMP-2 has also been shown to have erythroid potentiating activity and cell growth-promoting activity. However, TIMP-2 complexed with proMMP-2 has no cell growth-promoting activity at all. TIMPs are known to inhibit invasion and metastasis in animal models and, hence, are capable of altering the metastatic potential of cancer cells. The balance between the level of active MMPs and available TIMPs determines the net MMP activity and is therefore a pivotal determinant of ECM turnover. This balance among MMPs and TIMPs is thought to be important in several disease states including arthritis, cancer, tumor invasion, and metastasis.
      Principles of the assayThe TIMP-2 ELISA is a "sandwich" enzyme immunoassay employing two monoclonal antibodies. A monoclonal antibody, specific for human TIMP-2 protein, has been immobilized onto the surface of the plastic wells provided in the kit. The sample to be assayed (test samples and standards) is mixed with TIMP-2 conjugate (a second anti-TIMP-2 monoclonal antibody conjugated to horseradish peroxidase) and this mixture is added to the anti-TIMP-2 coated well. Following this incubation and a wash step, TMB is added to the wells. The horseradish peroxidase catalyses the conversion of TMB from a colorless solution to a blue solution (or yellow after the addition of an acid stopping reagent), the intensity of which is proportional to the amount of human TIMP-2 protein in the test sample. The colored reaction product is quantified using a spectrophotometer.

      Quantitation is achieved by the construction of a standard curve using known concentrations of human TIMP-2 protein (provided lyophilized). By comparing the absorbance obtained from a sample containing an unknown amount of human TIMP-2 protein with that obtained from the standards, the concentration of human TIMP-2 protein in the test 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 TIMP-2 ELISA contains one plate with sufficient reagents to run 96 tests (including standard curves). 96 tests are sufficient to construct a standard curve plus the measurement of 42 samples in duplicate.

      • COATED 96-WELL PLATE (kit Component No. JA8118-1EA): 12 x 8 well strips coated with mouse anti-TIMP-2 monoclonal antibody. A lid is also provided.
      • TIMP-2 PROTEIN STANDARD (Kit Component No. JA8119-1EA): 256 ng of lyophilized human TIMP-2 lyophilized with preservatives. Upon reconstitution with 1 ml of distilled water, this is 256 ng/ml TIMP-2 in 0.03 M phosphate buffer, pH 7.0, containing 0.1 M NaCl, 0.3% BSA, and 10 mM EDTA.
      • TIMP-2 CONJUGATE (Kit Component JA8120-1EA): Lyophilized anti-TIMP-2 monoclonal antibody conjugated to horseradish peroxidase (HRP). Upon reconstitution with 12 ml of distilled water, the buffer is 0.03 M phosphate buffer, pH 7.0, containing 0.1 M NaCl, 0.3% BSA, and 10 mM EDTA.
      • ASSAY BUFFER (Kit Component JA8121-10ML): 10 ml of phosphate buffer concentrate that upon dilution gives 0.03 M phosphate buffer, pH 7.0 containing 0.1 M NaCl, 0.3% BSA, and 10 mM EDTA.
      • WASH BUFFER (Kit Component JA8122-12.5ML): 12.5 ml of buffer concentrate which upon dilution gives a 10 mM phosphate buffer, pH 7.5, containing 50 mM NaCl and 0.05% Tween®-20 detergent.
      • COLOR REAGENT (Kit Component (JA8123-1EA): Contains the ready-to-use chromogenic substrate, tetra-methylbenzidine (TMB)/hydrogen peroxide in 20% dimethylformamide. This reagent is light sensitive and should be protected from direct sunlight or UV sources.
      Materials Required but not provided• 2-20 µl, 20-200 µl and 200-1000 µl precision pipettors with disposable tips; 1 ml, 5 ml, and 10 ml pipettes for reagent preparation.
      • Wash bottle or multi-channel dispenser for washing.
      • Deionized or distilled H2O.
      • Graduated cylinder (100 ml and 500 ml)
      • Disposable polypropylene test tubes.
      • Spectrophotometer capable of measuring absorbance at 630 nm or 450 nm.
      • 1.0 M sulfuric acid
      • Automatic plate washer or wash bottle
      Precautions and recommendations• Store unopened kit at -20°C. Do not expose reagents to excessive light.
      • Do not mix reagents from different kits.
      • Do not use metallic labware with the enclosed reagents. Color Reagent may react with the metal labware.
      • The incubation temperature range is critical (20°C-27°C).
      • Allow samples and all reagents to reach 20°C-27°C prior to performing assay.
      • Incubation times must be carried out exactly. If more than one plate is being assayed, each plate must be timed individually.
      • The total dispensing time for each plate should not exceed 20 min.
      • Standards and samples should be assayed in duplicate. A separate standard curve must be run on each plate.
      • Wear disposable gloves and eye protection.
      • The Stop Solution is an acid solution. Exercise caution when handling this corrosive solution.
      • Use only the wells provided with the kit.
      • Mix all samples and reagents thoroughly before use. Avoid excessive foaming of reagents.
      • Keep the wells covered with lids except when adding reagents and reading the plate.
      • Do not make direct contact with kit buffers and reagents. Do not mouth pipette or ingest any of the reagents.
      • Do not smoke, eat, or drink when performing the assay or in areas where samples/reagents are handled.
      • Samples may be contaminated with infectious agents. Do not ingest, expose to open wounds, or breathe aerosols. Dispose of samples properly.
      • It is recommended that samples falling above the range of the standard curve be diluted to fall within the mid-range of the curve. For samples that have been diluted, the TIMP-2 protein concentration must be multiplied by the dilution factor (e.g. if samples are diluted five fold, then the TIMP-2 protein concentration value obtained from the standard curve must be multiplied by five).
      • Do not use samples that have gone through multiple freeze/thaw cycles. If samples are to be stored at -20°C, it is recommended to divide samples into working aliquots and store at -20°C.
      • Avoid adding reagents to the side of the wells. Add samples directly to the center of the well.
      • Add Stop Solution in same well order as the Substrate Solution.
      • Change pipette tips between different sample additions to avoid cross-contamination.
      Preparation• Serum: Use a serum separator tube (SST) and allow blood samples to clot for at least 30 min. Once clotted, samples are centrifuged at 1000 x g for 10 min. Carefully remove serum and assay immediately or aliquot and store serum samples at ≤-20°C. Avoid freeze/thaw cycles. Note: Before assay, it is recommended to dilute serum samples 1:4 (4-fold dilution) or more with Assay Buffer (provided), so that the TIMP-2 concentration falls within the range spanned by the standard curve. • Plasma: Collect plasma using EDTA or citrate as an anticoagulant. Centrifuge at 1000 x g for 10 min and remove plasma. Assay immediately or aliquot and store plasma samples at ≤-20°C. Avoid freeze/thaw cycles. Note: Before assay, it is recommended to dilute plasma samples 1:4 (4-fold dilution) or more with Assay Buffer (provided), so that the TIMP-2 concentration falls within the range spanned by the standard curve. • Cell Culture Medium: Centrifuge all samples to remove particulate material before assaying. Assay immediately or aliquot and store samples at ≤20°C. Avoid freeze/thaw cycles. It may be necessary to dilute the samples with Assay Buffer (provided) if the levels of TIMP-2 are high. Tissue Samples: Users are advised to carefully validate any tissue extraction procedure employed. The following method is described for the extraction of placental TIMP-212. • Homogenize in 20 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl, 1 mM CaCl2, and 0.005% Brij™35 detergent at 4°C using 2 ml buffer per 1 g tissue. • Centrifuge at 18,000 x g at 4°C for 50 min. • Assay the supernatant for TIMP-2.
      Reagent preparationNote: Bring all reagents to 20°C-27°C before use. For convenience, the assay buffer may be thawed out overnight at 4°C before use. Either distilled or deionized water may be used for buffer, standard, and peroxidase conjugate preparation. The plate and enzyme substrate are supplied ready for use when equilibrated to 20°C-27°C. Once reconstituted, these components should be stored at 4°C and re-used within 7 days. • Assay Buffer: Warm the Assay Buffer Concentrate to room temperature before use (this will dissolve any crystals, which may be present in the concentrate). Transfer the contents of the bottle to a 100 ml cylinder by repeated washing with distilled water. Adjust the final volume to 100 ml with distilled water and mix thoroughly. Note: Solution may be cloudy on storage. This will disappear on dilution and does not affect the performance of the assay. • Stop Solution: 1 M sulfuric acid • TIMP-2 Conjugate: Add 12 ml of distilled water to the TIMP-2 Conjugate and replace the stopper. Gently mix until the contents are completely dissolved. Vigorous agitation and foaming should be avoided. • TIMP-2 Standard: Reconstitute the TIMP-2 Standard with 1 ml of distilled water and replace the stopper. Mix gently for 15 min until the contents are completely dissolved. Vigorous agitation and foaming should be avoided. • Wash Buffer: Warm the Wash Buffer Concentrate to room temperature before use (this will dissolve any crystals, which may be present in the concentrate). Transfer the contents of the bottle to a 500 ml cylinder by repeated washing with distilled water. Adjust the final volume to 500 ml with distilled water and mix thoroughly. Note: Solution may be cloudy on storage. This will disappear on dilution and does not affect the performance of the assay. Preparation of Working Standards TIMP-2 Standard Dilutions: Note: It is important to use a clean polypropylene pipette tip for each dilution. 1. Label 5 polypropylene tubes for 8, 19, 32, 64, and 128 ng/ml. 2. Pipette 500 µl of assay buffer into each tube. 3. Pipette 500 µl of the stock standard (256 ng/ml) into the 128 ng/ml tube. Vortex. 4. Pipette 500 µl from the 128 ng/ml tube into the 64 ng/ml tube. Vortex. 5. Repeat this doubling dilution step successively with the remaining tubes. 6. 100 µl aliquots from each serial dilution will give rise to 5 standard levels of TIMP-2 ranging from 8-128 ng/ml. Note: The stock solution (256 ng/ml) is NOT part of the standard curve. This may be stored at 4°C if running only part of a plate. Working standards should be prepared immediately before each assay and should not be reused.
      Detailed protocolThe TIMP-2 ELISA is provided with removable wells so the assay can be carried out on separate occasions. Since conditions may vary, a standard curve MUST be determined each time the assay is performed. Standards and test samples 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.

      Note: Bring all reagents to 20-27°C before use. This is especially important for the enzyme substrate TMB.
      Also note: Standards and samples are pre-mixed with conjugate in appropriate tubes prior to adding to the coated wells of the plate. See step 4.
      As an alternative protocol, users may wish to add 50 µl of standard/sample and 50 µl of conjugate directly to the wells. If so, omit step 4 and modify steps 5-9 below as appropriate. Although we have found little difference in results with this alternate protocol, users should properly validate the use of this method with their samples if they choose to use it. We do not routinely QC our assay by this method.
      1. Prepare all samples, controls, standards, and reagents as described in "Reagent Preparation".
      2. Prepare the working standards as described in the previous section.
      3. Set up the plate with sufficient wells for running all zero (blanks), standards, and samples, as required.
      4. Prepare a sufficient number of appropriately labeled polypropylene test tubes for mixing all zero (blanks), standards, and samples as required.
      5. Pipette 100 µl of assay buffer into the zero standard tubes.
      6. Pipette 100 µl of each standard into the appropriate tubes, using a clean polypropylene pipette tip for each standard.
      7. Pipette 100 µl of unknown sample into the appropriate tubes.
      8. Add 100 µl of TIMP-2 Conjugate into all tubes containing zero (blanks), standards, and samples. Vortex.
      9. Pipette 100 µl of the mixture of standards or samples and peroxidase conjugate into the appropriate wells.
      10. Cover the plate with the lid provided and incubate at 20-27°C for exactly 2 h.
      11. Aspirate each well and wash wells 4 times with 1X Wash Buffer. Each well is washed by filling with 400 µl of 1X Wash Buffer (using a multichannel pipette, automatic plate washer or wash bottle). It is essential to completely remove the Wash Buffer after each step and to ensure that the wells do not contain any buffer after the last wash.
      12. Blot the plate on tissue paper to remove residual solution in the wells.
      13. Immediately dispense 100 µl of room-temperature equilibrated Color Reagent into all wells.
      14. Cover the plate with the lid provided and stand for exactly 30 min at room temperature (20-27°C).
      15. The blue color that develops may be read spectrophotometrically at 630 nm.
      16. For greater sensitivity, add 100 µl of stop solution (1 M sulfuric acid) to all wells in the same order as the previously added Color Reagent and read the plate at 450 nm within 30 min.
      17. If the absorbance of the top standard is too high (e.g., >3.0), dilute it further with more 1 M sulfuric acid and read the absorbance of the color again. Do not allow greater than 30 min before reading the absorbance as the yellow color begins to fade.
      Calculations1. Calculate the average absorbance for each set of standard wells. 2. A standard curve is generated by plotting the mean optical density (y axis) against ng/ml standard (x axis). The curve shape should be similar to figure 1. The ng/ml value can be read directly from the graph. 3. For samples that have been diluted, the TIMP-2 protein concentration must be multiplied by the dilution factor (e.g. if samples are diluted five fold, then the TIMP-2 protein concentration value obtained from the standard curve must be multiplied by five).

      Figure 1: Standard Curve

      Table 1: Typical Assay Data
      Sensitivity3 ng/ml
      Sensitivity NotesThe sensitivity, defined as two standard deviations above the mean optical density of 20 zero standard replicates was determined. The corresponding concentration was calculated from a standard curve. The mean zero and standard values were then used to calculate the sensitivity.
      Assay Range8 - 128 ng/ml
      Precision

      Table 2: Intra-Assay Precision

      This reflects the precision within an assay (mean values as ng/ml).


      Table 3: Inter-Assay Precision

      This reflects the precision between assays (mean values as ng/ml).
      RecoveryThe average % recovery of TIMP-2 protein, spiked to levels throughout the range of the assay in various sample types diluted down to 1:4 (plasma, serum, and tissue samples) are shown:

      Table 4: Recovery

      The average % recovery of TIMP-2 protein, spiked to levels throughout the range of the assay in various sample types diluted down to 1:4 (plasma, serum, and tissue samples) are shown above.
      ReproducibilityLevels in normal human plasma and serum samples evaluated in the assay.

      Table 5: Expected Values

      Levels in normal human plasma and serum samples evaluated in the assay.
      LinearityThe assay linearity was assessed using one serum and one cell culture media sample spiked with known concentrations of TIMP-2 protein in various matrices and diluted with Assay/Wash Buffer. The samples generated values, which fell within the dynamic range of the assay. The linearity assay results are shown below:

      Table 6: Linearity

      The assay linearity was assessed using one serum and one cell culture media sample spiked with known concentrations of TIMP-2 protein in various matrices and diluted with Assay/Wash Buffer. The samples generated values, which fell within the dynamic range of the assay. The linearity assay results are shown abve.
      SpecificityThe assay recognizes free TIMP-2 and TIMP-2 complexed with the active form of MMPs but not TIMP-2 complexed with the precursor of MMP-2 (proMMP-2). The immunoreactivity is shown below.

      Table 7: Specificity


      The cross-reactivity with other human TIMPs was tested. The results are shown below (values as Abs):

      Table 8: Specificity

      The assay can detect TIMP-2 in other species including mouse, rat, guinea pig, rabbit, and bovine. The immunoreactivity between human and rat purified TIMP-2 is equivalent.
      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.