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KAC2411 Pr257 Reva4 Jun1608 (Hu Active MMP-13) (BSE)

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KAC2411 Pr257 Reva4 Jun1608 (Hu Active MMP-13) (BSE) Immunoassay Kit Catalog #KAC2411 Human Active MMP-13 www.invitrogen.com Invitrogen Corporation 542 Flynn Rd, Camarillo, CA 93012 Tel: 800-955-6288 E-mail: [email protected] 1 2 TABLE OF CONTENTS Introduction ........................................................................................ 4 Intended Use....................................................................................... 6 Principle of the Method...................................................................... 6 Reagents Provided.............................................................................. 7 Supplies Required But Not Provided ................................................ 8 Procedural Notes/Lab Quality Control.............................................. 8 Sample Handling and Storage ........................................................... 10 Safety.................................................................................................. 11 Directions for Washing...................................................................... 12 Reagent Preparation and Storage....................................................... 12 Reconstitution and Dilution of Standard ........................................... 12 Storage and Final Dilution of Streptavidin-Peroxidase .................... 15 Dilution of Wash Buffer .................................................................... 16 Assay Method..................................................................................... 16 Typical Data ....................................................................................... 19 Limitations of the Procedure.............................................................. 20 Performance Characteristics .............................................................. 20 Sensitivity........................................................................................... 20 Precision ............................................................................................. 21 Linearity of Dilution .......................................................................... 22 Recovery............................................................................................. 23 Specificity........................................................................................... 23 Expected Values................................................................................. 23 References .......................................................................................... 24 3 Rev. A4 06/16/08 PR257 INTRODUCTION The matrix metalloproteinases (MMPs) comprise a family of multi-domain, zinc-containing neutral endopeptidases that include collagenases, stromelysins, gelatinases, and membrane-type metalloproteinases. This family of enzymes plays an important role in normal and pathological tissue remodeling by contributing to the degradation of the extracellular matrix. Over 20 MMP family members have been described to date. MMP-13, known alternatively as collagenase-3, is one of the principal enzymes that degrades fibrillar collagen. The gene for human MMP-13 maps to chromosome 11 in a cluster that also contains MMP-1, MMP-7, and MMP-12. Northern blot analysis and RNA PCR reveal relatively high levels of MMP-13 gene expression in fetal tissues, while MMP-13 expression levels in normal adult tissues are usually low. Elevated MMP-13 expression levels are observed in degenerative joint diseases and certain cancers. Factors that regulate MMP-13 gene expression are currently under investigation. The 5’ flanking region of the MMP-13 gene contains a TATA box, an AP-1 motif, a runt domain (RD) binding site, a PEA-3 consensus sequence, an osteoblast specific element (OSE-2), and a TGF-β inhibitory site. The AP-1 site and the RD binding site, along with the transcription factor RUNX-2, appear to be important for maintaining basal transcription levels. Cytokines and growth factors, including IL-1α, IL-1β, TNF-α, IL-6, bFGF, IGF-1, and TGF-β1, as well as stimuli that activate JNK1/2, p38 MAPK, ERK, and NF-κB signaling, have been observed to modulate MMP-13 expression. 4 MMP-13 is secreted into the extracellular space as an inactive proenzyme with Mr=60 kDa, known as the latent form of MMP-13. MMP-13’s activation requires cleavage of the proenzyme by either plasmin, MMP-2, MMP-3, or MMP-14, yielding active fragments with Mr=48 kDa and below. MMP-13’s activity is further regulated by association with members of a family of inhibitory proteins, tissue inhibitors of metalloproteinase TIMP-1, TIMP-2, and TIMP-3. MMP-13 has numerous substrates. MMP-13 degrades collagen type I, II, and III at specific sites to yield large N terminal and smaller C terminal collagen fragments. Following cleavage, the fragments rapidly denature and are further hydrolyzed through the action of gelatinases. Other MMP-13 substrates include: tenascin, aggrecan core protein, CXCL12, and the MMP-9 proenzyme. By removing collagen from the bone growth plates during normal embryonic development, MMP-13 plays an important role in bone morphogenesis. MMP-13 dysregulation is implicated in numerous disease states. For example, MMP-13 produced by osteoarthritic chondrocytes is observed to co-localize with collagen type II and contributes to the disease-related cartilage destruction. Other disease states in which MMP-13 is implicated include rheumatoid arthritis, chronic cutaneous ulcers, and periodontitis. Specific cancers in which MMP-13 is implicated include: breast carcinoma, squamous cell carcinoma of the head and neck, cutaneous basal carcinomas, and chondrosarcomas. Compounds that inhibit MMP-13 expression or enzyme activity are currently in development as therapeutics for degenerative joint diseases and cancer. 5 INTENDED USE The Invitrogen Human Active MMP-13 (Hu Active MMP-13) ELISA is to be used for the in vitro quantitative determination of activated MMP-13 in serum and tissue culture medium. The assay will recognize both natural and recombinant Hu Active MMP-13. This kit has been configured for research use only and is not to be used in diagnostic procedures. READ ENTIRE PROTOCOL BEFORE USE PRINCIPLE OF THE METHOD The Invitrogen Hu Active MMP-13 ELISA kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for Hu Active MMP-13 has been coated onto the wells of the microtiter strips provided. Samples, including standards of known active MMP-13 content, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the active MMP-13 antigen binds to the immobilized (capture) antibody on one site. After washing, a biotinylated monoclonal antibody specific for Hu Active MMP-13 is added. During the second incubation, this antibody binds to the immobilized active MMP-13 captured during the first incubation. After removal of excess second antibody, Streptavidin-Peroxidase (enzyme) is added. The Streptavidin-Peroxidase binds to the biotinylated antibody to complete the four-member sandwich. After a third incubation and washing to remove all the unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to 6 produce color. The intensity of this colored product is directly proportional to the concentration of active MMP-13 present in the original specimen REAGENTS PROVIDED Note: Store all reagents at 2 to 8°C. 96 Reagent Test Kit Hu Active MMP-13 Standard. Each vial contains 20 ng 1 vial lyophilized active MMP-13 in Assay Buffer. Reconstitute each vial with 1 mL deionized water. Once reconstituted, aliquot and store at -20 ° C or below. Avoid repeated freeze-thaw cycles. Assay Buffer. Each bottle contains 25 mL phosphate buffer with 1 bottle additives. Serum Standard Diluent. Each bottle contains 20 mL of certified 1 bottle human serum in buffer with additives. Hu Active MMP-13 Antibody-Coated Wells. Each plate contains 1 plate 6 x 16 strips, for a total of 96 wells Hu Active MMP-13 Biotin Conjugate. Each bottle contains 12 mL 1 bottle of Biotin-labeled anti-Hu Active MMP-13 in buffer with additives. Streptavidin-Peroxidase (HRP), (40x), in stabilizer with additives. 1 vial Each vial contains 300 µ L. Streptavidin-Peroxidase (HRP) Diluent. Each bottle contains 1 vial 12 mL phosphate buffer with additives. Wash Buffer Concentrate (20x); 25 mL per bottle. 2 bottles Stabilized Chromogen, Tetramethylbenzidine (TMB); 12 mL per 1 bottle bottle. Stop Solution; 12 mL per bottle. 1 bottle Plate Covers, adhesive strips. 2 7 SUPPLIES REQUIRED BUT NOT PROVIDED 1. Microtiter plate reader capable of measurement at or near 450 nm. A reference filter (either 540, 570, or 620 nm) is recommended. 2. Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi-channel pipette is desirable for large assays.) 3. Distilled or deionized water. 4. Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.). 5. Data analysis and graphing software. Graph paper: linear (Cartesian), log-log, or semi-log, as desired. 6. Glass or plastic tubes for diluting and aliquoting standard. 7. Absorbent paper towels. 8. Calibrated beakers and graduated cylinders in various sizes. PROCEDURAL NOTES/LAB QUALITY CONTROL 1. When not in use, kit components should be refrigerated. All reagents should be warmed to room temperature before use (20 to 25°C). Do not allow any of the components to exceed a temperature of 26°C. The Stabilized Chromogen is especially sensitive to elevated temperatures. 2. Microtiter plates should be allowed to
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