Parameter™

PGE2 Assay

Catalog Number KGE004

For the quantitative determination of Prostaglandin E2 (PGE2)in cell culture supernates, serum, plasma, and urine.

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 CONTENTSContents Page

INTRODUCTION 2PRINCIPLE OF THE ASSAY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3LIMITATIONS OF THE PROCEDURE 3TECHNICAL HINTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3REAGENTS 4STORAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4OTHER SUPPLIES REQUIRED 5PRECAUTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5SAMPLE COLLECTION AND STORAGE 5SAMPLE PREPARATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6SAMPLE EXTRACTION 6REAGENT PREPARATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7ASSAY PROCEDURE, REGULAR SENSITIVITY ASSAY OPTION 8ASSAY PROCEDURE, HIGH SENSITIVITY ASSAY OPTION . . . . . . . . . . . . . . . . . . . . . . 9ASSAY PROCEDURE SUMMARY, REGULAR SENSITIVITY ASSAY OPTION 10ASSAY PROCEDURE SUMMARY, HIGH SENSITIVITY ASSAY OPTION . . . . . . . . . . . . . . . 11CALCULATION OF RESULTS 12TYPICAL DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12PRECISION 13RECOVERY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13LINEARITY 14SENSITIVITY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14SAMPLE VALUES 15ASSAY CORRELATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16SPECIFICITY 16REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17PLATE LAYOUT 18

MANUFACTURED AND DISTRIBUTED BY:R&D Systems, Inc. TELEPHONE: (800) 343-7475614 McKinley Place NE (612) 379-2956Minneapolis, MN 55413 FAX: (612) 656-4400United States of America E-MAIL: info@RnDSystems.com

DISTRIBUTED BY:R&D Systems Europe, Ltd.19 Barton Lane TELEPHONE: +44 (0)1235 529449Abingdon Science Park FAX: +44 (0)1235 533420Abingdon, OX14 3NB E-MAIL: info@RnDSystems.co.ukUnited KingdomR&D Systems GmbHBorsigstrasse 7 TELEPHONE: +49 (0)6122 9098065205 Wiesbaden-Nordenstadt FAX: +49 (0)6122 909819Germany E-MAIL: infogmbh@RnDSystems.co.ukR&D Systems Europe77 boulevard Vauban FREEPHONE: +0800 90 72 4959041 LILLE CEDEX FAX: +0800 77 16 68France E-MAIL: info@RnDSystems.co.uk

INTRODUCTIONProstaglandins (PG), thromboxanes, and leukotrienes belong to the class of prostanoid fattyacid derivatives of arachidonic acid. Arachidonic acid is liberated from membranephospholipids by the action of phospholipases, metabolized into PGG2 and PGH2 by thecyclooxygenases COX-1 and COX-2, and converted into PGE2 by prostaglandin E synthetase(PGES) (1 - 3). PGE2 is found in many bodily fluids and is inactivated in the lungs and liver byprostaglandin dehydrogenase and cytochrome P-450 monooxygenases. COX and PGES existin different isoforms that are constitutively active or inducible by inflammatory stimuli. PGE2synthesis can be blocked by corticosteroids that inhibit the phospholipases or by nonsteroidalanti-inflammatory drugs (NSAIDs) that inhibit the cyclooxygenases (4, 5). PGE2 is produced bya wide variety of tissues, including bronchiolar, gastrointestinal, vascular, uterine, and bladdersmooth muscle, fetal ductus arteriosus, placenta, brain, renal macula densa cells, testicularLeydig cells, mesenchymal stem cells, monocytes, and macrophages (6 - 17). Increasedamounts of PGE2 are produced in several pathologic conditions, including inflammation andarthritis, fever, tissue injury, endometriosis, and a wide variety of cancers (1, 7, 18 - 21).

The broad range of tissues that produce PGE2 and multiple differentially expressed receptorsand isoforms predict the complexity of PGE2-induced effects. In the kidney, this includesstimulation of renin release and both promotion and inhibition of salt and water absorption(10, 15, 16). PGE2 promotes either smooth muscle relaxation or contraction depending on thetissue (10, 22, 23). It is important in normal joint physiology and is a principal mediator of theinflammatory response to tissue damage (18, 19, 24). It sensitizes peripheral nociceptivenerves and exaggerates inflammatory and neuropathic pain by direct inhibition of spinal cordglycinergic neurotransmission (25). PGE2 also plays a role in hippocampal synaptic plasticityand febrile responses (1, 8). PGE2 activity is linked to the synthesis and release of severalhormones and the fetal hypothalamus-pituitary-adrenal axis (7, 26). PGE2 also stimulatestumor cell proliferation and differentiation as well as tumor-associated neovascularization(27, 28).

R&D Systems’ PGE2 Immunoassay offers two options. The regular sensitivity assay option is a2.5 hour competitive enzyme immunoassay designed to measure PGE2 in cell culturesupernates, urine, serum, and plasma. An overnight high sensitivity assay option is alsoincluded.

2

PRINCIPLE OF THE ASSAYThis assay is based on the competitive binding technique in which PGE2 present in a samplecompetes with a fixed amount of horseradish peroxidase (HRP)-labeled PGE2 for sites on amouse monoclonal antibody. During the incubation, the mouse monoclonal antibody becomesbound to the goat anti-mouse antibody coated onto the microplate. Following a wash toremove excess conjugate and unbound sample, a substrate solution is added to the wells todetermine the bound enzyme activity. The color development is stopped, and the absorbanceis read at 450 nm. The intensity of the color is inversely proportional to the concentration ofPGE2 in the sample.

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, further dilute the samples

with 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 thisassay, the possibility of interference cannot be excluded.

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 separatereservoirs for each reagent.

� Pre-rinse the pipette tips when pipetting.� Pipette standards and samples to the bottom of the wells. Add all other reagents to the

side of the wells to avoid contamination.� To ensure accurate results, proper adhesion of plate sealers during incubation steps is

necessary.� 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 mayimprove assay precision.

� Substrate Solution should remain colorless until added to the plate. Keep SubstrateSolution protected from light. Substrate Solution should change from colorless togradations 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 StopSolution. Wells that are green in color indicate that the Stop Solution has not mixedthoroughly with the Substrate Solution. Gently tap the side of the plate to ensure thoroughmixing.

3

REAGENTSGoat Anti-mouse Microplate (Part 892575) - 96 well polystyrene microplate (12 strips of 8 wells)coated with a goat anti-mouse polyclonal antibody.

PGE2 Conjugate (Part 892926) - 6 mL of PGE2 conjugated to horseradish peroxidase with red dyeand preservatives.

PGE2 Standard (Part 892928) - 0.5 mL (125 ng/mL) of PGE2 in buffer with preservatives.

Primary Antibody Solution (Part 892927) - 6 mL of a mouse monoclonal antibody to PGE2 in bufferwith blue dye and preservatives.

Calibrator Diluent RD5-39 (Part 895882) - 21 mL of a buffered protein base with preservatives.

Wash Buffer Concentrate (Part 895003) - 21 mL of a 25-fold concentrated solution of bufferedsurfactant 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.

STORAGE

Unopened Kit Store at � -20° C in a manual defrost freezer. Do not use past kit expiration date.

Opened/ReconstitutedReagents

Diluted Wash Buffer

May be stored for up to 1 month at 2 - 8° C.*

Stop Solution

Calibrator Diluent RD5-39

Unmixed Color Reagent A

Unmixed Color Reagent B

Primary Antibody Solution

Conjugate

Standard

Microplate WellsReturn unused wells to the foil pouch containing thedesiccant 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.

4

OTHER SUPPLIES REQUIRED� Microplate reader capable of measuring absorbance at 450 nm, with the correction

wavelength set at 540 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.� Indomethacin (Sigma # I7378 or equivalent).� PGE2 Controls (optional; available from R&D Systems).

If extracting PGE2 from the sample matrix, the following supplies are also required:� 2 N HCl, ethanol, hexane, and ethyl acetate.� 100 mg C18 Reverse Phase Columns (Amersham Biosciences, Catalog # RPN1900).� Centrifuge capable of achieving 16,000 x g.

PRECAUTIONSThe Stop Solution provided with the kit is an acid solution. Wear eye, hand, face, and clothingprotection when using this material.

Care should be taken when handling the PGE2 Standard because of the known and unknowneffects of prostaglandins.

SAMPLE COLLECTION AND STORAGEMouse IgG may interfere with this assay. Samples suspected of containing mouse IgGshould be extracted prior to assay.

Cell Culture Supernates - Remove particulates by centrifugation and assay immediately oraliquot 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 beforecentrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot andstore samples at � -20° C. Avoid repeated freeze-thaw cycles.

Plasma - Collect plasma using heparin or EDTA as an anticoagulant. Centrifuge for15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and storesamples at � -20° C. Avoid repeated freeze-thaw cycles.

Note: Citrate plasma has not been validated for use in this assay. Do not use lipemic samples.

Urine - Aseptically collect the first urine of the day (mid-stream), voided directly into a sterilecontainer. Centrifuge to remove particulate matter, assay immediately or aliquot and store at� -20° C� Avoid repeated freeze-thaw cycles. For more information on collecting and handlingurine specimens, refer to the National Committee for Clinical Laboratory Standards (NCCLS)publication GP16-T.

A prostaglandin synthetase inhibitor, such as indomethacin, should be added to serumand plasma samples at approximately 10 �g/mL final concentration.

5

SAMPLE PREPARATIONCell culture supernate samples require a 3-fold dilution. A suggested 3-fold dilution is 150 �Lsample + 300 �L of Calibrator Diluent RD5-39.

All serum, plasma, and urine samples require a 10-fold dilution. A suggested 10-fold dilution is40 �L sample + 360 �L Calibrator Diluent RD5-39.

Note: To avoid dilution of samples containing low levels of PGE2, PGE2 may be extracted fromthe sample matrix as outlined in the section below.

SAMPLE EXTRACTION1. Acidify the sample with 50 �L of 2 N HCl per 1.0 mL of sample. Allow to sit at 2 - 8° C for

15 minutes. Centrifuge the sample at 16,000 x g for 1 minute to remove any precipitate.

2. Prepare the C18 reverse phase column by washing with 10 mL of ethanol followed by10 mL of deionized water.

3. Apply the sample under a slight positive pressure to obtain a flow rate of about0.5 mL/minute. Wash the column with 10 mL of cold deionized water, followed by 10 mL of15% ethanol, and 10 mL of hexane. Elute the sample from the column by the addition of10 mL of ethyl acetate.

4. If analysis is to be delayed, store the sample as the eluted ethyl acetate solutionat � -70° C until the assay is to be run. To prepare the eluate for assay, evaporate thesample under a stream of nitrogen. Reconstitute with at least 250 �L of Calibrator DiluentRD5-39. Vortex well. Extracted samples may be concentrated by reconstituting in less thanthe original volume.

Note: It is recommended that a control spike be run with each sample to determine theextraction efficiency. The PGE2 Standard can be used as the spiking material.

6

Concentration factor = initial volumefinal volume

REAGENT PREPARATIONBring all reagents to room temperature before use.

Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gentlyuntil the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionizedor distilled water to prepare 500 mL of Wash Buffer.

Substrate Solution - Color Reagents A and B should be mixed together in equal volumes within15 minutes of use. Protect from light. 200 �L of the resultant mixture is required per well.

PGE2 Standard, Regular Sensitivity Option - Pipette 980 �L of Calibrator Diluent RD5-39 into the2500 pg/mL tube. Pipette 500 �L of Calibrator Diluent RD5-39 into the remaining tubes. Use the125,000 pg/mL standard stock to produce a dilution series (below). Mix each tube thoroughly andchange pipette tips between each transfer. The 2500 pg/mL standard serves as the high standard andCalibrator Diluent RD5-39 serves as the zero standard (B0) (0 pg/mL). Use diluted standards within60 minutes of preparation.

PGE2 Standard, High Sensitivity Option - Pipette 990 �L of Calibrator Diluent RD5-39 into the1250 pg/mL tube. Pipette 500 �L of Calibrator Diluent RD5-39 into the remaining tubes. Use the125,000 pg/mL standard stock to produce a dilution series (below). Mix each tube thoroughly andchange pipette tips between each transfer. The 1250 pg/mL standard serves as the high standard andCalibrator Diluent RD5-39 serves as the zero standard (B0) (0 pg/mL). Use diluted standards within60 minutes of preparation.

7

ASSAY PROCEDUREREGULAR SENSITIVITY ASSAY OPTION

Bring all reagents and samples to room temperature before use. It is recommendedthat 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 pouchcontaining the desiccant pack, reseal.

3. Add 150 �L of Calibrator Diluent RD5-39 to the NSB wells.

4. Add 100 �L of Calibrator Diluent RD5-39 to the zero standard (B0) wells.

5. Add 100 �L of Standard or sample* to the remaining wells.

6. Add 50 �L of the Primary Antibody Solution to each well (excluding the NSB wells). All wellsexcept the NSB wells will now be blue in color.

7. Add 50 �L of PGE2 Conjugate to each well. All wells except the NSB wells will now beviolet in color. Cover with the adhesive strip provided.

8. Incubate for 2 hours at room temperature on a horizontal orbital microplate shaker(0.12" orbit) set at 500 � 50 rpm.

9. 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, manifolddispenser or autowasher. Complete removal of liquid at each step is essential to goodperformance. After the last wash, remove any remaining Wash Buffer by aspirating ordecanting. Invert the plate and blot it against clean paper towels.

10. Add 200 �L of Substrate Solution to each well. Incubate for 30 minutes at roomtemperature on the benchtop. Protect from light.

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

12. Determine the optical density of each well within 30 minutes, using a microplate reader setto 450 nm. If wavelength correction is available, set to 540 or 570 nm. If wavelengthcorrection is not available, subtract readings at 540 or 570 nm from the readings at450 nm. This subtraction will correct for optical imperfections in the plate. Readings madedirectly at 450 nm without correction may be higher and less accurate.

*Samples require dilution or extraction. See Sample Preparation section.

8

ASSAY PROCEDUREHIGH SENSITIVITY ASSAY OPTION

Bring all reagents and samples to room temperature before use. It is recommendedthat 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 pouchcontaining the desiccant pack, reseal.

3. Add 200 �L of Calibrator Diluent RD5-39 to the NSB wells.

4. Add 150 �L of Calibrator Diluent RD5-39 to the zero standard (B0) wells.

5. Add 150 �L of Standard or sample* to the remaining wells.

6. Add 50 �L of the Primary Antibody Solution to each well (excluding the NSB wells). All wellsexcept the NSB wells will now be blue in color.

7. Add 50 �L of PGE2 Conjugate to each well. All wells except the NSB wells will now beviolet in color. Cover with the adhesive strip provided.

8. Incubate for 16 - 20 hours at 2 - 8° C.

9. 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, manifolddispenser or autowasher. Complete removal of liquid at each step is essential to goodperformance. After the last wash, remove any remaining Wash Buffer by aspirating ordecanting. Invert the plate and blot it against clean paper towels.

10. Add 200 �L of Substrate Solution to each well. Incubate for 20 minutes at roomtemperature on the benchtop. Protect from light.

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

12. Determine the optical density of each well within 30 minutes, using a microplate reader setto 450 nm. If wavelength correction is available, set to 540 or 570 nm. If wavelengthcorrection is not available, subtract readings at 540 or 570 nm from the readings at450 nm. This subtraction will correct for optical imperfections in the plate. Readings madedirectly at 450 nm without correction may be higher and less accurate.

*Samples require dilution or extraction. See Sample Preparation section.

9

ASSAY PROCEDURE SUMMARYREGULAR SENSITIVITY ASSAY OPTION

10

ASSAY PROCEDURE SUMMARYHIGH SENSITIVITY ASSAY OPTION

11

CALCULATION OF RESULTSAverage the duplicate readings for each standard, control, and sample then subtract the average NSBoptical density.

Create a standard curve by reducing the data using computer software capable of generating a fourparamater logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the meanabsorbance for each standard on a linear y-axis against the concentration on a logarithmic x-axis anddraw the best fit curve through the points on the graph. Do not include the B0 in the standard curve.

If desired, % B/B0 can be calculated by dividing the corrected OD for each standard or sample by thecorrected B0 OD and multiplying by 100.

Calculate the concentration of PGE2 corresponding to the mean absorbance from the standard curve.

If samples have been diluted, the concentration read from the standard curve must be multiplied by thedilution factor.

If samples have been extracted, the concentration read from the standard curve must be divided by theconcentration factor.

TYPICAL DATAThese standard curves are provided for demonstration only. A standard curve should be generated foreach set of samples assayed.

12

10 100 1000 100000.00

0.25

0.50

0.75

1.00

1.25

0

20

40

60

80

100

10 100 1000 100000.00

0.50

1.00

1.50

2.00

2.50

0

20

40

60

80

100

pg/mL

NSB

0(B0)

19.6

39

78

156

312

625

1,250

O.D.0.0170.0232.4832.5392.0202.2071.8992.0151.5781.6791.1671.1900.7560.8440.4360.4690.2490.285

Average

0.020

2.501

2.114

1.957

1.629

1.179

0.800

0.453

0.267

Corrected

___

2.481

2.094

1.937

1.609

1.159

0.780

0.433

0.247

% B/B0

___

___

84.4

78.1

64.9

46.7

31.4

17.5

10.0

pg/mL

NSB

0(B0)

39

78

156

312

625

1,250

2,500

O.D.0.0070.0111.2411.2611.1621.2321.0851.1520.9431.0130.7960.8380.5820.5530.3610.3460.2030.198

Average

0.009

1.251

1.197

1.119

0.978

0.817

0.568

0.354

0.201

Corrected

___

1.242

1.188

1.110

0.969

0.808

0.559

0.345

0.192

% B/B0

___

___

95.7

89.4

78.0

65.1

45.0

27.8

15.5

PRECISIONIntra-assay Precision (Precision within an assay)Three samples of known concentration were tested twenty times on one plate to assessintra-assay precision.

Inter-assay Precision (Precision between assays)Three samples of known concentration were tested in forty separate assays to assessinter-assay precision.

Regular Sensitivity Assay Option

Intra-assay Precision Inter-assay Precision

Sample 1 2 3 1 2 3

n 20 20 20 40 40 40

Mean(pg/mL)

100 238 717 114 259 720

Standarddeviation

10.2 21.9 42.5 11.9 23.0 52.0

CV (%) 10.3 9.2 5.9 10.4 8.9 7.2

High Sensitivity Assay Option

Intra-assay Precision Inter-assay Precision

Sample 1 2 3 1 2 3

n 20 20 20 40 40 40

Mean(pg/mL)

90 163 424 88 174 456

Standarddeviation

9.4 17.0 36.1 9.4 18.9 46.3

CV (%) 10.4 10.4 8.5 10.7 10.9 10.2

RECOVERYThe recovery of PGE2 spiked to levels throughout the range of the assay in various matriceswas evaluated in the regular sensitivity assay option. Similar results were obtained with thehigh sensitivity assay option.

Sample Average % Recovery Range

Cell culture media* (n=4) 96 88 - 104%

Serum* (n=4) 96 86 - 109%

EDTA plasma* (n=4) 98 87 - 121%

Heparin plasma* (n=4) 97 87 - 108%

Urine* (n=4) 104 88 - 120%

*Samples were diluted prior to assay as directed in the Sample Preparation section.

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LINEARITYTo assess the linearity of the assay, samples containing and/or spiked with high concentrationsof PGE2 were serially diluted with Calibrator Diluent RD5-39 to produce samples with valueswithin the dynamic range of the assay. The results below were obtained with the regularsensitivity assay option. Similar results were obtained with the high sensitivity assay option.

Cell culturemedia*(n=4)

Serum*(n=4)

Heparinplasma*(n=4)

EDTAplasma*(n=4)

Urine*(n=4)

1:2Average % of Expected 103

100 - 10810197 - 104

102101 - 104

10299 - 104

104102 - 109Range (%)

1:4Average % of Expected 107

105 - 108106100 - 111

10495 - 112

10396 - 108

10593 - 111Range (%)

1:8Average % of Expected 98

90 - 115109100 - 115

9284 - 95

10095 - 111

110101 - 115Range (%)

*Samples were diluted prior to assay as directed in the Sample Preparation section.

SENSITIVITYTwenty-one assays were evaluated in the regular sensitivity assay option and the minimumdetectable dose (MDD) of PGE2 ranged from 18.2 - 36.8 pg/mL. The mean MDD was27.5 pg/mL.

Thirteen assays were evaluated in the high sensitivity assay option and the MDD of PGE2ranged from 8.5 - 13.9 pg/mL. The mean MDD was 10.1 pg/mL.

The MDD was determined by subtracting two standard deviations from the mean opticaldensity value of twenty zero standard replicates and calculating the correspondingconcentration.

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SAMPLE VALUESRegular Sensitivity Assay Option - Serum/Plasma/Urine samples were evaluated for thepresence of PGE2 in the regular sensitivity assay option.

Sample TypeMean of Detectable

(pg/mL) % DetectableRange(pg/mL)

Serum (n=34) 1360 6 ND - 1578

EDTA plasma (n=34) 1138 6 ND - 1247

Heparin plasma (n=34) 1059 6 ND - 1088

Urine (n=27) 1135 33 ND - 1869

ND = Non-detectable

Note: Five extracted serum and plasma samples were tested in the regular sensitivity assayoption and were 100% detectable. The average extraction efficiency was 64%.

High Sensitivity Assay Option - Serum/Plasma/Urine samples were evaluated for thepresence of PGE2 in the high sensitivity assay option.

Sample TypeMean of Detectable

(pg/mL) % DetectableRange(pg/mL)

Serum (n=32) 419 75 ND - 1695

EDTA plasma (n=32) 347 81 ND - 1245

Heparin plasma (n=32) 347 75 ND - 1123

Urine (n=27) 623 93 ND - 1874

ND = Non-detectable

Cell Culture Supernates - Human peripheral blood cells (1 x 106 cells/mL) were cultured inRPMI supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and100 �g/mL streptomycin sulfate. Cells were cultured unstimulated or stimulated with 10 �g/mLPHA. Aliquots of the cell culture supernate were removed and assayed for levels of PGE2.

Regular Sensitivity Assay Option

Condition Day 1 (pg/mL) Day 6 (pg/mL)

Stimulated 874 1280

Unstimulated ND ND

High Sensitivity Assay Option

Condition Day 1 (pg/mL) Day 6 (pg/mL)

Stimulated 721 1242

Unstimulated 71.4 75.9

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ASSAY CORRELATIONSamples were tested in both assay options (all sample types) and graphed to show thecorrelation between the two methods.

SPECIFICITYThe factors listed below were prepared at 50 ng/mL in Calibrator Diluent RD5-39 and assayedfor cross-reactivity. Preparations of the following factors at 300 pg/mL in a mid-range PGE2control were assayed for interference. No interference was observed. Cross-reactivity is listedbelow.

Compound % Cross-reactivity

PGE3 17.5

PGE1 11.9

PGF1� 7.0

PGF2� 6.0

6-keto-PGF1� 2.5

PGA2 < 0.1

PGB1 < 0.1

Thromboxane B2 < 0.1

Arachidonic acid < 0.1

Arachidonylethanolamide < 0.1

16

0 500 1000 1500 20000

500

1000

1500

2000

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17

PLATE LAYOUTUse this plate layout as a record of standards and samples assayed.

18

NOTES

© 2007 R&D Systems, Inc.

01.06 751393.3 1/07

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