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CBA053 COX-2 ELISA Kit

COX-2 ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms:  Cyclooxygenase-2 ELISA Kit

Detection Methods: Colorimetric
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CBA053
  
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      Overview

      Replacement Information

      Key Specifications Table

      Species ReactivityDetection Methods
      HColorimetric
      Description
      Overview

      This product has been discontinued.



      This kit detects and quantifies human COX-2 in cell lysates and tissue extracts. Cyclooxygenase (COX) enzymes catalyze the synthesis of prostaglandins from arachidonic acid. COX-2 expression induced by various inflammatory and/or mitogenic stimuli such as cytokines, growth factors or tumor promoters.
      Catalogue NumberCBA053
      Brand Family Calbiochem®
      Synonyms Cyclooxygenase-2 ELISA Kit
      Application Data
      Example standard curve using the COX-2 Standard. A standard curve was generated according to the COX-2 Standard preparation and the Detailed Protocol above. The results represent a typical standard curve. Assay range: 2.5-200 ng/ml. Lower limit of detection: ~2 ng/ml.
      Materials Required but Not Delivered• Precision pipettors with disposable tips
      • Wash bottle or multi-channel dispenser for washing
      • COX-2 inducer (see Preparation of Samples below)
      • CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) or equivalent cell lysis buffer, preferably with protease inhibitors (e.g. Protease Inhibitor Cocktail Set III, Cat. No. 539134)
      • Protein assay (e.g. Non-Interfering Protein Assay™, Cat. No. 488250 or BCA Protein Assay Kit, Cat. No. 71285)
      • Spectrophotometer capable of measuring absorbance in 96-well plates at 450 nm, preferably with a reference wavelength of 540-600 nm
      References
      ReferencesBlanke, S.R., et al. 2005. Semin. Oncol. 32, 69.
      Dannenberg, A.J., et al. 2005. J. Clin. Oncol. 23, 254.
      Sanborn, R. and Blake, C.D., 2005. Semin. Oncol. 32, 69.
      Kim, H.J., et al. 2003. Int. J. Oncol. 22, 99.
      Shigemasa, K., et al. 2003. Int. J. Oncol. 22, 99.
      El-Bayoumy, K., et al. 2002. Int. J. Oncol. 20, 557.
      Hwang, D., et al. 1998. J. Natl. Cancer Inst. 90, 455.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsAnti-COX-2 Coated 96-Well Plate, COX-2 Standard, COX-2 Detector Antibody, HRP Conjugate, Assay Diluent, ELISA 20X Plate Wash Concentrate, TMB Substrate, ELISA Stop Solution, Plate Sealers, and a user protocol
      Positive controlU937 cells
      Applications
      Biological Information
      Assay range2.5-200 ng/ml
      Sample TypeCell lysate, tissue extract
      Species Reactivity
      • Human
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® COX-2 ELISA Kit is intended for the quantitative measurement of COX-2 in human cell lysates. Serum and plasma samples are not recommended due to inherently low concentrations of COX-2 in biological fluids.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage -20°C
      Storage ConditionsUpon arrival store the entire contents of the kit at -20°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsAnti-COX-2 Coated 96-Well Plate, COX-2 Standard, COX-2 Detector Antibody, HRP Conjugate, Assay Diluent, ELISA 20X Plate Wash Concentrate, TMB Substrate, ELISA Stop Solution, Plate Sealers, and a user protocol
      Specifications
      Global Trade Item Number
      Catalog Number GTIN
      CBA053 0

      Documentation

      COX-2 ELISA Kit Certificates of Analysis

      TitleLot Number
      CBA053

      References

      Reference overview
      Blanke, S.R., et al. 2005. Semin. Oncol. 32, 69.
      Dannenberg, A.J., et al. 2005. J. Clin. Oncol. 23, 254.
      Sanborn, R. and Blake, C.D., 2005. Semin. Oncol. 32, 69.
      Kim, H.J., et al. 2003. Int. J. Oncol. 22, 99.
      Shigemasa, K., et al. 2003. Int. J. Oncol. 22, 99.
      El-Bayoumy, K., et al. 2002. Int. J. Oncol. 20, 557.
      Hwang, D., et al. 1998. J. Natl. Cancer Inst. 90, 455.

      Brochure

      Title
      COX-2 ELISA Kit Flyer
      User Protocol

      Revision23-August-2016 JSW
      Synonyms Cyclooxygenase-2 ELISA Kit
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival store the entire contents of the kit at -20°C.
      Intended useThe Calbiochem® COX-2 ELISA Kit is intended for the quantitative measurement of COX-2 in human cell lysates. Serum and plasma samples are not recommended due to inherently low concentrations of COX-2 in biological fluids.
      BackgroundCyclooxygenase-1 and -2 (COX-1 and COX-2) catalyze the synthesis of prostaglandins (PGs) from arachidonic acid. COX-1 is expressed constitutively in most tissues and appears to be responsible for the production of PGs that control normal physiologic functions, such as gastric mucosa maintenance and regulation of renal blood flow. Conversely, COX-2 is not constitutively expressed in most normal tissues, but is induced by various inflammatory and/or mitogenic stimuli such as cytokines, growth factors, and tumor promoters. However, COX-2 is constitutively expressed in the brain and kidneys. Numerous experimental studies suggest a relationship between COX-2 expression and carcinogenesis. In vivo treatment with selective inhibitors of COX-2 reduced the formation of tongue, esophageal, intestinal, breast, skin, lung, and bladder tumors in experimental animals. EGFR signaling involves AP-1-mediated induction of COX-2 transcription and increased amounts of COX-2 have been observed in breast cancers that overexpress HER-2/neu.
      Principles of the assayThe Calbiochem® COX-2 ELISA Kit is a complete kit for the quantitative determination of human COX-2 in cell lysates. The 96-well plate is coated with a rabbit anti-human COX-2 polyclonal antibody as the capture antibody. Standards and samples are added to the wells at which time COX-2 is bound to the coated antibody. COX-2 is then detected with a monoclonal anti-COX-2 antibody and a goat anti-mouse IgG HRP Conjugate. The addition of TMB Substrate results in a blue-colored product in the presence of the HRP Conjugate. Sensitivity is increased by the addition of ELISA Stop Solution (H₂SO₄), yielding a yellow color. The absorbance of each well is measured at 450 nm (preferably with reference wavelength set of 540-600 nm) and the level of COX-2 in each sample is calculated from the standard curve.

      Note: The use of tissue extracts may be limited to tissues that have induced COX-2 expression or COX-2 overexpression.
      Materials provided• Anti-COX-2 Coated 96-Well Plate (Kit Component No. JA9172): pre-coated with rabbit anti-human COX-2
      • COX-2 Standard (Kit Component No. JA9173): recombinant, human COX-2
      • COX-2 Detector Antibody (Kit Component No. JA9174): supplied as 1000X solution
      • HRP Conjugated Secondary Antibody (Kit Component No. JA9372): supplied as a 400X solution
      • Assay Diluent (Kit Component No. JA7644)
      • ELISA 20X Plate Wash Concentrate (Kit Component No. JA1617)
      • TMB Substrate (Kit Component No. JA1608): ready-to-use
      • ELISA Stop Solution (Kit Component No. JA1616): 2.5 N H₂SO₄ ready-to-use
      • Plate Sealers:
      Materials Required but not provided• Precision pipettors with disposable tips
      • Wash bottle or multi-channel dispenser for washing
      • COX-2 inducer (see Preparation of Samples below)
      • CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) or equivalent cell lysis buffer, preferably with protease inhibitors (e.g. Protease Inhibitor Cocktail Set III, Cat. No. 539134)
      • Protein assay (e.g. Non-Interfering Protein Assay™, Cat. No. 488250 or BCA Protein Assay Kit, Cat. No. 71285)
      • Spectrophotometer capable of measuring absorbance in 96-well plates at 450 nm, preferably with a reference wavelength of 540-600 nm
      PreparationCell lysates: Most mammalian cells do not express significant amounts of COX-2, so in order to detect COX-2 in lysates of most cell types; COX-2 must be induced by an appropriate stimulus. Below is a sample protocol for COX-2 induction in U937 cells: 1. Prepare all reagents immediately prior to use. 2. Grow U937 cells in complete RPMI 1640 medium. 3. Harvest the cells during the log phase of growth. 4. Adjust the cell density to 1 x 106 cells/ml and incubate for 3 days in the presence of 15 nM PMA. 5. Harvest the cells and incubate for 24 h in fresh complete RPMI 1640 medium. 6. Stimulate the cells with LPS for 6 h at a concentration of 250 ng/ml. 7. Lyse the cells with CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) according to the recommended protocol. 8. Determine the protein concentration. A concentration of 2-5 mg/ml is recommended. 9. Store the cell lysate at -70°C until use.
      Reagent preparationNote: Allow all kit components and samples to reach room temperature (18-25°C) prior to assay. Prepare all reagents immediately prior to use. The following table provides reagent preparation instructions to obtain a volume of reagent sufficient for one 2x8-well strip (16 wells).

      Table 1: Reagent Preparation

      4. Standards: a. Reconstitute the COX-2 Standard with 1000 µl deionized or distilled water to yield a stock solution of 2000 ng/ml. Dispense unused reconstituted COX-2 Standard into aliquots and freeze (-70°C). Aliquots are stable for several months at -70°C. b. Prepare a standard curve by making dilutions of the reconstituted COX-2 Standard in Assay Diluent as follows:

      Table 2: Sample Dilutions
      Detailed protocolIt is recommended that all samples and standards be assayed in duplicate.

      1. Remove the desired number of strips from the Anti-COX-2-Coated 96-Well Plate and place them in the frame. Return the unused strips to the foil pouch and reseal the entire edge with tape. Store the unused strips at 4°C or -20°C.
      2. Add 100 µl diluted Standards and samples (2-5 mg/ml total protein) to designated wells. Cover the plate with a Plate Sealer and incubate for 90 min at room temperature.
      3. Aspirate and discard the contents of the wells and wash by completely filling the wells with ELISA 1X Plate Wash. Aspirate and discard the contents of the wells; complete removal of liquid at each step is essential for good assay performance. Repeat for a total of 4 washes. Following the final wash, remove any remaining liquid by tapping the inverted plate on paper towels.
      4. Add 100 µl COX-2 Detector Antibody 1X to each well. Cover the plate with a Plate Sealer and incubate for 90 min at room temperature.
      5. Wash the plate as indicated in step 4.
      6. Add 100 µl HRP Conjugated Secondary Antibody 1X to each well. Cover the plate with a Plate Sealer and incubate for 60 min at room temperature.
      7. Wash the plate as indicated in step 4
      8. Add 100 µl TMB Substrate to each well and incubate for 30 min at room temperature.
      9. Add 100 µl ELISA Stop Solution to each well. Read the absorbance at 450 nm with a reference wavelength of 595 nm (Note: if the appropriate filter is not available, 540 or 550 nm can be used as alternative reference wavelengths). If a dual wavelength reading option is not available, read the absorbance at 450 nm only.
      10. Calculation of Results: several options are available for the calculation of the concentration of COX-2 in the samples. We recommend the use of an immunoassay software package capable of generating a four-parameter logistic (4-PL) curve fit. If data reduction software is not available, construct a standard curve by plotting the mean absorbance for each standard on the Y-axis against the protein concentration on the X-axis and draw a best-fit curve through the points on the graph. Values are expressed in ng/ml.
      Standard curve

      Figure 1: Standard Curve

      Example standard curve using the COX-2 Standard. A standard curve was generated according to the COX-2 Standard preparation and the Detailed Protocol above. The results represent a typical standard curve. Assay range: 2.5-200 ng/ml. Lower limit of detection: ~2 ng/ml.
      Example data

      Figure 2: LPS-induced COX-2 protein expression in U937 cells.

      U937 cells were stimulated and lysates were prepared as described in the Sample Preparation section above. The level of COX-2 was measured using 50 µg total protein according to the Detailed Protocol above.
      Assay Range2.5-200 ng/ml
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      CytoBuster™ InteractivePathways™ are trademarks of EMD Chemicals, Inc.
      Non-Interfering Protein Assay™ is a trademark of Geno Technology, Inc.