Parameter™

LTB4 Assay

Catalog Number KGE006

For the quantitative determination of Leukotriene B4 (LTB4)concentrations in cell culture supernates, serum, and plasma.

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 8ASSAY PROCEDURE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9CALCULATION OF RESULTS 10TYPICAL DATA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10PRECISION 11RECOVERY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11LINEARITY 12SENSITIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12SAMPLE VALUES 13SPECIFICITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13REFERENCES 14PLATE LAYOUT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

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 Kingdom

R&D Systems GmbHBorsigstrasse 7 TELEPHONE: +49 (0)6122 9098065205 Wiesbaden-Nordenstadt FAX: +49 (0)6122 909819Germany E-MAIL: infogmbh@RnDSystems.co.uk

R&D Systems Europe77 boulevard Vauban FREEPHONE: +0800 90 72 4959041 LILLE CEDEX FAX: +0800 77 16 68France E-MAIL: info@RnDSystems.co.uk

INTRODUCTIONLeukotriene B4 (LTB4) is a potent pro-inflammatory molecule that belongs to a family ofeicosanoid lipid mediators (1, 2). It is synthesized from arachidonic acid that is generated fromnuclear membrane phospholipid (3). In general, and upon cell activation, phospholipase A2 isdirected to the nuclear membrane. Here, it generates arachidonic acid that is bound by one 18kDa membrane-bound protein termed Five-Lipooxygenase Activating Protein (FLAP).FLAP-bound arachidonic acid is subjected to two distinct, sequential actions by 80 kDa5-lipooxygenase (5-LO). The first 5-LO action generates 5(S)-HETE, an oxygenated form ofarachidonic acid. The newly generated 5(S)-HETE is then acted on again by 5-LO to generateLTA4 (1 - 4). The LTB4 synthetic pathway has two key checkpoints. The first is at the level ofarachidonic acid formation, where COX enzymes offer an alternative to 5-LO in the generationof prostaglandins. The second is at the level of LTA4, where 69 kDa LTA4 hydrolase activitywill generate LTB4 (a pro-inflammatory molecule), and 18 kDa LTC4 synthetase activity willgenerate LTC/D/E4 (smooth muscle contractants) (4, 5). The key enzyme in LTB4 synthesis is5-LO, which has limited cell expression. Cells known to express 5-LO include B cells,macrophages, monocytes, mast cells, neutrophils, neurons, eosinophils and dendritic cells(1, 2). Notably, all cells are potential sources for LTB4 production. This is due to the fact thatLTA4 hydrolase is ubiquitously expressed, and excess LTA4 generated by 5-LO expressingcells can diffuse into neighboring LTA4 hydrolase-containing cells for conversion into LTB4 (5).

LTB4 has two known membrane receptors, and one intracellular receptor. The membranereceptors are named BLT1 and BLT2 (2, 6). Both are 7-transmembrane receptors that induceCa2+ flux when activated. BLT1 is a high-affinity receptor for LTB4 expressed by hematopoieticcells and keratinocytes, while BLT2 is a low-affinity receptor that is widely expressed (6, 7).The intracellular receptor is PPAR-�, which becomes active upon binding to LTB4. Thisactivation promotes the expression of LTB4 degradative enzymes, creating a negativefeedback loop for LTB4 production (1, 8).

LTB4 functions are considered proinflammatory, but vary depending upon the target cell. Onhematopoietic stem cells, LTB4 may induce differentiation into CFU-GEMM and CFU-GMprogenitor cells (9). B cells are induced to differentiate by LTB4, and in the presence of IL-4, torelease IgE (2). LTB4 is also considered essential for Th2-type responses. It drives IL-4 andIL-5 production, and promotes CD4+ cell proliferation while inhibiting CD8+ cell expansion(2, 6, 10). Under the influence of LTB4, endothelial cells bind and promote neutrophiltransmigration (1, 6). LTB4 is perhaps best known for its activity on neutrophils. It promotespolymorphonuclear (PMN) cell migration, blocks PMN apoptosis, and induces neutrophilgranule release in conjunction with reactive oxygen species generation (2, 6). Finally, itactivates macrophage phagocytosis and drives mononuclear proinflammatory cytokine release(2, 6).

R&D Systems’ LTB4 Immunoassay is a 3.5 hour forward sequential competitive enzymeimmunoassay designed to measure LTB4 in cell culture supernates, serum, and plasma.

2

PRINCIPLE OF THE ASSAYThis assay is based on the forward sequential competitive binding technique in which LTB4present in a sample competes with a fixed amount of horseradish peroxidase (HRP)-labeledLTB4 for sites on a chicken polyclonal antibody. During the incubations, the chicken polyclonalantibody becomes bound to the rabbit anti-chicken antibody coated onto the microplate.Following a wash to remove excess conjugate and unbound sample, a substrate solution isadded to the wells to determine the bound enzyme activity. Immediately following colordevelopment, the absorbance is read at 450 nm. The intensity of the color is inverselyproportional to the concentration of LTB4 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 tip 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

REAGENTSRabbit Anti-chicken Microplate (Part 893074) - 96 well polystyrene microplate (12 strips of 8 wells)coated with a rabbit anti-chicken polyclonal antibody.

LTB4 Conjugate (Part 893075) - 6 mL of LTB4 conjugated to horseradish peroxidase with red dye andpreservatives.

LTB4 Standard (Part 893077) - 25 ng of LTB4 in buffer with preservatives. Lyophilized.

Primary Antibody Solution (Part 893076) - 6 mL of a chicken polyclonal antibody to LTB4 in bufferwith blue dye and preservatives.

Calibrator Diluent RD5-44 (Part 895904) - 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 RD5-44

Unmixed Color Reagent A

Unmixed Color Reagent B

Primary Antibody Solution

Conjugate Aliquot and store for up to 1 month at � -20° C in amanual defrost freezer.* Avoid repeated freeze-thawcycles.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.� LTB4 Controls (optional; available from R&D Systems).

If extracting LTB4 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 LTB4 Standard because of the known and unknowneffects of eicosinoids.

SAMPLE COLLECTION AND STORAGESamples containing chicken IgY may interfere with this assay.

Cell Culture Supernates - Remove particulates by centrifugation and assay immediately oraliquot and store samples at � -20° C. Avoid repeated freeze-thaw cycles.

Note: Animal serum used in the preparation of cell culture media may contain high levels ofLTB4. For best results, do not use animal serum for growth of cell cultures when assaying forLTB4 production. If animal serum is used as a supplement in the media, precautions should betaken to prepare the appropriate control and run the control in the immunoassay to determinethe baseline concentration of LTB4.

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 for 15minutes 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 samplesin this assay.

5

SAMPLE PREPARATIONCell culture supernate, serum and plasma samples require a 2-fold dilution. A suggested 2-folddilution is 150 �L sample + 150 �L of Calibrator Diluent RD5-44.

Note: For serum and plasma samples containing low levels of LTB4, LTB4 may be extractedfrom the 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 samples 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 samples as the eluted ethyl acetate solutions at � -70° Cuntil the assay is to be run. To prepare the eluate for assay, evaporate samples under astream of nitrogen. Reconstitute with at least 250 �L of Calibrator Diluent RD5-44. Vortexwell.

Note: It is recommended that a control spike be run with each sample to determine theextraction efficiency. The LTB4 Standard can be used as spiking material.

6

REAGENT PREPARATIONBring all reagents to room temperature before use.Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mixgently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrateinto deionized or distilled water to prepare 500 mL of Wash Buffer.

Substrate Solution - Color Reagents A and B should be mixed together in equal volumeswithin 15 minutes of use. Protect from light. 200 �L of the resultant mixture is required perwell.

LTB4 Standard - Reconstitute the LTB4 Standard with 0.5 mL of deionized or distilled water.This reconstitution produces a stock solution of 50,000 pg/mL. Mix the standard to ensurecomplete reconstitution and allow the standard to sit for a minimum of 15 minutes with gentleagitation prior to making dilutions.

Pipette 950 �L of Calibrator Diluent RD5-44 into the 2500 pg/mL tube. Pipette 500 �L ofCalibrator Diluent RD5-44 into the remaining tubes. Use the 50,000 pg/mL standard stock toproduce a dilution series (below). Mix each tube thoroughly and change pipette tips betweeneach transfer. The 2500 pg/mL standard serves as the high standard and the CalibratorDiluent RD5-44 serves as the zero standard (B0) (0 pg/mL). Use diluted standards within60 minutes of preparation.

7

ASSAY PROCEDUREBring 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 RD5-44 to the NSB wells.

4. Add 100 �L of Calibrator Diluent RD5-44 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. Cover with the adhesive strip provided.Incubate for 1 hour at room temperature on a horizontal orbital microplate shaker (0.12" orbit)set at 500 � 50 rpm.

7. Do not wash the plate. Add 50 �L of LTB4 Conjugate to each well. All wells except theNSB wells will now be violet in color. Cover with a new adhesive strip. Incubate for 2 hoursat room temperature on the shaker.

8. 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.

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

10. 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.

11. 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 nm or 570 nm. If wavelengthcorrection is not available, subtract readings at 540 nm 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 PROCEDURE SUMMARY

9

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

Create a standard curve by reducing the data using computer software capable of generating afour paramater logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plottingthe mean absorbance for each standard on a linear y-axis against the concentration on alogarithmic x-axis and draw the best fit curve through the points on the graph. Do not includethe B0 in the standard curve.

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

Calculate the concentration of LTB4 corresponding to the mean absorbance from the standardcurve.

If samples have been diluted, the concentration read from the standard curve must bemultiplied by the dilution factor.

If samples have been extracted, the concentration read from the standard curve must bedivided by the dilution factor.

TYPICAL DATAThis standard curve is provided for demonstration only. A standard curve should be generatedfor each set of samples assayed.

10

10 100 1000 100000.00

0.25

0.50

0.75

1.00

1.25

0

20

40

60

80

100pg/mL

NSB

0(B0)

39

78

156

312

625

1,250

2,500

O.D.0.0090.0091.2511.2631.1481.1041.0140.9960.8830.8630.6920.6780.4410.4330.2330.2300.1240.110

Average

0.009

1.257

1.126

1.005

0.873

0.685

0.437

0.232

0.117

Corrected

___

1.248

1.117

0.996

0.864

0.676

0.428

0.223

0.108

% B/B0

___

___

90

80

69

54

34

18

9

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.

Intra-assay Precision Inter-assay Precision

Sample 1 2 3 1 2 3

n 20 20 20 40 40 40

Mean(pg/mL)

252 711 2237 205 590 1958

Standarddeviation

13.2 66.4 132 28.2 61.8 177

CV (%) 5.2 9.3 5.9 13.8 10.5 9.0

RECOVERYThe recovery of LTB4 spiked to levels throughout the range of the assay in various matriceswas evaluated.

Sample Average % Recovery Range

Cell culture media (n=4) 100 82 - 116%

Serum (n=4) 93 75 - 108%

EDTA plasma (n=4) 91 78 - 111%

Heparin plasma (n=4) 94 82 - 115%

11

LINEARITYTo assess the linearity of the assay, samples containing and/or spiked with high concentrationsof LTB4 were serially diluted with Calibrator Diluent RD5-44 to produce samples with valueswithin the dynamic range of the assay.

Cell culturemedia(n=4)

Serum(n=4)

Heparinplasma(n=4)

EDTAplasma(n=4)

1:2Average % of Expected 102

96 - 10810194 - 120

10390 - 115

9686 - 107Range (%)

1:4Average % of Expected 100

97 - 1029988 - 105

10083 - 108

9588 - 102Range (%)

1:8Average % of Expected 109

93 - 120106103 - 110

10489 - 121

9791 - 108Range (%)

SENSITIVITYThirty-five assays were evaluated and the minimum detectable dose (MDD) of LTB4 rangedfrom 13.7 - 39.0 pg/mL. The mean MDD was 27.6 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.

12

SAMPLE VALUESSerum/Plasma - Samples were evaluated for the presence of LTB4 in this assay.

Sample Type*Mean of Detectable

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

Serum (n=36) 343 67 ND - 1335

EDTA plasma (n=36) 145 14 ND - 254

Heparin plasma (n=36) 152 28 ND - 235

ND = Non-detectable*Samples were diluted 2-fold as directed in the Sample Preparation section.

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 LTB4.

Condition Day 1 (pg/mL) Day 5 (pg/mL)

Stimulated 245 ND

Unstimulated ND ND

ND = Non-detectable

SPECIFICITYThe factors listed below were prepared at 50 ng/mL in Calibrator Diluent RD5-44 and assayedfor cross-reactivity. Preparations of the following factors at 500 pg/mL in a mid-range LTB4control were assayed for interference. No significant cross-reactivity or interference wasobserved.

13

5(S)-HETE12(S)-HETE

15(S)-HETELeukotriene C4

Leukotriene D4Leukotriene E4

REFERENCES1. Crooks, S.W and R.A. Stockley (1998) Int. J. Biochem. Cell Biol. 30:173.

2. Hedi, H. and G. Norbert (2004) J. Biomed. Biotechnol. 4:99.

3. Yokomizo, T. et al. (2001) Arch. Biochem. Biophys. 385:231.

4. Haeggstrom, J.Z. and A. Wetterholm (2002) Cell. Mol. Life Sci. 59:742.

5. Werz, O. (2002) Curr. Drug Targets Inflamm. Allergy 1:23.

6. Tager, A.M. and A.D. Luster (2003) Prostaglandins Leukot. Essent. Fatty Acids 69:123.

7. Iizuka, Y. et al. (2005) J. Biol. Chem. 280:24816.

8. Kikuta, Y. et al. (2002) Prostaglandins Other Lipid Mediat. 68-69:345.

9. Chung, J.W. et al. (2005) Exp. Mol. Med. 37:45.

10. Terawaki, K. et al. (2005) J. Immunol. 175:4217.

14

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

01.06 751573.0 1/06

15