human inflammation panel 1 mosaic elisa · 2015. 6. 26. · mosaic kits employ multiplex microarray...

Human Inflammation Panel 1

MosaicTM ELISA

This package insert must be read in its entirety before using this product. For research use only. Not for use in diagnostic procedures.

Catalog Number MEA004

For the simultaneous quantitative determination of concentrations of multiple human inflammation markers in cell culture supernates, serum, and plasma.

MANUFACTURED AND DISTRIBUTED BY:

USA & Canada | R&D Systems, Inc. 614 McKinley Place NE, Minneapolis, MN 55413, USATEL: (800) 343-7475 (612) 379-2956 FAX: (612) 656-4400E-MAIL: [email protected]

DISTRIBUTED BY:

UK & Europe | R&D Systems Europe, Ltd.19 Barton Lane, Abingdon Science Park, Abingdon OX14 3NB, UKTEL: +44 (0)1235 529449 FAX: +44 (0)1235 533420E-MAIL: [email protected]

China | R&D Systems China Co., Ltd.24A1 Hua Min Empire Plaza, 726 West Yan An Road, Shanghai PRC 200050TEL: +86 (21) 52380373 FAX: +86 (21) 52371001E-MAIL: [email protected]

TABLE OF CONTENTS

SECTION PAGE

INTRODUCTION .....................................................................................................................................................................1PRINCIPLE OF ASSAY ............................................................................................................................................................1TECHNICAL HINTS AND LIMITATIONS...........................................................................................................................2MATERIALS PROVIDED & STORAGE CONDITIONS ...................................................................................................3OTHER SUPPLIES REQUIRED .............................................................................................................................................3SAMPLE COLLECTION & STORAGE .................................................................................................................................4SAMPLE PREPARATION........................................................................................................................................................4REAGENT PREPARATION .....................................................................................................................................................5ASSAY PROCEDURE .............................................................................................................................................................6INSTRUMENTATION ..............................................................................................................................................................7SENSITIVITY .............................................................................................................................................................................7CALIBRATION ..........................................................................................................................................................................7CALCULATION OF RESULTS ...............................................................................................................................................7TYPICAL DATA .........................................................................................................................................................................8PRECISION ............................................................................................................................................................................. 11RECOVERY.............................................................................................................................................................................. 13LINEARITY .............................................................................................................................................................................. 15SAMPLE VALUES .................................................................................................................................................................. 17REFERENCES ......................................................................................................................................................................... 18SPECIFICITY ........................................................................................................................................................................... 19PLATE LAYOUT ..................................................................................................................................................................... 20

www.RnDSystems.com 1

INTRODUCTIONInflammation is an adaptive response triggered by injury or exposure to pathogens, and is crucial for survival. It is comprised of a series of cellular and biochemical events that culminate in enhanced vascular permeability and increased movement of plasma and immune cells from the blood into the injured tissues. This facilitates the accumulation of leukocytes at the site of injury and the subsequent elimination of the harmful agent. This acute inflammatory response is well-regulated by pro-inflammatory and anti-inflammatory cytokines, and is typically terminated when the infectious agent is cleared from the body. However, if the activities of pro-inflammatory and anti-inflammatory cytokines become unbalanced, prolonged inflammation and tissue damage can occur. Chronic inflammation has been linked to many disorders including arthritis (1,2), diabetes (3,4), heart disease (1,5,6), and Alzheimer’s disease (7,8). Thus, investigating the activity of cytokines is useful for understanding the mechanisms underlying a wide range of human diseases. The Mosaic Human Inflammation Panel 1 is an excellent tool for the detection of 8 different inflammation biomarkers in the same sample. Mosaic kits employ multiplex microarray technology to provide an accurate, efficient, and economical alternative to conducting multiple traditional ELISA experiments.

IL-10

IL-1β

IL-18 BPa TNF-α ReferenceSpot

IL-1ra IL-6

G-CSF IFN-γ

PRINCIPLE OF ASSAYThe Mosaic Human Inflammation Panel 1 employs a two-site sandwich ELISA technique to simultaneously detect eight inflammation markers in cell culture supernates, serum, and plasma. Multiple capture antibodies that specifically recognize the target inflammation markers have been pre-spotted into each well of a 96 well microplate. Standards and samples are added, and inflammation markers present in the samples are bound by the immobilized antibodies. After washing away unbound material, biotinylated detection antibodies are used to detect the specific inflammation markers. Unbound detection antibodies are washed away and streptavidin-HRP is added. Following an additional wash, chemiluminescent substrate reagents are added to the wells, and a signal proportional to the amount of each cytokine bound in the initial step is produced. Plates are read using a digital camera imaging system, and pixel intensity is measured using an analytical software package.

Figure 1: A visualization of the spot layout per well.

For research use only. Not for use in diagnostic procedures.2

TECHNICAL HINTS AND LIMITATIONS• FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

• This 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.

• Any variation in buffers, operator, pipetting technique, washing technique, instrumentation, and incubation time or temperature and kit age can alter the performance of the kit.

• Variations in sample collection, processing, and storage may cause sample value differences.

• 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.

• Avoid microbial contamination of reagents and buffers.

• To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

• If samples fall outside the dynamic range of the assay, further dilute the samples with the appropriate Calibrator Diluent and repeat the assay.

• Mosaic affords the user the benefit of multianalyte analysis of eight inflammation markers in a complex sample. Multipurpose diluents are used to optimize recovery, linearity, and reproducibility. These diluents may not optimize any single analyte to the same degree that a unique diluent selected for analysis of that analyte can optimize conditions. Therefore, some performance characteristics may be more variable than those for assays designed specifically for single analyte analysis.

• This assay is designed to eliminate interference by ligands, proteins, and other factors present in biological samples. Until all factors have been tested, the possibility of interference cannot be excluded.

• Discrepancies may exist in values obtained for the same analyte utilizing different technologies.

• Only the analytes listed on the enclosed Standard Value Card can be measured with this kit.


MATERIALS PROVIDED & STORAGE CONDITIONSStore the unopened kit at 2-8 °C. Do not use past kit expiration date.

PART PART # DESCRIPTION STORAGE OF OPENED/RECONSTITUTED MATERIAL

Inflammation Microplate

893432 96 well microplate spotted with 8 antibodies against specific cytokines.

Invert the plate, and blot it against clean paper towels to dry the plate. Mark the used wells. Return the plate to the foil pouch containing the desiccant pack, and reseal along entire edge of the zip-seal. May be stored for up to 1 month at 2-8 °C.*

Standard 893434 2 vials of a cocktail of recombinant human cytokines in a buffered protein base with preservatives; lyophilized.

Discard after use. Use a fresh standard for each assay.

Detection Mix 893433 6 mL of a cocktail of antibodies conjugated to biotin with preservatives.

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

Assay Diluent RD1-102

895939 6 mL of a buffered protein base with blue dye and preservatives.

Calibrator Diluent RD5K

895119 21 mL of a buffered protein base with preservatives.

Calibrator Diluent RD6-40

895817 21 mL of a buffered protein base with preservatives.

Wash Buffer Concentrate

895003 2 vials (21 mL/vial) of a 25-fold concentrated solution of buffered surfactant with preservative.

Streptavidin-HRP 895469 6 mL of a streptavidin-horseradish peroxidase conjugate with preservatives.

Substrate 1 895471 3 mL of a buffered solution.Substrate 2 895472 3 mL of a buffered solution.Plate Sealers N/A 8 adhesive strips.

Store at room temperature.Standard Value Card

749177 1 card listing the standard reconstitution volume and concentrations for this lot of standard.

* Provided this is within the expiration date of the kit.

OTHER SUPPLIES REQUIRED• Pipettes and pipette tips.• Deionized or distilled water.• Manifold dispenser, squirt bottle, or automated microplate washer.• 500 mL graduated cylinder for preparing Wash Buffer.• Horizontal orbital microplate shaker (0.12” orbit) capable of maintaining a speed of

500 ± 50 rpm.• Digital Imaging System (for details, visit www.RnDSystems.com/go/ImagingSystems).• Polypropylene test tubes for dilution of standards and samples.


SAMPLE COLLECTION & STORAGEThe sample collection and storage conditions listed below are intended as general guidelines. Sample stability has not been evaluated.

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

Serum - Allow blood samples to clot for 30 minutes at room temperature before centrifuging 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 using EDTA or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles.

Note: Citrate plasma has not been validated for use in this assay. Hemolyzed and icteric samples are not recommended for use in this assay.

SAMPLE PREPARATIONSerum and plasma samples require at least a 2-fold dilution. A suggested 2-fold dilution is 75 μL of sample + 75 μL of Calibrator Diluent RD6-40. Mix thoroughly.

Substrates 1 and 2 are comprised of TMA-6, a product of Lumigen, Inc., Southfield, Michigan, USA, and are covered by the following:

US Patent Numbers: 5,922,558 and 6,858,733

International Patent Numbers: 733,086, 1,019,525, 2,300,071, 1,015,461, 2,002,352,881, ZL02805225.0, and 1,456,716


REAGENT PREPARATIONBring all reagents to room temperature before use.

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

Substrate Solution - Substrates 1 and 2 should be mixed together in equal volumes 2-30 minutes prior to use. Protect from light. 50 μL of the resultant mixture is required per well.

Standard - Reconstitute the Standard Cocktail with Calibrator Diluent RD5K (for cell culture supernate samples) or Calibrator Diluent RD6-40 (for serum/plasma samples). Refer to the Standard Value Card for the reconstitution volume and assigned values. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.

Use polypropylene tubes. Pipette 500 μL of the reconstituted Standard into the Standard 1 tube. Pipette 200 μL of Calibrator Diluent RD5K (for cell culture supernate samples) or Calibrator Diluent RD6-40 (for serum/plasma samples) into the remaining tubes. Use Standard 1 to produce a 3-fold dilution series (below). Mix each tube thoroughly before the next transfer. Standard 1 serves as the high standard. The appropriate Calibrator Diluent serves as the zero standard.

500 µL Std.

Standard Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6

100 µL 100 µL 100 µL 100 µL 100 µL


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

Note: Protect Streptavidin-HRP and the Substrate from light at all times.

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

2. Add 50 μL of Assay Diluent RD1-102 to each well.

3. Add 50 μL of Standard or sample* per well. Securely cover with a plate sealer. 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 as a record of standards and samples assayed.

4. 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 μL) using a squirt bottle, manifold dispenser, or autowasher. 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.

5. Add 50 μL of the Detection Mix to all wells. For Cell Culture Supernate samples: Incubate for 1 hour at room temperature on the shaker set at 500 ± 50 rpm. For Serum/Plasma samples: Incubate for 2 hours at room temperature on the shaker set at 500 ± 50 rpm.

6. Repeat the wash as in step 4.

7. Add 50 μL of Streptavidin-HRP to all wells. Securely cover with a plate sealer and incubate for 30 minutes at room temperature on the shaker set at 500 ± 50 rpm. Protect from light.

8. Repeat the wash as in step 4.

9. Add 50 μL of Substrate Solution to each well. Protect from light.

10. Place the microplate in the imager. Wait no longer than 15 minutes to commence imaging.Note: For details, visit www.RnDSystems.com/go/ImagingSystems.

*Samples may require dilution. See the Sample Preparation section.


INSTRUMENTATIONThe Mosaic ELISA Kits have been validated on the Q-View™ Imager from Quansys Biosciences. Please visit www.RnDSystems.com/go/ImagingSystems for suitable imaging systems and their instructions for use.

SENSITIVITYThirty-nine assays were evaluated and the minimum detectable dose (MDD) was determined by adding two standard deviations to the mean pixel intensity of twenty zero standard replicates and calculating the corresponding concentration.

Analyte Mean (pg/mL) Range (pg/mL)

G-CSF 3.59 1.46-6.18IFN-γ 0.34 0.19-0.56IL-1β 0.11 0.05-0.25IL-1ra 9.51 3.41-18.3IL-6 0.19 0.09-0.42IL-10 0.26 0.07-0.44IL-18 BPa 7.76 3.71-16.9TNF-α 0.66 0.33-1.36

CALIBRATIONThis assay is calibrated against highly purified recombinant human cytokines produced at R&D Systems.

This assay has been correlated to the respective Quantikine® ELISA kits where applicable with slopes of 0.9-1.1 and R2 values greater than 0.9.

CALCULATION OF RESULTSUse the Standard concentrations on the Standard Value Card and calculate 3-fold dilutions for the remaining levels. Average the duplicate readings for each standard and sample and subtract the average zero standard pixel intensity (PI).

Create a standard curve for each analyte by reducing the data using computer software capable of generating a 5-PL curve fit.

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

All trademarks and registered trademarks are the property of their respective owners.


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

Standard (pg/mL) PI Average CorrectedBlank 0 1283 1306 —

1329Standard 1 2600 146,976 147,613 146,307

148,250Standard 2 867 95,892 97,200 95,894

98,508Standard 3 289 34,207 34,880 33,574

35,553Standard 4 96.3 10,632 10,994 9688

11,355Standard 5 32.1 3887 3901 2595

3914Standard 6 10.7 1955 2000 694

2045


1524Standard 1 1000 146,489 147,721 146,164

148,952Standard 2 333 116,248 117,319 115,763

118,390Standard 3 111 61,655 62,015 60,459

62,375Standard 4 37 24,627 24,958 23,401

25,288Standard 5 12.4 9017 9276 7720

9535Standard 6 4.12 3857 3896 2339

3934


1396Standard 1 180 138,003 138,115 136,792

138,226Standard 2 60 101,251 101,850 100,527

102,448Standard 3 20 49,289 48,170 46,847

47,050Standard 4 6.7 18,434 18,792 17,469

19,149Standard 5 2.22 6273 6655 5332

7037Standard 6 0.74 2829 2730 1407

2631

CALIBRATOR DILUENT RD6-40



3374Standard 1 6400 120,349 120,590 117,173

120,830Standard 2 2133 79,406 78,927 75,510

78,447Standard 3 711 36,188 36,040 32,623

35,891Standard 4 237 14,633 14,561 11,145

14,489Standard 5 79 7293 7394 3978

7495Standard 6 26.3 4435 4395 978

4354


1227Standard 1 490 146,961 147,346 146,149

147,730Standard 2 163 120,141 120,448 119,251

120,754Standard 3 54.4 66,213 66,183 64,986

66,152Standard 4 18.1 26,559 26,673 25,477

26,787Standard 5 6.05 8657 8872 7676

9087Standard 6 2.02 3267 3347 2150

3426


1239Standard 1 600 145,478 146,442 145,174

147,406Standard 2 200 116,361 118,476 117,208

120,590Standard 3 66.7 65,293 66,711 65,443

68,128Standard 4 22.2 26,829 27,778 26,510

28,726Standard 5 7.41 9057 9439 8171

9820Standard 6 2.47 3582 3590 2322

3597

TYPICAL DATA CONTINUEDCALIBRATOR DILUENT RD6-40


TYPICAL DATA CONTINUEDCALIBRATOR DILUENT RD6-40


1179Standard 1 11,400 151,824 151,860 150,682

151,896Standard 2 3800 122,648 122,895 121,717

123,142Standard 3 1267 61,284 60,489 59,311

59,693Standard 4 422 22,399 22,419 21,241

22,439Standard 5 141 7130 7412 6234

7694Standard 6 46.9 2664 2663 1485

2661


1293Standard 1 680 151,042 150,878 149,574

150,714Standard 2 227 116,864 116,584 115,280

116,304Standard 3 75.6 53,856 53,480 52,176

53,104Standard 4 25.2 18,258 18,346 17,042

18,433Standard 5 8.4 6253 6321 5017

6389Standard 6 2.8 2773 2717 1413

2661


PRECISIONIntra-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 seventy-five separate assays to assess inter-assay precision.

G-CSF Intra-Assay Precision Inter-Assay Precision

Sample 1 2 3 1 2 3

n 20 20 20 75 75 75Mean (pg/mL) 28.3 137 634 28.8 135 613Standard deviation 1.88 3.40 12.9 3.22 9.18 27.6CV (%) 6.7 2.5 2.0 11.2 6.8 4.5

IFN-γ Intra-Assay Precision Inter-Assay Precision

Sample 1 2 3 1 2 3

n 20 20 20 75 75 75Mean (pg/mL) 9.50 46.4 214 9.73 46.7 208Standard deviation 0.54 1.72 8.32 1.58 5.52 24.5CV (%) 5.7 3.7 3.9 16.3 11.8 11.8

IL-1β Intra-Assay Precision Inter-Assay Precision

Sample 1 2 3 1 2 3

n 20 20 20 75 75 75Mean (pg/mL) 2.40 12.2 57.2 2.47 11.5 54.5Standard deviation 0.13 0.32 1.89 0.26 1.09 3.32CV (%) 5.5 2.7 3.3 10.4 9.4 6.1

IL-1ra Intra-Assay Precision Inter-Assay Precision

Sample 1 2 3 1 2 3



PRECISION CONTINUED

IL-6 Intra-Assay Precision Inter-Assay Precision

Sample 1 2 3 1 2 3

n 20 20 20 75 75 75Mean (pg/mL) 5.70 27.4 134 5.60 26.3 124Standard deviation 0.23 1.02 4.72 0.50 1.74 6.03CV (%) 4.0 3.7 3.5 8.8 6.6 4.9

IL-10 Intra-Assay Precision Inter-Assay Precision

Sample 1 2 3 1 2 3


IL-18 BPa Intra-Assay Precision Inter-Assay Precision

Sample 1 2 3 1 2 3

n 20 20 20 75 75 75Mean (pg/mL) 125 585 2880 116 552 2491Standard deviation 6.40 22.8 117 9.68 32.1 139CV (%) 5.1 3.9 4.0 8.3 5.8 5.6

TNF-α Intra-Assay Precision Inter-Assay Precision

Sample 1 2 3 1 2 3

n 20 20 20 75 75 75Mean (pg/mL) 24.8 104 385 25.0 99.3 347Standard deviation 1.04 3.59 18.5 2.61 7.74 24.9CV (%) 4.2 3.5 4.8 10.4 7.8 7.2


RECOVERYThe recovery of cytokines spiked to levels throughout the range of the assay in various matrices was evaluated.

G-CSF

Sample Type Average % Recovery Range

Cell culture supernates 100 94-105%Serum* 87 83-92%EDTA plasma* 88 82-93%Heparin plasma* 90 86-98%

IFN-γ



IL-1β



IL-1ra


Cell culture supernates 103 94-106%Serum* 108 100-116%EDTA plasma* 107 98-114%Heparin plasma* 110 105-114%*Samples were diluted prior to assay as directed in the Sample Preparation section.


RECOVERY CONTINUEDIL-6



IL-10



IL-18 BPa


Cell culture supernates 106 102-110%

TNF-α


Cell culture supernates 100 95-105%Serum* 89 87-94%EDTA plasma* 92 80-100%Heparin plasma* 91 85-94%*Samples were diluted prior to assay as directed in the Sample Preparation section.


LINEARITYTo assess the linearity of the assay, samples containing and/or spiked with high concentrations of cytokines were serially diluted with the appropriate Calibrator Diluent to produce samples with values within the dynamic range of the assay.

G-CSF Cell culture supernates

Serum*

EDTA plasma*

Heparin plasma*

1:2Average % of Expected 103 105 110 108Range (%) 100-105 101-107 108-113 104-112



IFN-γ Cell culture supernates

Serum*

EDTA plasma*

Heparin plasma*




IL-1β Cell culture supernates

Serum*

EDTA plasma*

Heparin plasma*




IL-1ra Cell culture supernates

Serum*

EDTA plasma*

Heparin plasma*




*Samples were diluted prior to assay.


LINEARITY CONTINUED

IL-6 Cell culture supernates

Serum*

EDTA plasma*

Heparin plasma*




IL-10 Cell culture supernates

Serum*

EDTA plasma*

Heparin plasma*




IL-18 BPa Cell culture supernates

Serum*

EDTA plasma*

Heparin plasma*




TNF-α Cell culture supernates

Serum*

EDTA plasma*

Heparin plasma*




*Samples were diluted prior to assay.


SAMPLE VALUESSerum/Plasma - Samples from apparently healthy volunteers were evaluated in this assay. No medical histories were available for the donors used in this study.

G-CSFSample Type Mean of Detectable (pg/mL) % Detectable Range (pg/mL)

Serum* (n=20) 29.9 95 ND-59.6EDTA plasma* (n=20) 28.2 85 ND-57.0Heparin plasma* (n=20) 31.1 45 ND-49.6

IL-1βSample Type Mean of Detectable (pg/mL) % Detectable Range (pg/mL)

Serum* (n=20) 1.67 15 ND-1.80EDTA plasma* (n=20) ND 0 ____

Heparin plasma* (n=20) 1.40 5 ND-1.40

IL-1raSample Type Mean (pg/mL) Range (pg/mL) Standard Deviation (pg/mL)

Serum* (n=20) 368 152-1209 233EDTA plasma* (n=20) 255 83-865 178Heparin plasma* (n=20) 329 107-966 199

IL-6Sample Type Mean of Detectable (pg/mL) % Detectable Range (pg/mL)

Serum* (n=20) 5.04 45 ND-8.80EDTA plasma* (n=20) 5.15 40 ND-7.60Heparin plasma* (n=20) 4.82 45 ND-7.00

IL-10Sample Type Mean of Detectable (pg/mL) % Detectable Range (pg/mL)

Serum* (n=20) ND 0 ____

EDTA plasma* (n=20) 2.60 10 ND-2.60Heparin plasma* (n=20) 2.87 15 ND-3.00

IL-18 BPaSample Type Mean (pg/mL) Range (pg/mL) Standard Deviation (pg/mL)

Serum* (n=20) 13,675 10,826-22,608 2561EDTA plasma* (n=20) 13,343 9462-17,928 2389Heparin plasma* (n=20) 12,397 8999-17,985 2557*Samples were diluted prior to assay as directed in the Sample Preparation section. ND=Non-detectable


SAMPLES VALUES CONTINUEDTNF-α

Sample Type Mean of Detectable (pg/mL) % Detectable Range (pg/mL)

Serum* (n=20) 8.79 75 ND-12.4EDTA plasma* (n=20) 7.40 55 ND-9.60Heparin plasma* (n=20) 8.22 55 ND-10.2*Samples were diluted prior to assay as directed in the Sample Preparation section. ND=Non-detectable

Twenty serum and plasma samples were tested for IFN-γ and no detectable levels were observed.

Cell Culture Supernates - Human peripheral blood mononuclear cells (PBMC; 1 x 106 cells/mL) were cultured in RPMI supplemented with 10% fetal calf serum, 50 µM β-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin sulfate. The cells were cultured stimulated with 10 µg/mL PHA for 1 day. An aliquot of the cell culture supernate was removed and assayed in the Mosaic ELISA.

Cell Line

(pg/mL)

G-CSF IFN-γ IL-1β IL-1ra IL-6 IL-10 IL-18 BPa TNF-α

PBMC + PHA 52.4 436 46.4 1998 6300 201 84.7 1170

REFERENCES1. Libby, P. (2008) Am. J. Med. 121:s21.2. Taylor, P.C. and M. Feldmann (2009) Nat. Rev. Rheumatol. 5:578.3. Goldberg, R.B. (2009) J. Clin. Endocrinol. Metab. 94:3174.4. Ehses, J.A. et al. (2009) Arch Physiol. Biochem. 115:240.5. Packard R.R. and P. Libby (2008) Clin. Chem. 54:24.6. Ward, J.R. (2009) Clin. Exp. Immunol. 156:386.7. Miklossy, J. and R.N. Martins (2008) J. Alzheimers Dis. 13:1875.8. Holmes, C. et al. (2009) Neurology 73.768.


SPECIFICITYThis assay recognizes natural and recombinant proteins. This assay also recognizes recombinant viral IL-10 (EBV).

The following factors were assayed for cross-reactivity and interference in the Mosaic Human Inflammation Panel. Less than 1% cross-reactivity or interference was observed.

Recombinant human:4-1BB Ligand4-1BBAPRILBAFF RBCMACCL2/MCP-1CCL23/MPIF-1CCL27/CTACKCD30 LigandCLC/CNTF RαCNTFCT-1CXCL12/SDF-1αCXCL12/SDF-1βEDAEDA-A2Fas LigandGITRGITR LigandIL-1αIL-1F7/FIL-1ζIL-1F10/IL-1HY2IL-2IL-11IL-13IL-15 RαIL-18IL-21IL-22IL-36γ/IL-1F9IL-36 Rα/IL-1F5LIGHT

LIFLIF RαLymphotoxin-α1/β2Lymphotoxin-α2/β1MSP β chainOLFM2OSMOX40 LigandTACITL1ATNF-βTNFR1TNFR2TPOTRAILTRANCE/RANK LTWEAKVEGF206

Recombinant mouse:G-CSFIFN-γIL-1αIL-1βIL-1raIL-6IL-10IL-18BPcIL-18BPdTNF-α

Recombinant rat:IFN-γIL-1αIL-1βIL-1raIL-6IL-10TNF-α

Recombinant cotton rat:IFN-γIL-1βIL-6IL-10TNF-α

Recombinant guinea pig:IL-1βIL-10TNF-α

Recombinant feline:IFN-γIL-6IL-10TNF-α

Recombinant canine:G-CSFIFN-γIL-1βIL-6IL-10TNF-α

Recombinant porcine:IFN-γIL-1αIL-1βIL-1raIL-6IL-10TNF-α

Recombinant equine:IFN-γIL-1βIL-1raIL-6IL-10TNF-α

Recombinant bovine:IFN-γTNF-α

Recombinant viral:IL-10 (HCMV)MCV-type II Chemokine-like proteinMIP-1

Other recombinants:primate TNF-αrabbit TNF-α

The following factors cross-react at the percentages listed below:

Factor IFN-γ IL-1β

Recombinant primate IFN-γ 6.90% ___

Recombinant feline IL-1β ___ 1.10%Recombinant primate IL-1β ___ 26.9%

Recombinant mouse IL-18 interferes with IL-18 BPa at levels ≥ 10 ng/mL.


PLATE LAYOUTUse this plate layout to record standards and samples assayed.


NOTES

human inflammation panel 1 mosaic elisa · 2015. 6. 26. · mosaic kits employ multiplex microarray...

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