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 (MMPs) comprise a family of multi-domain, zinc-containing neutral endopeptidases that include , stromelysins, , and membrane-type metalloproteinases. This family of 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 -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) , 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 , tissue inhibitors of 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, core , 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 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 come to room temperature before opening the foil bags. Once the desired number of strips has been removed, immediately reseal the bag and store at 2 to 8°C to maintain plate integrity. 3. It is recommended that all standards and samples be run in duplicate. 4. During all incubation steps, seal the plates with the adhesive plate covers provided.

8 5. Samples that are >2000 pg/mL should be diluted, as appropriate for sample type, and reanalyzed. 6. When pipetting reagents, maintain a consistent order of addition from well-to-well. This ensures equal incubation times for all wells. 7. Cover or cap all reagents when not in use. 8. Do not mix or interchange different reagent lots from various kit lots. 9. Do not use reagents after the kit expiration date. 10. Read absorbances within 30 minutes of assay completion. 11. All residual wash liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells. 12. Because the Stabilized Chromogen is light sensitive, avoid prolonged exposure to light. Avoid contact between the Stabilized Chromogen and metal. Avoid exposing the Stabilized Chromogen to temperatures above 26°C.

9 SAMPLE HANDLING AND STORAGE Note: EDTA and other chelating agents inhibit the assay. All samples should be free from chelating agents. Serum 1. Samples should be collected in pyrogen/endotoxin-free tubes. When possible, avoid use of badly hemolyzed or lipemic sera. 2. Serum samples should be apportioned into aliquots and stored in polypropylene tubes at -80°C until analyzed. Avoid repeated freeze/thaw cycles. 3. Upon thawing, mix serum samples well. 4. For serum samples containing relatively low levels of active MMP-13, dilute the samples 1:10 in Assay Buffer. 5. For serum samples containing higher levels of active MMP-13, dilute the samples 1:10 in Assay Buffer, then perform further dilutions in Serum Standard Diluent to maintain a serum concentration of 10% in the sample.

10 Tissue Culture Supernatants Note: Fetal or neonatal bovine serum may contain MMP-13. Medium only controls are recommended when analyzing serum-supplemented tissue culture supernatant samples. 1. Samples should be collected in pyrogen/endotoxin-free tubes. 2. Clarify samples by centrifugation for 15 minutes at 4,000 x g or greater. 3. Clarified tissue culture supernatant samples should be apportioned into working aliquots and stored in polypropylene tubes at -80°C until analyzed. Avoid repeated freeze-thaw cycles. 4. Tissue culture supernatant samples must be diluted by a factor of 1:3 or greater in Assay Buffer. SAFETY All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing of infectious agents.

11 DIRECTIONS FOR WASHING Incomplete washing will adversely affect the test outcome. All washing must be performed with Wash Buffer provided. Washing can be performed manually as follows: completely aspirate the liquid from all wells by gently lowering an aspiration tip (aspiration device) into the bottom of each well. Take care not to scratch the inside of the well. After aspiration, fill the wells with at least 0.4 mL of diluted wash solution. Let soak for 15 to 30 seconds, then aspirate the liquid. Repeat as directed under ASSAY METHOD. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue. Alternatively, the wash solution may be put into a squirt bottle. If a squirt bottle is used, flood the plate with wash buffer, completely filling all wells. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue. If using an automated washer, the operating instructions for washing equipment should be carefully followed. REAGENT PREPARATION AND STORAGE Reconstitution and Dilution of Hu Active MMP-13 Standard Either glass or plastic tubes may be used for standard dilutions. A. Reconstitution and Dilution of Hu Active MMP-13 Standard for Serum Sample Analysis.

12 1. Reconstitute standard to 20,000 pg/mL by pipetting 1 mL of deionized water into standard vial. Swirl or mix gently and allow to sit for 30 minutes to ensure complete reconstitution. Avoid foaming. 2. Label 8 tubes as follows: 2000 pg/mL, 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31 pg/mL, and 0 pg/mL. 3. Into the tube labeled 2000 pg/mL, pipette 900 µ L Serum Standard Diluent followed by 100 µ L of the reconstituted standard and mix thoroughly. 4. Generate the standard curve by performing the serial dilutions indicated in the table presented below. Standard: Add: Into:

2000 pg/mL Prepare as described in Step 3.

1000 pg/mL 500 µL of the 500 µL of the 2000 pg/mL std. Serum Standard Diluent 500 pg/mL 500 µL of the 500 µL of the 1000 pg/mL std. Serum Standard Diluent 250 pg/mL 500 µL of the 500 µL of the 500 pg/mL std. Serum Standard Diluent 125 pg/mL 500 µL of the 500 µL of the 250 pg/mL std. Serum Standard Diluent 62.5 pg/mL 500 µL of the 500 µL of the 125 pg/mL std. Serum Standard Diluent 31 pg/mL 500 µL of the 500 µL of the 62.5 pg/mL std. Serum Standard Diluent 0 pg/mL 500 µL of the An empty tube Serum Standard Diluent

13 B. Reconstitution and Dilution of Hu Active MMP-13 Standard for Tissue Culture Supernatant Samples. 1. Reconstitute standard to 20,000 pg/mL by pipetting 1 mL of deionized water into standard vial. Swirl or mix gently and allow to sit for 30 minutes to ensure complete reconstitution. Avoid foaming. 2. Label 8 tubes as follows: 2000 pg/mL, 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31 pg/mL, and 0 pg/mL. 3. Into the tube labeled 2000 pg/mL, pipette 900 µ L Assay Diluent followed by 100 µ L of the reconstituted standard and mix thoroughly. 4. Generate the standard curve by performing the serial dilutions indicated in the table presented below. Standard: Add: Into: 2000 pg/mL Prepare as described in Step 3. 1000 pg/mL 500 µL of the 500 µL of the 2000 pg/mL std. Assay Diluent 500 pg/mL 500 µL of the 500 µL of the 1000 pg/mL std. Assay Diluent 250 pg/mL 500 µL of the 500 µL of the 500 pg/mL std. Assay Diluent 125 pg/mL 500 µL of the 500 µL of the 250 pg/mL std. Assay Diluent 62.5 pg/mL 500 µL of the 500 µL of the 125 pg/mL std. Assay Diluent 31 pg/mL 500 µL of the 500 µL of the 62.5 pg/mL std. Assay Diluent 0 pg/mL 500 µL of the An empty tube Assay Diluent

14 The unused portion of the reconstituted standard may be stored at -20°C for subsequent assays, if desired. C. Storage and Final Dilution of Streptavidin-Peroxidase The Streptavidin-HRP (Conjugate) is provided in a concentrated form that must be diluted 1:40 with Streptavidin-HRP Diluent immediately prior to use in the assay. 1. Prepare sufficient Streptavidin-HRP Working Solution for each assay according to the table presented below: For Example:

Volume of # of 16-Well Streptavidin-Peroxidase Strips Concentrate Volume of Diluent 2 100 µL 4 mL 3 150 µL 6 mL 6 300 µL 12 mL

Label as Streptavidin-HRP Working Solution. 2. Return the unused Streptavidin-Peroxidase Concentrate to the refrigerator.

15 D. Dilution of Wash Buffer Allow the 20x concentrate to reach room temperature and mix to ensure that any precipitated salts have redissolved. Dilute the 50 mL of Wash Buffer Concentrate to a final volume of 1 liter. Label as Working Wash Buffer. Store both the concentrate and the Working Wash Buffer in the refrigerator. The diluted buffer should be used within 14 days. ASSAY METHOD: PROCEDURE AND CALCULATIONS Be sure to read the Procedural Notes/Lab Quality Control section before carrying out the assay. Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use, but avoid foaming. Note: A standard curve must be run with each assay. 1. Determine the number of 16-well strips needed for the assay. Insert these in the frame(s) for current use. (Re-bag extra strips and frame. Store these in the refrigerator for future use.) 2. Pipette 100 µ L of standards into the appropriate microtiter wells. Pipette 100 µ L of samples into appropriate wells. Well(s) reserved for chromogen blank(s) should be left empty. 3. Cover plate with plate cover and incubate on a shaker at room temperature for exactly 120 minutes. 4. Thoroughly aspirate solution from wells and discard the liquid. Wash wells 4 times with Working Wash Solution. See DIRECTIONS FOR WASHING. Blot the plate on paper towels to remove residual liquid.

16 5. Pipette 100 µ L of Hu Active MMP-13 Biotin Conjugate into each well except the chromogen blank(s). Tap gently on the side of the plate to mix. 6. Cover plate with plate cover and incubate on a shaker at room temperature for exactly 90 minutes. 7. Thoroughly aspirate solution from wells and discard the liquid. Wash wells 4 times. See DIRECTIONS FOR WASHING. Blot the plate on paper towels to remove residual liquid. 8. Add 100 µ L Streptavidin-Peroxidase Working Solution to each well except the chromogen blank(s). (Prepare the working dilution as described in REAGENT PREPARATION AND STORAGE, Section C.) 9. Cover plate with the plate cover and incubate on a shaker at room temperature for exactly 30 minutes. 10. Thoroughly aspirate solution from wells and discard the liquid. Wash wells 5 times. See DIRECTIONS FOR WASHING. Blot the plate on paper towels to remove residual liquid. 11. Add 100 µ L of Stabilized Chromogen to each well. The liquid in the wells will begin to turn blue. 12. Incubate for 15 minutes at room temperature and in the dark. Please Note: Do not cover the plate with aluminum foil or metalized mylar. The incubation time for chromogen substrate is often determined by the microtiter plate reader used. Many plate readers have the capacity to record a maximum optical density (O.D.) of 2.0. The O.D. values should be monitored and the substrate reaction stopped before the O.D. of the positive wells exceed the limits of the instrument. The O.D. values at 450 nm can only be read after the Stop Solution has been added to each well. If using a reader that records only to 2.0 O.D., stopping the assay after 10 to 15 minutes is suggested.

17 13. Add 100 µ L of Stop Solution to each well. Tap side of plate gently to mix. The solution in the wells should change from blue to yellow. 14. Read the absorbance of each well at 450 nm having blanked the plate reader against a chromogen blank composed of 100 µ L each of Stabilized Chromogen and Stop Solution. Read the plate within 30 minutes after adding the Stop Solution. 15. Plot on graph paper the absorbance of the standards against the standard concentration. (Optimally, the background absorbance may be subtracted from all data points, including standards, unknowns, and controls prior to plotting.) Draw the best smooth curve through these points to construct the standard curve. If using curve fitting software, the four parameter algorithm provides the best curve fit. 16. Read the Hu Active MMP-13 concentrations for unknown samples and controls from the standard curve plotted in step 15. Multiply value(s) obtained for sample(s) by the appropriate dilution factor. (Samples producing signals greater than that of the highest standard (2000 pg/mL) should be further diluted, as appropriate for sample type, and reanalyzed, multiplying the concentration found by the appropriate dilution factor.)

18 TYPICAL DATA The following data were obtained for the various standards over the range of 0 to 2000 pg/mL Active MMP-13.

Standard Hu Active MMP-13 Optical Density (pg/mL) (450 nm) 2000 2.445

1000 1.396

500 0.764

250 0.433

125 0.269

62.5 0.168

31 0.128

0 0.088

19 LIMITATIONS OF THE PROCEDURE Do not extrapolate the standard curve beyond the 2000 pg/mL standard point; the dose-response is non-linear in this region and accuracy is difficult to obtain. Dilute samples >2000 pg/mL as appropriate for sample type (please see the section on sample handling and storage); reanalyze these and multiply results by the appropriate dilution factor. The influence of various drugs, aberrant sera (hemolyzed, hyperlipidemic, jaundiced, etc.) and the use of biological fluids in place of serum or tissue culture supernatant samples have not been thoroughly investigated. The rate of degradation of native MMP-13 in various matrices has not been investigated. The immunoassay literature contains frequent references to aberrant signals seen with some sera, attributed to heterophilic antibodies. Though such samples have not been seen to date, the possibility of this occurrence cannot be excluded. This kit is for research use only. Not for human therapeutic or diagnostic use.

PERFORMANCE CHARACTERISTICS SENSITIVITY The minimum detectable dose of Human Active MMP-13 is <7 pg/mL. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 48 times.

20 PRECISION 1. Intra-Assay Precision Samples of known Hu Active MMP-13 concentration were prepared in Assay Buffer and assayed in replicates of 20 to determine precision within an assay.

Sample 1 Sample 2 Sample 3 Sample 4 Mean (pg/mL) 63 254 516 1017 SD 4.7 13 19.0 32 %CV 7.5 5.0 3.6 3.1 SD = Standard Deviation CV = Coefficient of Variation

2. Inter-Assay Precision Samples of known Hu Active MMP-13 concentration were prepared in Assay Buffer and assayed in replicates of 16 on different plates to determine precision between assays.

Sample 1 Sample 2 Sample 3 Sample 4 Mean (pg/mL) 68 242 490 1000 SD 4.7 16 25 48 %CV 7.0 6.6 5.1 4.8 SD = Standard Deviation CV = Coefficient of Variation

21 LINEARITY OF DILUTION Human serum and tissue culture medium were spiked with Hu Active MMP-13 and serially diluted in Serum Standard Diluent and Assay Buffer, respectively, over the range of the assay. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 1.0 for each of the sample types.

Serum Dilution Measured Expected % Factor (pg/mL) (pg/mL) Expected 1:10 351 -- -- 1:20 165 176 94 1:40 85 88 97 1:80 42 44 95 1:160 26 22 118

Tissue Culture Medium Dilution Measured Expected % Factor (pg/mL) (pg/mL) Expected 1:3 479 -- -- 1:6 226 240 94 1:12 113 120 94 1:24 61 60 102 1:48 38 30 129

22 RECOVERY The recovery of Hu Active MMP-13 added to human serum, diluted 1:10 with Assay Buffer, averaged 109.5%. The recovery of Hu Active MMP-13 added to tissue culture medium, diluted 1:10 with Assay Buffer, averaged 110.5. SPECIFICITY The Hu Active MMP-13 ELISA recognizes the activated form of MMP-13 (collagenase-3). This assay does not cross-react with latent or activated forms of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and the catalytic domains of MT1 MMP, MT2 MMP, MT4 MMP, and MT5 MMP. This assay does not recognize the latent form of MMP-13. EXPECTED VALUES A limited number of serum samples was evaluated in this assay. Hu Active MMP-13 values measured with this assay ranged from 0 to 402 pg/mL.

23 REFERENCES 1. Bluteau, G., et al. (2001) -1, -3, -13 and aggrecanase-1 and -2 are differentially expressed in experimental osteoarthritis. Biochim. Biophys. Acta 1526:147-158. 2. Brew, K., et al. (2000) Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim. Biophys. Acta 1477:267-282. 3. Knauper, V., et al. (1996) Cellular mechanisms for human procollagenase-3 (MMP-13) activation. Evidence that MT1-MMP (MMP-14) and a (MMP-2) are able to generate active enzyme. J. Biol. Chem. 271:17124-17131. 4. Mitchell, P.G., et al. (1996) Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J. Clin. Invest. 97:761-768. 5. Nagase, H. and F. Woesner, Jr. (1999) Matrix metalloproteinases. J. Biol. Chem. 274:21491-21494. 6. Pendás, A.M., et al. (2000) An overview of collagenase-3 expression in malignant tumors and analysis of its potential value as a target in antitumor therapies. Clin. Chim. Acta 291:137-155. 7. Selvamurugan. N. and N.C. Partridge (2000) Constitutive expression and regulation of collagenase-3 in human breast cancer cells. Mol. Cell. Biol. Res. Commun. 3(4):218-223. 8. Selvamurugan, N., et al. (2004) Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-beta1-stimulated collagenase 3 expression in human breast cancer cells. J. Biol. Chem. 279(26):27764-27773.

24 9. Westhoff, C.S., et al. (1999) Characterization of collagenase 3 (matrix metalloproteinase 13) messenger RNA expression in the synovial membrane and synovial fibroblasts of patients with rheumatoid arthritis. Arthritis Rheum. 42:1517-1527. Important Licensing Information - These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen Catalog or our website, www.invitrogen.com). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.

25 NOTES

26 27 28 Rev. A4 06/16/08 PR257