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QIA94 Cathepsin L ELISA Kit

Cathepsin L ELISA Kit: Ficha de datos de seguridad (MSDS o SDS), certificado de análisis y de calidad (CoA y CoQ), expedientes, folletos y otros documentos disponibles.
Detection Methods: Colorimetric
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      Descripción

      Replacement Information

      Tabla espec. clave

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.





      Can be used to measure cathepsin L in serum and cell culture supernatant.
      Catalogue NumberQIA94
      Brand Family Calbiochem®
      Application Data
      Using the Cathepsin L Standard provided with the assay and the Detailed Protocol outlined above, an example standard curve was generated.
      Materials Required but Not Delivered• 5 and 10 ml graduated pipettes
      • 10 to 1,000 µl adjustable single channel micropipettes with disposable tips
      • 50 to 300 µl adjustable multichannel micropipette with disposable tips
      • Multichannel micropipette reservoir
      • Beakers, flasks, and cylinders necessary for preparation of reagents
      • Multichannel wash bottle or automatic wash system for delivery of wash solution
      • Microwell strip reader capable of reading at 450 nm with 620 nm as optional reference wavelength
      • Glass-distilled or deionized water
      • Calculator with program to perform linear regression analysis
      References
      ReferencesSantamaria, I., et al. 1999. J. Biol. Chem. 274, 13800.
      Nakagawa, T., et al. 1998. Science 280, 450.
      Xing, R.Y., et al. 1998. Biochem. J. 332, 499.
      Kagegawa, H., et al. 1993. FEBS Lett. 321, 247.
      Chambers, A.F., et al. 1992. Mol. Carcinog. 5, 238.
      Chauhan, S.S., et al. 1991. Cancer Res. 51, 1478.
      Rozhin, J., et al. 1989. Biochem. Biophys. Res. Comm. 164, 556.
      Denhardt, D.T., et al. 1987. Oncogene 2, 55.
      Kirschke, H., et al. 1977. Eur. J. Biochem. 74, 293.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsCathepsin L Monoclonal Antibody Coated 96-Well Plate, Anti-Cathepsin L/Biotin Conjugate, Steptavidin HRP, Cathepsin L Standard, Wash Buffer Concentrate, Assay Buffer Concentrate, Sample Diluent, Substrate Solution, Stop Solution, Plate Covers, and a user protocol.
      Applications
      Biological Information
      Assay range3 - 50 ng/ml
      Assay time3.5 h
      Sample TypeHuman serum, or other biological fluids or cell culture supernatants
      Physicochemical Information
      Sensitivity≤1.7 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 36/37/38

      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® Cathepsin L ELISA Kit is an enzyme-linked immunosorbent assay that can be used to specifically quantitate human cathepsin L in body fluids like serum or in cell culture supernatants.
      Storage and Shipping Information
      Ship Code Blue 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 +2°C to +8°C
      Storage ConditionsUpon arrival, store the entire contents of the kit at 4°C. Following initial use, all components should continue to be stored at 4°C.
      Do not freeze Yes
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCathepsin L Monoclonal Antibody Coated 96-Well Plate, Anti-Cathepsin L/Biotin Conjugate, Steptavidin HRP, Cathepsin L Standard, Wash Buffer Concentrate, Assay Buffer Concentrate, Sample Diluent, Substrate Solution, Stop Solution, Plate Covers, and a user protocol.
      Specifications
      Global Trade ITEM Number
      Número de referencia GTIN
      QIA94 0

      Documentation

      Referencias bibliográficas

      Visión general referencias
      Santamaria, I., et al. 1999. J. Biol. Chem. 274, 13800.
      Nakagawa, T., et al. 1998. Science 280, 450.
      Xing, R.Y., et al. 1998. Biochem. J. 332, 499.
      Kagegawa, H., et al. 1993. FEBS Lett. 321, 247.
      Chambers, A.F., et al. 1992. Mol. Carcinog. 5, 238.
      Chauhan, S.S., et al. 1991. Cancer Res. 51, 1478.
      Rozhin, J., et al. 1989. Biochem. Biophys. Res. Comm. 164, 556.
      Denhardt, D.T., et al. 1987. Oncogene 2, 55.
      Kirschke, H., et al. 1977. Eur. J. Biochem. 74, 293.

      Folleto

      Cargo
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      Protocolo de usuario

      Revision03-Febuary-2011 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival, store the entire contents of the kit at 4°C. Following initial use, all components should continue to be stored at 4°C.
      Intended useThe Calbiochem® Cathepsin L ELISA Kit is an enzyme-linked immunosorbent assay that can be used to specifically quantitate human cathepsin L in body fluids like serum or in cell culture supernatants.
      BackgroundCathepsin L is a lysosomal endopeptidase belonging to the papain cysteine protease superfamily. Macrophages and osteoclasts secrete the precursor of cathepsin L, procathepsin L, which is processed to some form of active enzyme by acid, surface activation, or proteolysis. It is then involved in connective tissue degradation and the turnover of extracellular matrix proteins. Procathepsin L is also secreted by many malignantly transformed cells, mRNA, and its protein expression levels, as well as the extent to which it is secreted are correlated to the malignant potential of these cells. As cathepsin L is capable of degrading protein constituents of the extracellular matrix, it may play a crucial role in tumor progression, metastasis, and other disorders involving the destruction of the extracellular matrix. Inhibition of the enzyme or the proenzyme by low molecular weight inhibitors or antibodies in vitro and in vivo lead to a suppression of the invasiveness of malignant cells or a decline in their ability to form tumors.
      Materials provided• Cathepsin L Monoclonal Antibody Coated 96-Well Plate: 1 plate, 96 wells, pre-coated with monoclonal antibody specific for human cathepsin L
      • Biotin-Conjugate Concentrate: 1 vial, 100 µl, anti-cathepsin L antibody, contains preservative
      • Streptavidin-HRP: 1 vial, 200 µl
      • Cathepsin L Standard: 2 vials, lyophilized, 100 ng/ml upon reconstitution
      • 20X Wash buffer Concentrate: 1 bottle, 50 ml, PBS with 1% Tween®-20 detergent
      • 20X Assay Buffer Concentrate: 1 vial, 5 ml, PBS with 1% Tween®-20 detergent and 10% BSA
      • Sample Diluent: 1 vial, 12 ml, contains preservative
      • Substrate Solution: 1 vial, 15 ml
      • Stop Solution: 1 vial, 15 ml, 1 M phosphoric acid
      • Blue-dye: 1 vial, 400 µl
      • Green-dye: 1 vial, 400 µl
      • Red-dye: 1 vial, 400 µl
      • Adhesive Plate Cover: 4 plate covers
      Materials Required but not provided• 5 and 10 ml graduated pipettes
      • 10 to 1,000 µl adjustable single channel micropipettes with disposable tips
      • 50 to 300 µl adjustable multichannel micropipette with disposable tips
      • Multichannel micropipette reservoir
      • Beakers, flasks, and cylinders necessary for preparation of reagents
      • Multichannel wash bottle or automatic wash system for delivery of wash solution
      • Microwell strip reader capable of reading at 450 nm with 620 nm as optional reference wavelength
      • Glass-distilled or deionized water
      • Calculator with program to perform linear regression analysis
      Precautions and recommendations• In some, very rare cases, an insoluble precipitate of stabilizing protein has been seen in the sample diluent vial. This precipitate does not interfere in any way with the performance of the test and can thus be ignored.
      • Immediately after use remaining reagents should be stored at 4°C.
      • As conditions may vary from assay to assay, a standard curve must be established for every run.
      • Bacterial, fungal, or cross-contamination of either samples or reagents or may cause erroneous results.
      • While disposable pipette tips, flasks and glassware are preferred, reusable glassware must be thoroughly washed and rinsed before use.
      • Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty and fill wells as indicated for each wash cycle and do not allow wells to dry out at any time.
      • The limit of detection of cathepsin L defined as the analyte concentration resulting in an absorption significantly higher than that of the dilution medium (mean plus three standard deviations) was determined to be less than 2.6 ng/ml (mean of 6 independent assays).
      PreparationCell culture supernatant and serum are suitable samples for this assay. Remove serum from the clot as soon as possible after clotting. Samples containing visible precipitate must be clarified prior to performing the assay. Do not use grossly hemolyzed or lipemic samples. Samples should be aliquotted and frozen at -20°C. Samples can be stored at 2-8°C for up to 24 hr. Avoid freeze/thaw cycles. Samples should be thawed and warmed to room temperature prior to performing tha assay.
      Reagent preparationNote: Buffer concentrates should be warmed to room temperature and diluted prior to starting the assay. If buffer concentrate contains crystals, warm gently until dissolved.

      • 1X Wash Buffer: Add distilled or deionized water to 50 ml Wash buffer Concentrate for a final volume of 1,000 ml. Mix gently to avoid foaming and transfer to a clean wash bottle. Bacterial or fungal contamination of samples or reagents may cause false results. The pH of the final solution will be 7.4. 1X Wash Buffer is stable for 30 days at 2-25°C. Prepare the amount needed for the assay.
      • 1X Assay Buffer: Mix the contents of the Assay Buffer Concentrate and add to 95 ml distilled or deionized water. Mix gently to avoid foaming. 1X Assay Buffer is stable for 30 days at 4°C. Prepare the amount needed for the assay.
      • Biotin-Conjugate: Dilute the Biotin-Conjugate Concentrate 1:100 with 1X Assay Buffer in a clean plastic tube as needed.
      • Cathepsin L Standard: Reconstitute the Cathepsin L Standard with distilled water according to the reconstitution volume stated on the label of the standard vial. Dissolve the contents entirely by swirling gently.
      • Streptavidin-HRP: Dilute the concentrated Streptavidin-HRP 1:200 with 1X Assay buffer as needed.

      Mistakes in pipetting may be avoided by the use of distinctive colors in each step of the ELISA procedure. Dye solutions (Blue-dye, Green-dye, Red-dye) can be added to the reagents according to the following guidelines. (This technique is optional and does not interfere with the test results. It can therefore be omitted.) • Blue-dye: Prior to sample dilution, dilute the Blue-dye 1:250 in the Sample Diluent. • Green-dye: Prior to diluting the Biotin-Conjugate Concentrate (as outlined above), dilute the Green-dye 1:100 using the 1X Assay Buffer that is to be used for the final Biotin-Conjugate dilution. • Red-dye: Prior to diluting the concentrated Streptavidin-HRP, dilute the Red-dye 1:250 in the 1X Assay Buffer that is to be used for the final Streptavidin-HRP dilution.
      Detailed protocol1. Mix all reagents gently (to avoid foaming) prior to use.
      2. Determine the number of Microwell strips required to test the desired number of samples and the appropriate number of blanks and standards. Each sample, standard, blank, and optional control sample should be assayed in duplicate. Remove extra Microwell Strips coated with Antibody to human Cathepsin L from holder and store in sealed foil bag with the desiccant at 4°C.
      3. Wash the Microwell strips twice with approximately 400 µl 1X Wash buffer per well with thorough aspiration of microwell contents between washes. Please note that insufficient washing at any step in the assay will result in false results. Completely empty wells before each wash. Take care not to scratch the surface of the wells. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess buffer. Use the microwell strips immediately after washing or place upside down on a wet absorbent paper for not longer than 15 min. Do not allow wells to dry.
      4. Add 100 µl Sample Diluent, in duplicate, to all standard wells. Prepare standard dilutions by pipetting 100 µl reconstituted Cathepsin L Standard, in duplicate, into wells A1 and A2. Mix the contents of wells A1 and A2 by repeated aspiration and transfer 100 µl to wells B1 and B2, respectively. Take care not to scratch the inner surface of the microwells. Continue this procedure three times, creating two rows of Cathepsin L Standard dilutions ranging from 50 ng to 3.1 ng/ml. Discard 100 µl of the contents from the last wells used.
      5. Add 100 µl Sample Diluent in duplicate to the blank wells.
      6. Add 50 µl Sample Diluent, in duplicate, to the sample wells.
      7. Add 50 µl of each sample, in duplicate, to the designated wells.
      8. Add 50 µl diluted Biotin-Conjugate to all wells including blanks.
      9. Cover with a plate cover and incubate at room temperature (18-25°C) for 2 h on a rotator set at 100 rpm.
      10. Remove plate cover and empty wells. Wash Microwell strips 3 times as outlined above.
      11. Add 100 µl of diluted Streptavidin-HRP to all wells including blanks.
      12. Cover with a plate cover and incubate at room temperature (18-25°C) for 1 h on a rotator set at 100 rpm.
      13. Remove plate cover and empty wells. Wash Microwell strips 3 times as outlined above.
      14. Pipette 100 µl Substrate Solution to all wells including blanks.
      15. Incubate at room temperature (18-25°C) for approximately 10 min. Avoid direct exposure to intense light. The point at which the substrate reaction should be stopped may be determined by the ELISA reader being used. Many ELISA readers record absorbance only up to 2.0. Therefore the color development and the substrate reaction must be stopped before positive wells are no longer properly recordable.
      16. Stop the enzyme reaction by quickly pipetting 100 µl Stop solution into each well including the blanks. It is important that the Stop Solution be spread quickly and uniformly throughout the wells to completely inactivate the enzyme. Results must be read immediately after the Stop Solution is added or within 1 h if the Microwell strips are stored at 4°C in the dark.
      17. Read absorbance of each well on a spectrophotometer at 450 nm as the primary wave length (620 nm as the reference wave length, 610 nm to 650 nm is acceptable). Blank the plate reader using the blank wells. Determine the absorbance of the samples and the cathepsin L standards. Note: incubation without shaking may result in lower than expected abs values.
      Calculations1. Calculate the average absorbance values for each set of duplicate standards and samples.
      2. Create a standard curve by plotting the mean absorbance for each standard concentration on the ordinate against the cathepsin L concentration on the abscissa.
      3. Determine the concentration of circulating cathepsin L for each sample by finding the mean absorbance value on the ordinate and extending a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding cathepsin L concentration. Calculation of samples with an abs exceeding the range of the standard curve may result in incorrect/low cathepsin L levels. Such samples should be re-analyzed at higher dilution to accurately quantitate the cathepsin L level. Values obtained should be within the expected range of known cathepsin L controls.
      4. A sample standard curve is shown below.

      Figure 1: Cathepsin L Standard Curve

      Using the Cathepsin L Standard provided with the assay and the Detailed Protocol outlined above, an example standard curve was generated.
      Sensitivity≤1.7 ng/ml
      Assay Range3 - 50 ng/ml
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
      Tween® is a registered trademark of ICI Americas, Inc.