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QIA54 TIMP-1 ELISA Kit

QIA54
  
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      Overview

      Replacement Information

      Key Spec Table

      Species ReactivityDetection Methods
      HColorimetric
      Description
      Overview

      This product has been discontinued.



      A sandwich ELISA based kit specific for human TIMP-1.

      A non-isotopic colorimetricin vitroassay for the measurement of human TIMP-1 protein in tissue culture media or serum.
      Catalogue NumberQIA54
      Brand Family Calbiochem®
      Application Data
      Materials Required but Not Delivered 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips; 1 ml, 5 ml, 10 ml and 25 ml pipettes for reagent preparation.
      Wash bottle or multi-channel dispenser for washing.
      Deionized or distilled H2O.
      Graduated cylinder (500 ml)
      12 mm x 75 mm polypropylene test tubes.
      Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/540 nm or 450/595 nm.
      References
      ReferencesBorden, P., and Heller, R. A. 1997. Crit. Rev. Eukaryot. Gen. Expression 7, 159.
      Gomis-Ruth, F. X., et al. 1997. Nature (London) 389, 77.
      Cottam, D. W. and Rees, R. C. 1993. Intl J. Oncol. 2, 861.
      Fridman, R., et al. 1993. Biochem J. 289, 411.
      Stetler-Stevenson, W. G., et al. 1993. FASEB J. 7, 1434.
      Woessner, J. F. 1991. FASEB J. 5, 2145.
      Product Information
      Detection methodColorimetric
      DeclarationManufactured by Daiichi Fine Chemical Co., Ltd. Not available for sale in Japan.
      Form96 Tests
      Format96-well plate
      Kit contains96-Well Coated Plate, Human TIMP-1 Standard, Detector Antibody, Assay Buffers, Color Reagent, Stop Solution, Plate Cover, and a user protocol.
      Applications
      Biological Information
      Assay range0.05 - 1.6 ng/ml
      Assay time2 h
      Sample TypeTissue culture media and serum
      Species Reactivity
      • Human
      Physicochemical Information
      Sensitivity9.6 pg/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 35-36/37/38

      Causes severe burns.
      Irritating to eyes, respiratory system and skin.
      S PhraseS: 26-36-45

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Intended useThe Calbiochem® TIMP-1 ELISA Kit is a non-isotopic immunoassay for the in vitro quantitation of human TIMP-1 protein in tissue culture media and serum.
      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 of the kit at 4°C. 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, Human TIMP-1 Standard, Detector Antibody, Assay Buffers, Color Reagent, Stop Solution, Plate Cover, and a user protocol.
      Specifications

      Documentation

      TIMP-1 ELISA Kit Certificates of Analysis

      TitleLot Number
      QIA54

      References

      Reference overview
      Borden, P., and Heller, R. A. 1997. Crit. Rev. Eukaryot. Gen. Expression 7, 159.
      Gomis-Ruth, F. X., et al. 1997. Nature (London) 389, 77.
      Cottam, D. W. and Rees, R. C. 1993. Intl J. Oncol. 2, 861.
      Fridman, R., et al. 1993. Biochem J. 289, 411.
      Stetler-Stevenson, W. G., et al. 1993. FASEB J. 7, 1434.
      Woessner, J. F. 1991. FASEB J. 5, 2145.
      User Protocol

      Revision16-September-2010 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival store the entire contents of the kit at 4°C. Do not expose reagents to excessive light.
      Intended useThe Calbiochem® TIMP-1 ELISA Kit is a non-isotopic immunoassay for the in vitro quantitation of human TIMP-1 protein in tissue culture media and serum.
      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-1, a 184 amino acid glycoprotein of 28.5 kDa, exhibits a 41% sequence homology with the non-glycosylate 21.5 kDa TIMP-2. It is more widely distributed and inhibits the activity of all the active MMPs. Its binding to MMP-9 and MMP-1 occurs via a reversible non-covalent binding to the catalytic domain of the MMP protein. (Kd~10-10M). TIMP-1 is not cleaved by this binding and can be recovered with full activity from complexes with MMP-3. The activity of MMPs can also be inhibited by α2-macroglobulin. However, it has been shown that transfer of MMP-1 to α2-macroglobulin does not occur if MMP is already complexed with TIMP-1. In addition to its inhibitory function, TIMP-1 has been shown to have erythroid potentiating cell growth-promoting activities. 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.
      Principles of the assayThe TIMP-1 ELISA is a "sandwich" enzyme immunoassay employing two monoclonal antibodies. A monoclonal antibody, specific for human TIMP-1 protein, has been immobilized onto the surface of the plastic wells provided in the kit. The sample to be assayed (test samples and standards) are pipetted into the wells and any human TIMP-1 protein present binds to the capture antibody. Unbound material is washed away and a monoclonal, horseradish peroxidase (HRP)-conjugated anti-TIMP-1 antibody is added to the wells. Following this incubation and a wash step, a chromogenic substrate is added to the wells. The horseradish peroxidase catalyses 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 human TIMP-1 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-1 protein (provided lyophilized). By comparing the absorbance obtained from a sample containing an unknown amount of human TIMP-1 protein with that obtained from the standards, the concentration of human TIMP-1 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-1 ELISA contains one plate with sufficient reagents to run 96 tests (including standard curves).

      • COATED 96-WELL PLATE (Kit Component No. JA1681): 1 EA, 96 removable wells coated with mouse anti-bovine TIMP-1 monoclonal antibody.
      • TIMP-1 PROTEIN STANDARD (Kit Component No. JA1682): 1 EA, 1.6 ng of human TIMP-1 lyophilized with preservatives.
      • TIMP-1 CONJUGATE (Kit Component No. JA1683): 0.5 ml of anti-TIMP-1 monoclonal antibody conjugated to horseradish peroxidase (HRP). Dilute with 12 ml of diluted Wash/Assay Buffer.
      • WASH/ASSAY BUFFER (Kit Component No. JA1684): 20 ml of 25X sodium phosphate buffer containing BSA. The contents of the bottle will be reconstituted in 480 ml of deionized or distilled water.
      • COLOR REAGENT (Kit Component No. JA1608): 12 ml of the chromagenic substrate, tetra-methylbenzidine (TMB). This reagent is light sensitive and should be protected from direct sunlight or UV sources.
      • STOP SOLUTION (Kit Component No. JA1616): 12 ml, 2.5N sulfuric acid
      • PLATE SEALER: 1 lid to cover plate during incubations.
      Materials Required but not provided 2-20 µl, 20-200 µl and 200-1000 µl precision pipetters with disposable tips; 1 ml, 5 ml, 10 ml and 25 ml pipettes for reagent preparation.
      Wash bottle or multi-channel dispenser for washing.
      Deionized or distilled H2O.
      Graduated cylinder (500 ml)
      12 mm x 75 mm polypropylene test tubes.
      Spectrophotometer capable of measuring absorbance in 96-well plates using dual wavelength of 450/540 nm or 450/595 nm.
      Precautions and recommendations Store unopened kit at -20°C. Do not expose reagents to excessive light.
      Do not use metallic labware with the enclosed reagents. Color Reagent may react with the metal labware.
      Bring all reagents to room temperature before use. Gently stir the reagents prior to use.
      Wear disposable gloves and eye protection.
      The Stop Solution is an acid solution. Exercise caution when handling this solution.
      Use only the wells provided with the kit.
      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 which have been diluted, the TIMP-1 protein concentration must be multiplied by the dilution factor (e.g. if samples are diluted five fold, then the TIMP-1 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.
      PreparationCell 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. Serum: Use a serum separator tube (SST) and allow blood samples to clot for 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. Samples found to contain greater than 1.6 ng/ml TIMP-1 (i.e., outside the range of the standard curve) must be diluted with Wash/Assay Buffer (provided), so that the TIMP-1 concentration falls within the range spanned by the standard curve, and assayed again.
      Reagent preparationNote: Bring all reagents to room temperature before use.

      • Wash/Assay Buffer: Warm the Wash/Assay Buffer Concentrate to room temperature before use (this will dissolve any crystals, which may be present in the concentrate). Mix 20 ml of Wash / Assay Buffer Concentrate with 480 ml of deionized or distilled water to provide enough 1X Wash/Assay buffer (500 ml) for one plate. The diluted Wash/Assay Buffer may be stored at 4°C for up to 1 month (provided this is still within the shelf life stated on the kit).

      • Color Reagent: Ready to use

      • Stop Solution: Ready to use

      • TIMP-1 Conjugate: Add 12 ml of Wash / Assay Buffer to the TIMP-1 Conjugate. The diluted TIMP-1 Conjugate may be stored at 4°C for up to 1 week (provided this is still within the shelf life stated on the kit).

      • TIMP-1 Standard: Reconstitute the TIMP-1 Standard with 1 ml of Wash/Assay Buffer and allow to mix gently for 15 min before making serial dilutions. This reconstitution makes a stock solution of 1.6 ng/ml. Prepare serially diluted standards just before use as follows:

      • TIMP-1 Standard Dilutions: Note: Use polypropylene tubes. To make serial dilutions of the standard, obtain 5 tubes and label them 0.8, 0.4, 0.2, 0.1, 0.05 ng/ml. Add 250 µl of Wash/Assay Buffer into each tube. The undiluted standard (1.6 ng/ml) serves as the high point of the standard curve. Wash / Assay Buffer alone acts as the zero standard (0 ng/ml). Remove 250 µl from the undiluted, reconstituted standard vial (1.6 ng/ml) and add it to the first tube (0.8 ng/ml). Mix gently before the next transfer. Remove 250 µl from the first tube (0.8 ng/ml) and add it to the second tube (0.4 ng/ml) and mix gently. Repeat this procedure until you reach the fifth tube (0.05 ng/ml). Any remaining undiluted standard (1.6 ng/ml) may be stored for 1 week at 4°C. Serially diluted standards should be discarded after one use.
      Detailed protocolThe TIMP-1 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 room temperature before use.
      1. Prepare all samples, controls, standards and reagents as described in the previous sections.
      2. Remove the appropriate number of wells from the pouch and place them into the empty well holder. Return any unused wells to the original pouch, refold, seal and store at 4°C. Unused wells may be stored at 4°C for up to 1 week (provided this is still within the shelf life stated on the kit).
      3. Pipette 100 µl of Standard or Sample into each well.
      4. Cover the plate with the lid provided and incubate the plate at room temperature for 1 h without shaking.
      5. Aspirate each well and wash wells 4 times with 1X Wash/Assay Buffer making sure each well is filled completely. Each well is washed by filling with 400 µl of 1X Wash/Assay Buffer (using a multichannel pipette, automatic plate washer or wash bottle). It is essential to completely remove the Wash/Assay Buffer after each step and to ensure that the wells do not contain any buffer after the last wash. This can be achieved by inverting the plate and tapping it on paper towels.
      6. Pipette 100 µl of the TIMP-1 Conjugate into each well. Cover the plate with the lid provided and incubate the plate at room temperature for 30 minutes without shaking.
      7. Aspirate each well and wash wells 4 times with 1X Wash/Assay Buffer making sure each well is filled completely, following procedure as outlined in Step 5.
      8. Pipette 100 µl of Color Reagent into each well. Cover the plate with the lid provided and incubate the plate in the dark at room temperature for 30 min without shaking.
      9. Stop the reaction by adding 100 µl of Stop Solution (2.5 N Sulfuric Acid) into each well in the same order as the previously added Color Reagent. If color change does not appear uniform in the well, gently tap the plate frame to ensure thorough mixing.
      10. Measure absorbance in each well using a spectrophotometric plate reader at dual wavelengths of 450/595 nm (540 nm can be used as an alternative reference wavelength). If wavelength reference is not available, subtract the readings at 595 nm or 540 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate. Wells must be read within 30 min of adding the Stop Solution.

      Note: Samples, which give values higher than the highest standard should be diluted with the Wash/Assay Buffer and re-evaluated in the assay.
      CalculationsEvaluation of Results

      1. Average the duplicate absorbance values for the standards (including the zero) and all samples. Subtract the average zero standard absorbance value from each averaged standard and sample value.
      2. On graph paper, plot the mean absorbance values (Abs) for each of the standards on the Y-axis, versus the concentration of each standard (ng/ml) on the X-axis. Alternatively, the data may be linearized by plotting the log of TIMP-1 concentration versus the log of the Abs and the best fit line can be determined by regression analysis.
      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 simplify this process. Interpolation of the sample values from the standard curve will represent TIMP-1 concentrations since TIMP-1 protein is used as the standard for this ELISA.
      4. For samples which have been diluted, the TIMP-1 protein concentration must be multiplied by the dilution factor (e.g. if samples are diluted five fold, then the TIMP-1 protein concentration value obtained from the standard curve must be multiplied by five).
      Standard curveA typical standard curve generated by this kit is shown below. The standard curve is for demonstration only and should not be used for data extrapolation purposes. Data should be extrapolated from standard curves run within, and in conjunction with the test samples. A standard curve should be run in duplicate each time an assay is performed.

      Figure 1: TIMP-1 ELISA: Sample Standard Curve

      Example data

      Table 1: TIMP-1 levels in various samples

      Sensitivity9.6 pg/ml
      Sensitivity NotesThe minimum dose of TIMP-1 protein as detectable in this assay is typically less than 0.0096 ng/ml. This value is calculated by adding two standard deviations to the mean absorbance of 30 zero standard replicates and calculating the corresponding concentration.
      Assay Range0.05 - 1.6 ng/ml
      Precision

      Table 2: Intra-Assay Precision

      This reflects the precision within an assay. Samples containing three known concentrations of TIMP-1 protein were analyzed 12 times each, in replicates of two in a single assay to assess intra-assay precision.


      Table 3: Inter-Assay Precision

      This reflects the precision between assays. Repeated measurements of serum samples containing three known concentrations of TIMP-1 protein were analyzed in replicates of two in ten separate assays to assess inter-assay precision.

      RecoveryThe average % recovery of TIMP-1 protein, spiked to three levels in one serum and one cell culture media sample, throughout the range of the assay in various matrices was evaluated.

      Table 4: Recovery

      LinearityThe assay linearity was assessed using one serum and one cell culture media sample spiked with known concentrations of TIMP-1 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 5: Linearity

      SpecificityAssay specificity was measured by evaluating the cross-reactivity of TIMP-1 ELISA to TIMP-2 protein.

      Table 6: Specificity

      This assay system recognizes TIMP-1 but not TIMP-2.

      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.