Quantikine

Human OX40 /TNFSF4 Immunoassay

Catalog Number DOXL00

For the quantitative determination of human OX40 Ligand concentrations in cell culture supernates, serum, plasma and cell lysates.

This package insert must be read in its entirety before using this product.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. TABLE OF CONTENTS Contents Page INTRODUCTION 2 PRINCIPLE OF THE ASSAY ...... 3 LIMITATIONS OF THE PROCEDURE 3 REAGENTS ...... 3 STORAGE 4 OTHER SUPPLIES REQUIRED ...... 4 PRECAUTION 4 SAMPLE COLLECTION AND STORAGE ...... 5 SAMPLE PREPARATION 5 REAGENT PREPARATION ...... 6 ASSAY PROCEDURE 7 ASSAY PROCEDURE SUMMARY...... 8 CALCULATION OF RESULTS 9 TYPICAL DATA...... 9 TECHNICAL HINTS 10 PRECISION ...... 10 RECOVERY 11 LINEARITY ...... 11 SENSITIVITY 11 CALIBRATION ...... 12 SAMPLE VALUES 12 SPECIFICITY ...... 13 REFERENCES 14 PLATE LAYOUT ...... 15

MANUFACTURED AND DISTRIBUTED BY: R&D Systems, Inc. TELEPHONE: (800) 343-7475 614 McKinley Place N.E. (612) 379-2956 Minneapolis, MN 55413 FAX: (612) 379-6580 United States of America E-MAIL: [email protected]

DISTRIBUTED BY: R&D Systems Europe 19 Barton Lane TELEPHONE: (0)1235 529449 Abingdon Science FAX: (0)1235 533420 Abingdon, Oxon OX14 3NB E-MAIL: [email protected] United Kingdom R&D Systems GmbH FREEPHONE: (0)800 909 4455 Borsigstrasse 7 TELEPHONE: (0)6122 90980 5205 Wiesbaden-Nordenstadt FAX: (0)6122 909819 Germany E-MAIL: [email protected] R&D Systems Europe 77 Vauban FREEPHONE: (0)800 90 72 49 59800 Lille FAX: (0)800 77 16 68 France E-MAIL: [email protected] INTRODUCTION OX40 Ligand (OX40L/TNFSF4/gp34) is a member of the superfamily (TNFSF), acting as a late costimulatory with roles in promoting the survival, proliferation, and migration of activated CD4+ T cells (1). Human OX40 Ligand cDNA encodes a 183 amino acid (aa) polypeptide with an N-terminal cytoplasmic domain (aa 1 - 23) and a C-terminal extracellular domain (aa 51 - 183). It shares 46% aa sequence identity with its mouse counterpart (2, 3). OX40 Ligand is a type II transmembrane glycoprotein and, similar to other TNFSF members, likely exists as a homotrimer. It is expressed on the surface of several cell types including activated B cells, T cells, dendritic cells, Langerhans cells, , NK cells, and endothelial cells (2, 4 - 8). Like other members of the TNFSF, OX40 Ligand may be shed from the membrane and exist in a soluble form (3, 9). OX40 Ligand binds to OX40 (CD134), a member of the TNF receptor superfamily that is expressed predominantly on activated CD4+ T cells but may be found on CD8+ cells as well (10). Several studies have utilized knockout or transgenic mice to illustrate the importance of OX40 Ligand and OX40, in the later stages of primary activation. Both OX40 Ligand and OX40 knockouts exhibit diminished T cell responses (10 - 14). Early CD4+ T cell proliferation is unimpaired in these mice, but decreases, and is followed by 4 to 5 days following activation. Decreases in the number of effector T cells are accompanied by the restricted development of memory T cells (12). In contrast, transgenic expression of OX40 Ligand results in increased numbers of both antigen-specific CD4+ T cells and memory T cells (15). The survival effects of OX40/OX40 Ligand may be mediated by regulated expression of anti-apoptotic Bcl-2 family members (16). Studies suggest that OX40/OX40 Ligand interactions are important in the pathogenesis of several immune disorders. For instance, the OX40 receptor is expressed at sites of inflammation (17, 18), and disrupting OX40/OX40 Ligand function results in the suppression of several models of immune-related disease including graft versus host disease (19), experimental autoimmune encephalomyelitis (20, 21), inflammatory bowel disease (22), and rheumatoid arthritis (23). OX40 Ligand-deficient mice also exhibit reduced responses to allergen in models of allergic asthma (24, 25), and an increased susceptibility to parasitic infection (26). Transgenic over-expression of OX40 Ligand can result in autoimmune-like reactions (27), enhanced contact hypersensitivity responses (8), and confers resistance to parasitic infection (28). In addition, OX40/OX40 Ligand interactions have been shown to augment T cell priming, leading to the suppression of tumor growth in several cancer models (29 - 31). The Quantikine OX40 Ligand Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human OX40 Ligand in cell culture supernates, serum, plasma and cell lysates. It contains NS0-expressed recombinant human OX40 Ligand and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human OX40 Ligand showed linear curves that were parallel to the standard curves obtained using the Quantikine standards. These results indicate that the Quantikine OX40 Ligand kit can be used to determine relative mass values for naturally occurring OX40 Ligand.

2 PRINCIPLE OF THE ASSAY This assay employs the quantitative sandwich enzyme immunoassay technique. A polyclonal antibody specific for OX40 Ligand has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any OX40 Ligand present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for OX40 Ligand is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of OX40 Ligand bound in the initial step. The color development is stopped and the intensity of the color is measured.

LIMITATIONS OF THE PROCEDURE FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. The kit should not be used beyond the expiration date on the kit label. Do not mix or substitute reagents with those from other lots or sources. If samples generate values higher than the highest standard, dilute cell lysates with Cell Lysis Buffer 3 and all other samples with Calibrator Diluent. Repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. This assay is designed to eliminate interference by soluble receptors, binding , and other factors present in biological samples. Until all factors have been tested in the Quantikine Immunoassay, the possibility of interference cannot be excluded.

REAGENTS OX40 Ligand Microplate (Part 892405) - 96 well polystyrene microplate (12 strips of 8 wells) coated with a goat polyclonal antibody against OX40 Ligand. OX40 Ligand Conjugate (Part 892406) - 21 mL of a goat polyclonal antibody against OX40 Ligand conjugated to horseradish peroxidase with preservatives. OX40 Ligand Standard (Part 892407) - 20 ng of recombinant human OX40 Ligand in a buffer with preservatives, lyophilized. Assay Diluent RD1-15 (Part 895424) - 11 mL of a buffer with preservatives. Calibrator Diluent RD5L (Part 895028) - 21 mL of a buffered protein base with preservatives. Cell Lysis Buffer 3 Concentrate (Part 895366) - 21 mL of a concentrated buffered solution with preservatives. Wash Buffer Concentrate (Part 895003) - 21 mL of a 25-fold concentrated solution of buffered surfactant with preservatives. Color Reagent A (Part 895000) - 12.5 mL of stabilized hydrogen peroxide. Color Reagent B (Part 895001) - 12.5 mL of stabilized chromogen (tetramethylbenzidine). Stop Solution (Part 895032) - 6 mL of 2 N sulfuric acid. Plate Covers - 4 adhesive strips.

3 STORAGE

Unopened Kit Store at 2 - 8° C. Do not use past kit expiration date. Diluted Wash Buffer Stop Solution Assay Diluent RD1-15 Calibrator Diluent RD5L Cell Lysis Buffer 3 May be stored for up to 1 month at 2 - 8° C.* Opened/ Reconstituted Conjugate Reagents Unmixed Color Reagent A Unmixed Color Reagent B Standard Return unused wells to the foil pouch containing the Microplate Wells desiccant pack, reseal along entire edge of zip-seal. May be stored for up to 1 month at 2 - 8° C.* *Provided this is within the expiration date of the kit.

OTHER SUPPLIES REQUIRED Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Centrifuge. Multi-channel pipette, squirt bottle, manifold dispenser, or automated microplate washer. 100 mL and 500 mL graduated cylinder. Horizontal orbital microplate shaker (0.12" orbit) capable of maintaining a speed of 500 50 rpm. Phosphate-Buffered Saline (for cell lysis). Human OX40 Ligand Controls (optional; available from R&D Systems).

PRECAUTION The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

4 SAMPLE COLLECTION AND STORAGE Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Plasma - Collect plasma using heparin or EDTA as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay. Grossly hemolyzed or lipemic samples are not suitable for use in this assay.

SAMPLE PREPARATION Cell lysis procedure Cells from culture extracts must be lysed before assaying according to the following directions. 1. Centrifuge cells at 500 x g for 15 minutes and remove supernates. The supernate may be saved for assay in this kit. 2. Wash the cells three times in cold PBS. 3. Prepare Cell Lysis Buffer 3 (1X) as directed in the Reagent Preparation section. Add 1.0 mL of Cell Lysis Buffer 3 (1X) per 5.0 x 106 cells. 4. Vortex to resuspend the pellet and incubate at 37° C with gentle agitation for 30 minutes. 5. Centrifuge at 500 x g for 15 minutes to remove cellular debris, pour off the cell lysate supernate, and assay immediately or aliquot and store at -70° C. Note: If the value is above the high standard, further dilute in Cell Lysis Buffer 3 (1X).

5 REAGENT PREPARATION Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 mL of Wash Buffer. Substrate Solution - Color Reagents A and B should be mixed together in equal volumes within 15 minutes of use. Protect from . 200 L of the resultant mixture is required per well. Cell Lysis Buffer 3 (1X) - Dilute 20 mL of Cell Lysis Buffer 3 Concentrate with 80 mL deionized or distilled water to prepare 100 mL of Cell Lysis Buffer 3 (1X). OX40 Ligand Standard - Reconstitute the OX40 Ligand Standard with 1.0 mL of deionized or distilled water. This reconstitution produces a stock solution of 20,000 pg/mL. Mix the standard to ensure complete reconstitution and allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.

Pipette 540 L of Calibrator Diluent RD5L into the 2000 pg/mL tube. Pipette 300 L of Calibrator Diluent RD5L into each of the remaining tubes. Use the stock solution to produce a dilution series (below). Mix each tube thoroughly before the next transfer. The 2000 pg/mL standard serves as the high standard. Calibrator Diluent RD5L serves as the zero standard (0 pg/mL).

300 L 300 L 300 L 300 L 300 L 300 L

60 L std.

20,000 2000 1000 500 250 125 62.5 31.2 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL

6 ASSAY PROCEDURE Bring all reagents and samples to room temperature before use. It is recommended that all samples, controls and standards be assayed in duplicate.

1. Prepare all reagents, working standards and samples as directed in the previous sections.

2. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, reseal.

3. Add 100 L of Assay Diluent RD1-15 to each well.

4. Add 50 L of Standard, control or sample per well. Cover with the adhesive strip provided. Incubate for 2 hours at room temperature on a horizontal orbital microplate shaker (0.12" orbit) set at 500 50 rpm. A plate layout is provided to record standards and samples assayed.

5. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (400 L) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

6. Add 200 L of OX40 Ligand Conjugate to each well. Cover with a new adhesive strip. Incubate for 2 hours at room temperature on the shaker.

7. Repeat the aspiration/wash as in step 5.

8. Add 200 L of Substrate Solution to each well. Incubate for 30 minutes at room temperature on the benchtop. Protect from light.

9. Add 50 L of Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.

10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 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.

7 ASSAY PROCEDURE SUMMARY

8 CALCULATION OF RESULTS Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Plot the optical density for the standards versus the concentration of the standards and draw the best curve. The data can be linearized by using log/log paper and regression analysis may be applied to the log transformation. To determine the OX40 Ligand concentration of each sample, first find the absorbance value on the y-axis and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the x-axis and read the corresponding OX40 Ligand concentration. If the samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

TYPICAL DATA This standard curve is provided for demonstration only. A standard curve should be generated for each set of samples assayed.

pg/mL O.D. Average Corrected 0.043 0 0.043 0.043 ___ 0.093 31.2 0.093 0.093 0.050 0.139 62.5 0.142 0.141 0.098 0.239 125 0.242 0.241 0.198 0.426 250 0.426 0.426 0.383 0.781 500 0.785 0.783 0.740 1.460 1000 1.479 1.470 1.427 2.697 2000 2.710 2.704 2.661

9 TECHNICAL HINTS Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue. Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution. When mixing or reconstituting protein solutions, always avoid foaming. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

PRECISION Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in forty separate assays to assess inter-assay precision.

Intra-assay Precision Inter-assay Precision Sample 1 2 3 1 2 3 n202020404040 Mean 198 491 1047 221 537 1124 (pg/mL) Standard 4.7 9.5 36.7 23.2 50.5 84.5 deviation CV (%) 2.4 1.9 3.5 10.5 9.4 7.5

10 RECOVERY The recovery of OX40 Ligand spiked to levels throughout the range of the assay in various matrices was evaluated.

Sample Average % Recovery Range Cell culture media (n=4) 101 91 - 107% Serum (n=5) 94 88 - 109% EDTA plasma (n=4) 97 87 - 107% Heparin plasma (n=4) 96 85 - 107%

LINEARITY To assess the linearity of the assay, samples containing and/or spiked with high concentrations of OX40 Ligand were serially diluted with Calibrator Diluent RD5L to produce samples with values within the dynamic range of the assay.

Cell culture Heparin EDTA Cell media Serum plasma plasma Lysates (n=4) (n=5) (n=4) (n=5) (n=1) Average % of Expected 103 104 99 103 97 1:2 ___ Range (%) 94 - 107 97 - 114 95 - 108 100 - 109 Average % of Expected 100 107 98 103 99 1:4 ___ Range (%) 90 - 106 98 - 116 87 - 110 97 - 117 Average % of Expected 100 104 100 102 95 1:8 ___ Range (%) 86 - 107 92 - 120 89 - 113 93 - 115 Average % of Expected 98 99 96 100 89 1:16 ___ Range (%) 86 - 103 85 - 110 85 - 110 86 - 114

SENSITIVITY Forty assays were evaluated and the minimum detectable dose (MDD) of OX40 Ligand ranged from 0.98 - 8.69 pg/mL. The mean MDD was 3.56 pg/mL. The MDD was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

11 CALIBRATION This immunoassay is calibrated against a highly purified NS0-expressed recombinant human OX40 Ligand produced at R&D Systems.

SAMPLE VALUES Serum/Plasma - Thirty-three matched sets of serum and plasma samples were evaluated for the presence of OX40 Ligand in this assay. Thirty-one matched sets measured below the low standard. The remaining seum and plasma sample sets averaged approximately 36 pg/mL and 2700 pg/mL, respectively. Cell Culture Supernates - Human peripheral blood cells (1 x 106 cells/mL) were cultured in DMEM supplemented with 5% fetal calf serum, 50 µM β-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin sulfate. Cells were cultured unstimulated or stimulated with 10 µg/mL PHA. Aliquots of the culture supernate were removed and assayed for levels of natural OX40 Ligand. No detectable levels were observed. Cell Lysates - 1186 human T cells were cultured in RPMI supplemented with 10% fetal calf serum, 2 mM L-glutamine and 10 ng/mL of rhIL-2. Cells were grown for ten days, lysed, and measured 1648 pg/mL.

12 SPECIFICITY This assay recognizes recombinant and natural human OX40 Ligand. The factors listed below were prepared at 50 ng/mL in Calibrator Diluent RD5L and assayed for cross-reactivity. Preparations of the following factors at 50 ng/mL in a mid-range recombinant human OX40 Ligand control were assayed for interference. No significant cross-reactivity or interference was observed.

Recombinant Recombinant Recombinant human: mouse: porcine: 4-1BB TNF-α CD27 Ligand TNF-α BAFF/BLyS TNF-β CD30 Ligand CD27 Ligand TWEAK Recombinant CD30 Ligand VEGI LT-α1/β2 rat: CD40 Ligand LT-α2/β1 TNF-α Fas Ligand OX40 GITR Ligand OX40 Ligand LT-α1/β2 TNF-α LT-α2/β1 TNF-α (truncated)

The factors below were found to cross-react in this assay.

Recombinant factor % Cross-reactivity human APRIL 1.4 human LIGHT 3.6 human 0.08 human TRANCE 4.5 mouse TRANCE 0.16

13 REFERENCES 1. Croft, M. (2003) Rev. 14:265. 2. Baum, P.R. et al. (1994) EMBO J. 13:3992. 3. Godfrey, W.R. et al. (1994) J. Exp. Med. 180:757. 4. Kashii, Y. et al. (1999) J. Immunol. 163:5358. 5. Stüber, E. et al. (1995) Immunity 2:507. 6. Imura, A. et al. (1996) J. Exp. Med. 183:2185. 7. Ohshima, Y. et al. (1997) J. Immunol. 159:3838. 8. Sato, T. et al. (2002) Eur. J. Immunol. 32:3326. 9. Baba, E. et al. (2001) J. Immunol. 167:875. 10. Al-Shamkhani, A. et al. (1996) Eur. J. Immunol. 26:1695. 11. Kopf, M. et al. (1999) Immunity 11:699. 12. Gramaglia, I. et al. (2000) J. Immunol. 165:3043. 13. Chen, A.I. et al. (1999) Immunity 11:689. 14. Murata, K. et al. (2000) J. Exp. Med. 191:365. 15. Brocker, T. et al. (1999) Eur. J. Immunol. 29:1610. 16. Rogers, P.R. et al. (2001) Immunity 15:445. 17. Weinberg, A.D. et al. (1994) J. Immunol. 152:4712. 18. Tittle, T.V. et al. (1997) Blood 89:4652. 19. Stüber, E. et al. (1998) Gastroenterology 115:1205. 20. Weinberg, A.D. et al. (1999) J. Immunol. 162:1818. 21. Ndhlovu, L.C. et al. (2001) J. Immunol. 167:2991. 22. Higgins, L.M. et al. (1999) J. Immunol. 162:486. 23. Yoshioka, T. et al. (2000) Eur. J. Imunnol. 30:2815. 24. Hoshino, A. et al. (2003) Eur. J. Immunol. 33:861. 25. Arestides, R.S.S. et al. (2002) Eur. J. Immunol. 32:2847. 26. Ekkens, M.J. et al. (2003) J. Immunol. 170:384. 27. Murata, K. et al. (2002) J. Immunol. 169:4628. 28. Ishii, N. et al. (2003) Eur. J. Immunol. 33:2372. 29. Weinberg, A.D. et al. (2000) J. Immunol. 164:2160. 30. Kjaergaard, J. et al. (2000) Cancer Res. 60:5514. 31. Pan, P.Y. et al. (2002) Mol. Ther. 6:528.

14 PLATE LAYOUT Use this plate layout as a record of standards and samples assayed.

11.03 751068.0 11/03

15