Quantikine? Human Semaphorin 3E Immunoassay Catalog Number DSM3E0 For the quantitative determination of human Semaphorin 3E (Sema3E) concentrations in cell culture supernates, cell lysates, serum, plasma, and human milk. 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 MATERIALS PROVIDED 3 STORAGE 4 OTHER SUPPLIES REQUIRED 4 PRECAUTIONS 4 SAMPLE COLLECTION AND STORAGE 5 CELL LYSIS PROCEDURE 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 11 SAMPLE VALUES 12 SPECIFICITY 13 REFERENCES 13 PLATE LAYOUT 14 MANUFACTURED AND DISTRIBUTED BY: R&D Systems, Inc. TELEPHONE: (800) 343-7475 614 McKinley Place NE (612) 379-2956 Minneapolis, MN 55413 FAX: (612) 656-4400 United States of America E-MAIL: info@RnDSystems.com DISTRIBUTED BY: R&D Systems Europe, Ltd. 19 Barton Lane TELEPHONE: +44 (0)1235 529449 Abingdon Science Park FAX: +44 (0)1235 533420 Abingdon, OX14 3NB E-MAIL: info@RnDSystems.co.uk United Kingdom R&D Systems China Co. Ltd. 24A1 Hua Min Empire Plaza TELEPHONE: +86 (21) 52380373 726 West Yan An Road FAX: +86 (21) 52371001 Shanghai PRC 200050 E-MAIL: info@RnDSystemsChina.com.cn INTRODUCTION Semaphorin 3E (Sema3E; also known as SemaH and Collectin-5) is an 85 - 90 kDa member of the semaphorin family of molecules (1 - 4). It is a covalently-linked, 170 kDa secreted homodimeric glycoprotein that is synthesized as a 775 amino acid (aa) precursor (1, 2). Human Sema3E contains a 25 aa signal sequence plus a 750 aa mature segment. The mature molecule contains one Sema domain that mediates ligand binding (aa 32 - 516), a plexin repeat that orients the ligand-binding motif (aa 519 - 566) and one C2-type Ig-like domain (aa 581 - 669). Cys729 mediates homodimerization (2). There are two potential splice variants. One shows an alternative start site at Met 61 (5), while a second shows a deletion of aa 753 - 761 (6). Proteolytic processing also generates multiple isoforms. Cleavage occurs between Arg560:Gln561 to create both a 61 kDa monomeric form (aa 26 - 560) and a 25 kDa C-terminal form (aa 561 - 775). The C-terminal fragment forms two disulfide-linked dimers; a 50 kDa complex with itself, and a 110 kDa complex with one full-length Sema3E molecule (2). Additional cleavage occurs at the extreme C-terminus between Arg770 and His771. Mature human Sema3E shares 91% aa sequence identity with mouse Sema3E (1, 2). Cells reported to secrete Sema3E include podocytes (7), melanocytes (8), embryonic mesoderm (9), thymic medullary cells (10), tufted and mitral cells of the olfactory bulb (11), and glutamatergic neurons of cortical layer VI (12) plus spinal motor neurons (13). Two receptors for Sema3E have been reported. The first is plexin D1, a 220 kDa type I transmembrane glycoprotein that is found on endothelial cells, neurons, and thymocytes (9, 10, 12, 14). Sema3E binds directly to plexin D1, and signaling occurs without any contribution from an accessory receptor component (9). Plexin D1 binding transmits a repulsive signal into D1-expressing cells. The second receptor is neuropilin-1/NP-1, a 120 - 130 kDa type I transmembrane glycoprotein that also serves as a component of the VEGF receptor (13, 15, 16). It is not clear if Sema3E binds directly to NP-1 (9, 12), but when linked to plexin D1, this receptor complex transmits permissive or attractive signals and counters the repulsive effects of D1 binding alone (13, 17). Notably, repulsion vs. attraction is not defined simply by the receptor. Under circumstances where full-length Sema3E signals cell repulsion, the 61 kDa isoform described above will signal attraction in the same system (2). Sema3E exhibits repulsive and attractive/permissive activities in multiple systems. In the circulatory system, Sema3E is reported to block angiogenesis initiated by VEGF. Although the exact mechanism is unclear, it is suggested that Sema3E is able to attract NP-1 to its D1 receptor, thereby removing a crucial component of the VEGF R2 signaling complex (18). During somite development, Sema3E is expressed by somatic mesoderm, thus deflecting in-growing endothelial cells away from the somite and into the intersomitic mesenchyme (9). During thymocyte development, Sema3E is expressed by undefined thymic medullary cells. At the double positive (DP) stage, cortical DP thymocytes are D1+ , and Sema3E is argued to keep such cells out of the medulla. Following maturation into single positive cells, membrane thymocyte D1 is down regulated, and an obstacle to thymocyte migration is removed (10). In the spinal cord, during development, in-growing axons from proprioceptive (joint sensation) sensory neurons are directed to appropriate motor neuron synapses by the coordinated efforts of Sema3E:D1 plus other undefined Sema:plexin combinations (13). The Quantikine Human Semaphorin 3E Immunoassay is a 4.5 hour solid phase ELISA designed to measure human Sema3E in cell culture supernates, cell lysates, serum, plasma, and human milk. It contains NS0-expressed recombinant human Sema3E and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human Sema3E showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that the Quantikine Human Semaphorin 3E kit can be used to determine relative mass values for naturally occurring human Sema3E. 2 PRINCIPLE OF THE ASSAY This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Sema3E has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Sema3E present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for Sema3E 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 Sema3E 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 the samples with the appropriate Calibrator Diluent and 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 proteins, and other factors present in biological samples. Until all factors have been tested in the Quantikine Immunoassay, the possibility of interference cannot be excluded. MATERIALS PROVIDED Semaphorin 3E Microplate (Part 893378) - 96 well polystyrene microplate (12 strips of 8 wells) coated with a mouse monoclonal antibody against Sema3E. Semaphorin 3E Conjugate (Part 893379) - 21 mL of a monoclonal antibody against Sema3E conjugated to horseradish peroxidase with preservatives. Semaphorin 3E Standard (Part 893380) - 200 ng of recombinant human Sema3E in a buffered protein solution with preservatives; lyophilized. Assay Diluent RD1-103 (Part 895950) - 11 mL of a buffered protein solution with preservatives. Calibrator Diluent RD5K (Part 895119) - 21 mL of a buffered protein solution with preservatives. For cell culture supernate samples. Calibrator Diluent RD6-15 (Part 895244) - 21 mL of animal serum with preservatives. For cell lysates, serum, plasma, and human milk samples. 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. Opened/ Reconstituted Reagents Diluted Wash Buffer May be stored for up to 1 month at 2 - 8° C.* Stop Solution Assay Diluent RD1-103 Calibrator Diluent RD5K Calibrator Diluent RD6-15 Conjugate Unmixed Color Reagent A Unmixed Color Reagent B Standard Microplate Wells Return unused wells to the foil pouch containing the 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. · Squirt bottle, manifold dispenser, or automated microplate washer. · 500 mL graduated cylinder. · Horizontal orbital microplate shaker (0.12" orbit) capable of maintaining a speed of 500 ± 50 rpm. · Polypropylene test tubes for dilution. · Human Semaphorin 3E Controls (optional; available from R&D Systems). If using cell lysate samples, the following are also required: · Cell Lysis Buffer 5 (R&D Systems, Catalog # 895890) · Aprotinin (Sigma, Catalog # A6279) · Leupeptin (Sigma, Catalog # L8511) · Sodium metavanadate (NaVO3) (Sigma, Catalog # 590088) PRECAUTIONS The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. Sema3E is detectable in saliva. Take precautionary measures to prevent contamination of kit reagents while running this assay. 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. Cell Lysates - Cells must be lysed prior to assay. Refer to the Cell Lysis Procedure below. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at ? -20° C. Avoid repeated freeze-thaw cycles. Plasma - Collect plasma on ice using EDTA or heparin as an anticoagulant. Centrifuge at 2 - 8° C 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. Human Milk - Centrifuge for 15 minutes at 10,000 x g at 2 - 8° C. Collect the aqueous fraction and repeat this process a total of 3 times. Filter through a 0.2 mm filter and assay immediately or aliquot and store samples at ? -20° C. Avoid repeated freeze-thaw cycles. CELL LYSIS PROCEDURE Use the following procedure for the preparation of cell lysate samples. 1. Resuspend 10 x 106 cells/mL with 1 mL of Cell Lysis Buffer 5, 5 mg/mL Aprotinin, 5 mg/mL Leupeptin, and 1 mM NaVO3. 2. Incubate for 30 minutes at room temperature with periodic vortexing. 3. Centrifuge for 10 minutes at top speed to remove cell debris. 4. Aliquot the lysis supernatant, and store at ? -20° C until ready for use. SAMPLE PREPARATION Cell lysates and human milk samples require a 2-fold dilution. A suggested 2-fold dilution is 100 mL of sample + 100 mL of Calibrator Diluent RD6-15. Cell culture supernate samples may require concentration. See the Sample Values section for more information. 5 REAGENT PREPARATION Bring all reagents to room temperature before use. Note: Sema3E is found in saliva. It is recommended that a face mask and gloves be used to protect kit reagents from contamination. 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 light. 200 mL of the resultant mixture is required per well. Semaphorin 3E Standard - Reconstitute the Semaphorin 3E Standard with 1.0 mL of deionized or distilled water. This reconstitution produces a stock solution of 200 ng/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. Use polypropylene tubes. Pipette 900 mL of Calibrator Diluent RD5K (for cell culture supernate samples) or Calibrator Diluent RD6-15 (for cell lysates/serum/plasma/human milk samples) into the 20 ng/mL tube. Pipette 500 mL of the appropriate Calibrator Diluent into the remaining tubes. Use the stock solution to produce a dilution series (below). Mix each tube thoroughly before the next transfer. The 20 ng/mL standard serves as the high standard. The appropriate Calibrator Diluent serves as the zero standard (0 ng/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. Note: Sema3E is found in saliva. It is recommended that a face mask and gloves be used to protect kit reagents from contamination. 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, and reseal. 3. Add 100 mL of Assay Diluent RD1-103 to each well. 4. Add 50 mL 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 mL) using a squirt bottle, 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 mL of Semaphorin 3E 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 mL of Substrate Solution to each well. Incubate for 30 minutes at room temperature on the benchtop. Protect from light. 9. Add 50 mL 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. *Samples may require dilution. See the Sample Preparation section. 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. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the Sema3E concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. TYPICAL DATA These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed. 9 ng/mL 0 0.313 0.625 1.25 2.5 5 10 20 O.D. 0.023 0.023 0.062 0.065 0.105 0.107 0.182 0.189 0.340 0.358 0.656 0.684 1.255 1.322 2.308 2.350 Average 0.023 0.064 0.106 0.186 0.349 0.670 1.289 2.329 Corrected ___ 0.041 0.083 0.163 0.326 0.647 1.266 2.306 ng/mL 0 0.313 0.625 1.25 2.5 5 10 20 O.D. 0.016 0.018 0.057 0.060 0.102 0.104 0.192 0.197 0.374 0.386 0.718 0.729 1.379 1.404 2.486 2.513 Average 0.017 0.059 0.103 0.195 0.380 0.724 1.392 2.500 Corrected ___ 0.042 0.086 0.178 0.363 0.707 1.375 2.483 TECHNICAL HINTS · 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. · 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. 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. Cell Culture Supernate Assay Intra-assay Precision Inter-assay Precision Sample 1 2 3 1 2 3 n 20 20 20 40 40 40 Mean (ng/mL) 2.84 5.74 11.3 3.02 6.08 11.8 Standard deviation 0.10 0.16 0.22 0.17 0.28 0.51 CV (%) 3.5 2.8 1.9 5.6 4.6 4.3 Cell Lysate/Serum/Plasma/Human Milk Assay Intra-assay Precision Inter-assay Precision Sample 1 2 3 1 2 3 n 20 20 20 40 40 40 Mean (ng/mL) 3.30 6.56 12.3 3.25 6.53 12.7 Standard deviation 0.13 0.22 0.46 0.23 0.34 0.60 CV (%) 3.9 3.4 3.7 7.0 5.1 4.7 10 RECOVERY The recovery of Sema3E spiked to levels throughout the range of the assay was evaluated. Sample Average % Recovery Range Cell culture media (n=4) 101 99 - 105% Serum (n=4) 102 93 - 113% Heparin plasma (n=4) 98 88 - 109% EDTA plasma (n=4) 101 91 - 111% LINEARITY To assess the linearity of the assay, samples containing and/or spiked with high concentrations of Sema3E were serially diluted with the appropriate Calibrator Diluent to produce samples with values within the dynamic range of the assay. Cell culture media (n=4) Cell lysates* (n=4) Serum (n=4) Heparin plasma (n=4) EDTA plasma (n=4) Human milk* (n=4) 1:2 Average % of Expected 99 97 - 102 109 102 - 118 100 95 - 102 101 97 - 105 99 96 - 102 104 95 - 111 Range (%) 1:4 Average % of Expected 99 96 - 100 106 93 - 113 102 95 - 106 103 96 - 109 103 96 - 107 101 89 - 112 Range (%) 1:8 Average % of Expected 98 93 - 102 114 112 - 115 101 92 - 109 102 94 - 112 103 94 - 112 110 ___ Range (%) 1:16 Average % of Expected 95 92 - 97 104 ___ 96 88 - 102 98 90 - 107 99 89 - 109 115 ___ Range (%) *Samples were diluted prior to assay as directed in the Sample Preparation section. SENSITIVITY Seventy-nine assays were evaluated and the minimum detectable dose (MDD) of Sema3E ranged from 0.009 - 0.134 ng/mL. The mean MDD was 0.037 ng/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. CALIBRATION This immunoassay is calibrated against a highly purified NS0-expressed recombinant human Sema3E produced at R&D Systems. 11 SAMPLE VALUES Serum/Plasma - Thirty-six samples from apparently healthy volunteers were evaluated for the presence of Sema3E in this assay. No medical histories were available for the donors used in this study. All samples measured less than the lowest standard, 0.313 ng/mL. Human Milk - Five samples from apparently healthy volunteers were evaluated for the presence of Sema3E in this assay. No medical histories were available for the donors used in this study. Sample Type Mean of Detectable (ng/mL) % Detectable Range (ng/mL) Human milk (n=5) 5.75 80 ND - 16.2 ND = Non-detectable Cell Culture Supernates* and Cell Lysates - Growth conditions are listed below. Cell lysates were prepared according to the Cell Lysis Procedure on page 5. Human peripheral blood lymphocytes (PBLs) were cultured in DMEM supplemented with 5% bovine calf serum, 5 mM b-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate. Cells were cultured unstimulated or stimulated with 10 mg/mL PHA for 1 and 5 days. Aliquots of the cell culture supernates were removed and assayed for levels of natural Sema3E. All samples measured less than the lowest standard, 0.313 ng/mL. Human breast cancer cells (MDA-MB-468) were cultured in L-15 medium supplemented with 10% bovine calf serum in an incubator with no CO2. An aliquot of the cell culture supernate was removed, assayed for levels of natural Sema3E, and measured 2.94 ng/mL. Lysates of the same cells measured 12.6 ng/mL. Human cervix carcinoma cells (ME-180) were cultured in McCoy's 5a media supplemented with 10% bovine calf serum. An aliquot of the cell culture supernate was removed, assayed for levels of natural Sema3E, and measured 1.11 ng/mL. Lysates of the same cells measured 4.80 ng/mL. Human mammary gland carcinoma cells (BT-20) were cultured in Eagle's MEM supplemented with 10% bovine calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate. After cells were split, 10 - 12 mL of media was added to a T75 flask, and the cells were grown to 90% confluence (3 - 5 days). An aliquot of the cell culture supernate was removed, concentrated, assayed for levels of natural Sema3E, and measured 1.83 ng/mL. Lysates of the same cells measured 4.40 ng/mL. Human breast carcinoma cells (ZR-75-1) were cultured in DMEM supplemented with 10% bovine calf serum, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate. After cells were split, 10 - 12 mL of media was added to a T75 flask, and the cells were grown to 90% confluence (3 - 5 days). An aliquot of the cell culture supernate was removed, concentrated, assayed for levels of natural Sema3E, and measured 6.46 ng/mL. Lysates of the same cells measured 1.31 ng/mL. Human melanoma, metastatic to lymph nodes (Hs294-T), were cultured in 90% DMEM supplemented with 10% bovine calf serum, 4.5 g/L glucose, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate. After cells were split, 10 - 12 mL of media was added to a T75 flask, and the cells were grown to 90% confluence (3 - 5 days). An aliquot of the cell culture supernate was removed, concentrated, assayed for levels of natural Sema3E, and measured 1.57 ng/mL. *Prior to assay, cell culture supernates from the BT-20, ZR-75-1, and Hs294-T cell lines were added to a centrifugal filter unit (15 mL; 30,000 molecular weight cut-off filter) and centrifuged at 2 - 8° C for 20 - 30 minutes at 4000 rpm. An aliquot of the retentate was assayed for levels of natural Sema3E. 12 SPECIFICITY This assay recognizes recombinant and natural human Sema3E. The factors listed below were prepared at 200 ng/mL in Calibrator Diluent and assayed for cross-reactivity. Preparations of the following factors at 200 ng/mL in a mid-range recombinant human Sema3E control were assayed for interference. No significant cross-reactivity or interference was observed. Recombinant human Sema3F cross-reacts at appoximately 0.15% in this assay. Recombinant mouse Sema3E cross-reacts at appoximately 15.3% in this assay. REFERENCES 1. Christensen, C. et al. (1998) Cancer Res. 58:1238. 2. Christensen, C. et al. (2005) Cancer Res. 65:6167. 3. Neufeld, G. and O. Kessler (2008) Nat. Rev. Cancer 8:632. 4. Flannery, E. and M. Duman-Scheel (2009) Curr. Drug Targets 10:611. 5. GenBank Accession # BAG64853. 6. GenBank Accession # BAG59693. 7. Guan, F. et al. (2006) Kidney Int. 69:1564. 8. Roodink, I. et al. (2008) Am. J. Pathol. 173:1873. 9. Gu, C. et al. (2005) Science 307:265. 10. Choi, Y.I. et al. (2008) Immunity 29:888. 11. Miyazaki, N. et al. (1999) Neuroscience 93:401. 12. Watakabe, A. et al. (2006) J. Comp. Neurol. 499:258. 13. Pecho-Vrieseling, E. et al. (2009) Nature 459:842. 14. van der Zwaag, B. et al. (2002) Dev. Dyn. 225:336. 15. Soker, S. et al. (1998) Cell 92:735. 16. He, Z. and M. Tessier-Lavigne (1997) Cell 90:739. 17. Chauvet, S. et al. (2007) Neuron 56:807. 18. Moriya, J. et al. (2010) Circ. Res. 106:391. 13 Recombinant human: Neuropilin-2 Plexin-D1 Sema3A Sema3C Sema3D Sema4A Sema4B Sema4G Sema6A Sema6B Sema6C Sema6D Sema7A PLATE LAYOUT Use this plate layout as a record of standards and samples assayed. 14 NOTES ? 2010 R&D Systems, Inc. 05.10 752045.0 5/10 15
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Quantikine? Human Semaphorin 3E Immunoassay Catalog Number DSM3E0 For the quantitative determination of human Semaphorin 3E (Sema3E) concentrations in cell culture supernates, cell lysates, serum, plasma, and human milk. 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 MATERIALS PROVIDED 3 STORAGE 4 OTHER SUPPLIES REQUIRED 4 PRECAUTIONS 4 SAMPLE COLLECTION AND STORAGE 5 CELL LYSIS PROCEDURE 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 11 SAMPLE VALUES 12 SPECIFICITY 13 REFERENCES 13 PLATE LAYOUT 14 MANUFACTURED AND DISTRIBUTED BY: R&D Systems, Inc. TELEPHONE: (800) 343-7475 614 McKinley Place NE (612) 379-2956 Minneapolis, MN 55413 FAX: (612) 656-4400 United States of America E-MAIL: info@RnDSystems.com DISTRIBUTED BY: R&D Systems Europe, Ltd. 19 Barton Lane TELEPHONE: +44 (0)1235 529449 Abingdon Science Park FAX: +44 (0)1235 533420 Abingdon, OX14 3NB E-MAIL: info@RnDSystems.co.uk United Kingdom R&D Systems China Co. Ltd. 24A1 Hua Min Empire Plaza TELEPHONE: +86 (21) 52380373 726 West Yan An Road FAX: +86 (21) 52371001 Shanghai PRC 200050 E-MAIL: info@RnDSystemsChina.com.cn INTRODUCTION Semaphorin 3E (Sema3E; also known as SemaH and Collectin-5) is an 85 - 90 kDa member of the semaphorin family of molecules (1 - 4). It is a covalently-linked, 170 kDa secreted homodimeric glycoprotein that is synthesized as a 775 amino acid (aa) precursor (1, 2). Human Sema3E contains a 25 aa signal sequence plus a 750 aa mature segment. The mature molecule contains one Sema domain that mediates ligand binding (aa 32 - 516), a plexin repeat that orients the ligand-binding motif (aa 519 - 566) and one C2-type Ig-like domain (aa 581 - 669). Cys729 mediates homodimerization (2). There are two potential splice variants. One shows an alternative start site at Met 61 (5), while a second shows a deletion of aa 753 - 761 (6). Proteolytic processing also generates multiple isoforms. Cleavage occurs between Arg560:Gln561 to create both a 61 kDa monomeric form (aa 26 - 560) and a 25 kDa C-terminal form (aa 561 - 775). The C-terminal fragment forms two disulfide-linked dimers; a 50 kDa complex with itself, and a 110 kDa complex with one full-length Sema3E molecule (2). Additional cleavage occurs at the extreme C-terminus between Arg770 and His771. Mature human Sema3E shares 91% aa sequence identity with mouse Sema3E (1, 2). Cells reported to secrete Sema3E include podocytes (7), melanocytes (8), embryonic mesoderm (9), thymic medullary cells (10), tufted and mitral cells of the olfactory bulb (11), and glutamatergic neurons of cortical layer VI (12) plus spinal motor neurons (13). Two receptors for Sema3E have been reported. The first is plexin D1, a 220 kDa type I transmembrane glycoprotein that is found on endothelial cells, neurons, and thymocytes (9, 10, 12, 14). Sema3E binds directly to plexin D1, and signaling occurs without any contribution from an accessory receptor component (9). Plexin D1 binding transmits a repulsive signal into D1-expressing cells. The second receptor is neuropilin-1/NP-1, a 120 - 130 kDa type I transmembrane glycoprotein that also serves as a component of the VEGF receptor (13, 15, 16). It is not clear if Sema3E binds directly to NP-1 (9, 12), but when linked to plexin D1, this receptor complex transmits permissive or attractive signals and counters the repulsive effects of D1 binding alone (13, 17). Notably, repulsion vs. attraction is not defined simply by the receptor. Under circumstances where full-length Sema3E signals cell repulsion, the 61 kDa isoform described above will signal attraction in the same system (2). Sema3E exhibits repulsive and attractive/permissive activities in multiple systems. In the circulatory system, Sema3E is reported to block angiogenesis initiated by VEGF. Although the exact mechanism is unclear, it is suggested that Sema3E is able to attract NP-1 to its D1 receptor, thereby removing a crucial component of the VEGF R2 signaling complex (18). During somite development, Sema3E is expressed by somatic mesoderm, thus deflecting in-growing endothelial cells away from the somite and into the intersomitic mesenchyme (9). During thymocyte development, Sema3E is expressed by undefined thymic medullary cells. At the double positive (DP) stage, cortical DP thymocytes are D1+ , and Sema3E is argued to keep such cells out of the medulla. Following maturation into single positive cells, membrane thymocyte D1 is down regulated, and an obstacle to thymocyte migration is removed (10). In the spinal cord, during development, in-growing axons from proprioceptive (joint sensation) sensory neurons are directed to appropriate motor neuron synapses by the coordinated efforts of Sema3E:D1 plus other undefined Sema:plexin combinations (13). The Quantikine Human Semaphorin 3E Immunoassay is a 4.5 hour solid phase ELISA designed to measure human Sema3E in cell culture supernates, cell lysates, serum, plasma, and human milk. It contains NS0-expressed recombinant human Sema3E and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human Sema3E showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that the Quantikine Human Semaphorin 3E kit can be used to determine relative mass values for naturally occurring human Sema3E. 2 PRINCIPLE OF THE ASSAY This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Sema3E has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Sema3E present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for Sema3E 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 Sema3E 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 the samples with the appropriate Calibrator Diluent and 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 proteins, and other factors present in biological samples. Until all factors have been tested in the Quantikine Immunoassay, the possibility of interference cannot be excluded. MATERIALS PROVIDED Semaphorin 3E Microplate (Part 893378) - 96 well polystyrene microplate (12 strips of 8 wells) coated with a mouse monoclonal antibody against Sema3E. Semaphorin 3E Conjugate (Part 893379) - 21 mL of a monoclonal antibody against Sema3E conjugated to horseradish peroxidase with preservatives. Semaphorin 3E Standard (Part 893380) - 200 ng of recombinant human Sema3E in a buffered protein solution with preservatives; lyophilized. Assay Diluent RD1-103 (Part 895950) - 11 mL of a buffered protein solution with preservatives. Calibrator Diluent RD5K (Part 895119) - 21 mL of a buffered protein solution with preservatives. For cell culture supernate samples. Calibrator Diluent RD6-15 (Part 895244) - 21 mL of animal serum with preservatives. For cell lysates, serum, plasma, and human milk samples. 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. Opened/ Reconstituted Reagents Diluted Wash Buffer May be stored for up to 1 month at 2 - 8° C.* Stop Solution Assay Diluent RD1-103 Calibrator Diluent RD5K Calibrator Diluent RD6-15 Conjugate Unmixed Color Reagent A Unmixed Color Reagent B Standard Microplate Wells Return unused wells to the foil pouch containing the 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. · Squirt bottle, manifold dispenser, or automated microplate washer. · 500 mL graduated cylinder. · Horizontal orbital microplate shaker (0.12" orbit) capable of maintaining a speed of 500 ± 50 rpm. · Polypropylene test tubes for dilution. · Human Semaphorin 3E Controls (optional; available from R&D Systems). If using cell lysate samples, the following are also required: · Cell Lysis Buffer 5 (R&D Systems, Catalog # 895890) · Aprotinin (Sigma, Catalog # A6279) · Leupeptin (Sigma, Catalog # L8511) · Sodium metavanadate (NaVO3) (Sigma, Catalog # 590088) PRECAUTIONS The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. Sema3E is detectable in saliva. Take precautionary measures to prevent contamination of kit reagents while running this assay. 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. Cell Lysates - Cells must be lysed prior to assay. Refer to the Cell Lysis Procedure below. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at ? -20° C. Avoid repeated freeze-thaw cycles. Plasma - Collect plasma on ice using EDTA or heparin as an anticoagulant. Centrifuge at 2 - 8° C 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. Human Milk - Centrifuge for 15 minutes at 10,000 x g at 2 - 8° C. Collect the aqueous fraction and repeat this process a total of 3 times. Filter through a 0.2 mm filter and assay immediately or aliquot and store samples at ? -20° C. Avoid repeated freeze-thaw cycles. CELL LYSIS PROCEDURE Use the following procedure for the preparation of cell lysate samples. 1. Resuspend 10 x 106 cells/mL with 1 mL of Cell Lysis Buffer 5, 5 mg/mL Aprotinin, 5 mg/mL Leupeptin, and 1 mM NaVO3. 2. Incubate for 30 minutes at room temperature with periodic vortexing. 3. Centrifuge for 10 minutes at top speed to remove cell debris. 4. Aliquot the lysis supernatant, and store at ? -20° C until ready for use. SAMPLE PREPARATION Cell lysates and human milk samples require a 2-fold dilution. A suggested 2-fold dilution is 100 mL of sample + 100 mL of Calibrator Diluent RD6-15. Cell culture supernate samples may require concentration. See the Sample Values section for more information. 5 REAGENT PREPARATION Bring all reagents to room temperature before use. Note: Sema3E is found in saliva. It is recommended that a face mask and gloves be used to protect kit reagents from contamination. 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 light. 200 mL of the resultant mixture is required per well. Semaphorin 3E Standard - Reconstitute the Semaphorin 3E Standard with 1.0 mL of deionized or distilled water. This reconstitution produces a stock solution of 200 ng/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. Use polypropylene tubes. Pipette 900 mL of Calibrator Diluent RD5K (for cell culture supernate samples) or Calibrator Diluent RD6-15 (for cell lysates/serum/plasma/human milk samples) into the 20 ng/mL tube. Pipette 500 mL of the appropriate Calibrator Diluent into the remaining tubes. Use the stock solution to produce a dilution series (below). Mix each tube thoroughly before the next transfer. The 20 ng/mL standard serves as the high standard. The appropriate Calibrator Diluent serves as the zero standard (0 ng/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. Note: Sema3E is found in saliva. It is recommended that a face mask and gloves be used to protect kit reagents from contamination. 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, and reseal. 3. Add 100 mL of Assay Diluent RD1-103 to each well. 4. Add 50 mL 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 mL) using a squirt bottle, 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 mL of Semaphorin 3E 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 mL of Substrate Solution to each well. Incubate for 30 minutes at room temperature on the benchtop. Protect from light. 9. Add 50 mL 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. *Samples may require dilution. See the Sample Preparation section. 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. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the Sema3E concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. TYPICAL DATA These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed. 9 ng/mL 0 0.313 0.625 1.25 2.5 5 10 20 O.D. 0.023 0.023 0.062 0.065 0.105 0.107 0.182 0.189 0.340 0.358 0.656 0.684 1.255 1.322 2.308 2.350 Average 0.023 0.064 0.106 0.186 0.349 0.670 1.289 2.329 Corrected ___ 0.041 0.083 0.163 0.326 0.647 1.266 2.306 ng/mL 0 0.313 0.625 1.25 2.5 5 10 20 O.D. 0.016 0.018 0.057 0.060 0.102 0.104 0.192 0.197 0.374 0.386 0.718 0.729 1.379 1.404 2.486 2.513 Average 0.017 0.059 0.103 0.195 0.380 0.724 1.392 2.500 Corrected ___ 0.042 0.086 0.178 0.363 0.707 1.375 2.483 TECHNICAL HINTS · 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. · 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. 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. Cell Culture Supernate Assay Intra-assay Precision Inter-assay Precision Sample 1 2 3 1 2 3 n 20 20 20 40 40 40 Mean (ng/mL) 2.84 5.74 11.3 3.02 6.08 11.8 Standard deviation 0.10 0.16 0.22 0.17 0.28 0.51 CV (%) 3.5 2.8 1.9 5.6 4.6 4.3 Cell Lysate/Serum/Plasma/Human Milk Assay Intra-assay Precision Inter-assay Precision Sample 1 2 3 1 2 3 n 20 20 20 40 40 40 Mean (ng/mL) 3.30 6.56 12.3 3.25 6.53 12.7 Standard deviation 0.13 0.22 0.46 0.23 0.34 0.60 CV (%) 3.9 3.4 3.7 7.0 5.1 4.7 10 RECOVERY The recovery of Sema3E spiked to levels throughout the range of the assay was evaluated. Sample Average % Recovery Range Cell culture media (n=4) 101 99 - 105% Serum (n=4) 102 93 - 113% Heparin plasma (n=4) 98 88 - 109% EDTA plasma (n=4) 101 91 - 111% LINEARITY To assess the linearity of the assay, samples containing and/or spiked with high concentrations of Sema3E were serially diluted with the appropriate Calibrator Diluent to produce samples with values within the dynamic range of the assay. Cell culture media (n=4) Cell lysates* (n=4) Serum (n=4) Heparin plasma (n=4) EDTA plasma (n=4) Human milk* (n=4) 1:2 Average % of Expected 99 97 - 102 109 102 - 118 100 95 - 102 101 97 - 105 99 96 - 102 104 95 - 111 Range (%) 1:4 Average % of Expected 99 96 - 100 106 93 - 113 102 95 - 106 103 96 - 109 103 96 - 107 101 89 - 112 Range (%) 1:8 Average % of Expected 98 93 - 102 114 112 - 115 101 92 - 109 102 94 - 112 103 94 - 112 110 ___ Range (%) 1:16 Average % of Expected 95 92 - 97 104 ___ 96 88 - 102 98 90 - 107 99 89 - 109 115 ___ Range (%) *Samples were diluted prior to assay as directed in the Sample Preparation section. SENSITIVITY Seventy-nine assays were evaluated and the minimum detectable dose (MDD) of Sema3E ranged from 0.009 - 0.134 ng/mL. The mean MDD was 0.037 ng/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. CALIBRATION This immunoassay is calibrated against a highly purified NS0-expressed recombinant human Sema3E produced at R&D Systems. 11 SAMPLE VALUES Serum/Plasma - Thirty-six samples from apparently healthy volunteers were evaluated for the presence of Sema3E in this assay. No medical histories were available for the donors used in this study. All samples measured less than the lowest standard, 0.313 ng/mL. Human Milk - Five samples from apparently healthy volunteers were evaluated for the presence of Sema3E in this assay. No medical histories were available for the donors used in this study. Sample Type Mean of Detectable (ng/mL) % Detectable Range (ng/mL) Human milk (n=5) 5.75 80 ND - 16.2 ND = Non-detectable Cell Culture Supernates* and Cell Lysates - Growth conditions are listed below. Cell lysates were prepared according to the Cell Lysis Procedure on page 5. Human peripheral blood lymphocytes (PBLs) were cultured in DMEM supplemented with 5% bovine calf serum, 5 mM b-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate. Cells were cultured unstimulated or stimulated with 10 mg/mL PHA for 1 and 5 days. Aliquots of the cell culture supernates were removed and assayed for levels of natural Sema3E. All samples measured less than the lowest standard, 0.313 ng/mL. Human breast cancer cells (MDA-MB-468) were cultured in L-15 medium supplemented with 10% bovine calf serum in an incubator with no CO2. An aliquot of the cell culture supernate was removed, assayed for levels of natural Sema3E, and measured 2.94 ng/mL. Lysates of the same cells measured 12.6 ng/mL. Human cervix carcinoma cells (ME-180) were cultured in McCoy's 5a media supplemented with 10% bovine calf serum. An aliquot of the cell culture supernate was removed, assayed for levels of natural Sema3E, and measured 1.11 ng/mL. Lysates of the same cells measured 4.80 ng/mL. Human mammary gland carcinoma cells (BT-20) were cultured in Eagle's MEM supplemented with 10% bovine calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate. After cells were split, 10 - 12 mL of media was added to a T75 flask, and the cells were grown to 90% confluence (3 - 5 days). An aliquot of the cell culture supernate was removed, concentrated, assayed for levels of natural Sema3E, and measured 1.83 ng/mL. Lysates of the same cells measured 4.40 ng/mL. Human breast carcinoma cells (ZR-75-1) were cultured in DMEM supplemented with 10% bovine calf serum, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, and 1.5 g/L sodium bicarbonate. After cells were split, 10 - 12 mL of media was added to a T75 flask, and the cells were grown to 90% confluence (3 - 5 days). An aliquot of the cell culture supernate was removed, concentrated, assayed for levels of natural Sema3E, and measured 6.46 ng/mL. Lysates of the same cells measured 1.31 ng/mL. Human melanoma, metastatic to lymph nodes (Hs294-T), were cultured in 90% DMEM supplemented with 10% bovine calf serum, 4.5 g/L glucose, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate. After cells were split, 10 - 12 mL of media was added to a T75 flask, and the cells were grown to 90% confluence (3 - 5 days). An aliquot of the cell culture supernate was removed, concentrated, assayed for levels of natural Sema3E, and measured 1.57 ng/mL. *Prior to assay, cell culture supernates from the BT-20, ZR-75-1, and Hs294-T cell lines were added to a centrifugal filter unit (15 mL; 30,000 molecular weight cut-off filter) and centrifuged at 2 - 8° C for 20 - 30 minutes at 4000 rpm. An aliquot of the retentate was assayed for levels of natural Sema3E. 12 SPECIFICITY This assay recognizes recombinant and natural human Sema3E. The factors listed below were prepared at 200 ng/mL in Calibrator Diluent and assayed for cross-reactivity. Preparations of the following factors at 200 ng/mL in a mid-range recombinant human Sema3E control were assayed for interference. No significant cross-reactivity or interference was observed. Recombinant human Sema3F cross-reacts at appoximately 0.15% in this assay. Recombinant mouse Sema3E cross-reacts at appoximately 15.3% in this assay. REFERENCES 1. Christensen, C. et al. (1998) Cancer Res. 58:1238. 2. Christensen, C. et al. (2005) Cancer Res. 65:6167. 3. Neufeld, G. and O. Kessler (2008) Nat. Rev. Cancer 8:632. 4. Flannery, E. and M. Duman-Scheel (2009) Curr. Drug Targets 10:611. 5. GenBank Accession # BAG64853. 6. GenBank Accession # BAG59693. 7. Guan, F. et al. (2006) Kidney Int. 69:1564. 8. Roodink, I. et al. (2008) Am. J. Pathol. 173:1873. 9. Gu, C. et al. (2005) Science 307:265. 10. Choi, Y.I. et al. (2008) Immunity 29:888. 11. Miyazaki, N. et al. (1999) Neuroscience 93:401. 12. Watakabe, A. et al. (2006) J. Comp. Neurol. 499:258. 13. Pecho-Vrieseling, E. et al. (2009) Nature 459:842. 14. van der Zwaag, B. et al. (2002) Dev. Dyn. 225:336. 15. Soker, S. et al. (1998) Cell 92:735. 16. He, Z. and M. Tessier-Lavigne (1997) Cell 90:739. 17. Chauvet, S. et al. (2007) Neuron 56:807. 18. Moriya, J. et al. (2010) Circ. Res. 106:391. 13 Recombinant human: Neuropilin-2 Plexin-D1 Sema3A Sema3C Sema3D Sema4A Sema4B Sema4G Sema6A Sema6B Sema6C Sema6D Sema7A PLATE LAYOUT Use this plate layout as a record of standards and samples assayed. 14 NOTES ? 2010 R&D Systems, Inc. 05.10 752045.0 5/10 15