QAM CYT 2000

Quantibody® Mouse Cytokine Array Q-2000 --- Quantitative measurement of 60 mouse cytokines Patent Pending Technology Us...

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Quantibody® Mouse Cytokine Array Q-2000 --- Quantitative measurement of 60 mouse cytokines

Patent Pending Technology User Manual (Version Aug 07)

Quantibody® Mouse Cytokine Array Q-2000 (Combination of Quantibody® mouse cytokine arrays 1, 2, & 3 to quantitatively measure the concentration of 60 mouse cytokines)

Cat # QAM-CYT-Q2000

Quantibody® Mouse Cytokine Array 1 (Cat# QAM-CYT-1) Quantibody® Mouse Cytokine Array 2 (Cat# QAM-CYT-2) Quantibody® Mouse Cytokine Array 3 (Cat# QAM-CYT-3)

RayBiotech, Inc. We Provide You With Excellent Protein Array Systems and Service Tel:(Toll Free) 1-888-494-8555 or 770-729-2992; Fax: 1-888-547-0580; Website:www.raybiotech.com Email: [email protected]

OVERVIEW Quantibody® Mouse Cytokine Array Q-2000 Cytokine Detected 60 Arrays Included

Quantibody® Mouse Cytokine Arrays 1, 2, and 3

Quantibody® Mouse Cytokine Array 1 (20)

GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-17, KC, MCP-1, MCSF, RANTES, TNFα, VEGF

Quantibody® Mouse Cytokine Array 2 (20)

Axl, BLC, CD30, CD40, CXCL16, Eotaxin-2, Fas Ligand, IGFBP-3, IGFBP-5, LIX, L-Selectin, MIG, MIP-1α, MIP-1γ, PF-4, P-Selectin, SDF-1α, TCA-3, sTNF RII, VCAM-1

Quantibody® Mouse Cytokine Array 3 (20)

Eotaxin, E-Selectin, G-CSF, GITR, ICAM-1, IGFBP2, IGFBP-6, IGF-I, IL-12p40p70, Leptin, MCP-5, MDC, MIP-2, MIP-3α, Osteopontin, OPG, Resistin, SCF, TPO, VEGF-D

Format

One standard glass slide is spotted with 16 wells of identical cytokine antibody arrays. Each antibody is arrayed in quadruplicate.

Detection Method

Fluorescence with laser scanner: Cy3 equivalent dye

Sample Volume

50 – 100 μl per array

Reproducibility

CV <20%

Assay duration

4 hrs

Quantibody® Mouse cytokine array Q-2000

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TABLE OF CONTENTS I.

Overview……………………………………….

1

Introduction….....................................................

3

How It Works………………………..…………

5

Materials Provided……………………………..

6

Additional Materials Required…………………

6

III. General Considerations…………………………

7

A. Preparation of Samples………………………

7

B. Handling Glass Chips………………………..

7

C. Incubation……………………………………

7

IV. Protocol…………………………………………

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A. Air Dry the Glass Chip………………………

8

B. Prepare Cytokine Standard Dilutions………..

8

C. Blocking and Incubation…………………….

9

D. Fluorescence Detection………………………

10

E. Data Analysis………………………………..

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Cytokine Array Map…………………………....

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II.

V.

VI. Six-Point Standard Curves & Detection Sensitivity. 13 VII. System Recovery …………………………………… 14 VIII. Troubleshooting Guide…………………………..

16

IX. Sample Raybio® Q Analyzer Output……………… 17 X.

Reference List………………………………….

XI. Experimental Record Form………………………. Quantibody® Mouse cytokine array Q-2000

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18 20

I. Introduction Cytokines play an important role in innate immunity, apoptosis, angiogenesis, cell growth and differentiation. They are involved in interactions between different cell types, cellular responses to environmental conditions, and maintenance of homeostasis. In addition, cytokines are also involved in most disease processes, including cancer and cardiac diseases. The traditional method for cytokine detection and quantification is through the use of an enzyme-linked immunosorbent array (ELISA). In this method, target protein is first immobilized to a solid support. The immobilized protein is then complexed with an antibody that is linked to an enzyme. Detection of the enzyme-complex can then be visualized through the use of a substrate that produces a detectable signal. While the traditional method works well for a single protein, the overall procedure is time consuming and requires a lot of sample. With little sample to work with, conservation of precious small quantities becomes a risky task. Take the advantage of advancement of microarray technology over the last decade; more and more choices are available to the scientist today. A long-standing leader in the field, Raybiotech, has pioneered the development of semi-quantitative cytokine antibody array, in which multiple cytokine antibodies are arrayed on solid support (membrane or glass slide). Detection of multiple cytokines is achieved through a sandwich-like ELISA procedure. Our current RayBio® Human Cytokine Antibody Array C or G series 2000 enables scientists to detect 174 human cytokines in a single experiment rapidly and inexpensively. The array data can be further validated and quantified by using RayBiotech ELISA kits. Our new multiplex Quantibody® Array is another quantum leap forward in protein microarray technology. This glass-chip-based multiplexed sandwich ELISA system enables researchers to accurately determine the concentration of multiple cytokines simultaneously. It combines the advantages of the high detection specificity / sensitivity of ELISA and the high throughput of the arrays. Like a traditional sandwich-based ELISA, it uses a pair of cytokine specific antibodies for detection. A capture antibody is first bound to the glass surface. After incubation with the sample, the target cytokine is trapped on the solid surface. A second biotin-labeled detection antibody is then Quantibody® Mouse cytokine array Q-2000

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added, which can recognize a different isotope of the target cytokine. The cytokine-antibody-biotin complex can then be visualized through the addition of the streptavidin-labeled cy3 equivalent dye using a laser scanner. Unlike the traditional ELISA, Quantibody products use array format. By arraying multiple cytokine specific capture antibodies onto a glass support, multiplex detection of cytokines in one experiment is made possible. In detail, one standard glass slide is spotted with 16 wells of identical cytokine antibody arrays. Each antibody, together with the positive and negative control is arrayed in quadruplicate. The slide comes with a 16-well removable gasket which allows for the process of 16 samples in one slide. Four slide chips can be nested into a tray, which matches a standard microplate and allows for automated robotic high throughput process of 64 arrays simultaneously. For cytokine quantification, the array specific cytokine standards, whose concentration has been predetermined, were provided to generate a five-point standard curve of each cytokine. In a real experiment, standard cytokines and samples will be assayed in each array simultaneously through a sandwich ELISA procedure. By comparing signals from unknown samples to the standard curve, the unknown cytokine concentration in the samples will be determined. Quantibody® Mouse Cytokine Array Q-2000 is the combination of Quantibody® Mouse Cytokine Arrays 1, 2, and 3, where each kit can independently detect 20 distinct mouse cytokines. With Q-2000, researchers can now measure the concentration of 60 different cytokines, chemokines, inflammation factors, and angiogenesis factors in a single experiment from as little as 150 μl samples. This is by far one of the most efficient products on the market for cytokine quantification. Quantibody® array kits have been confirmed to have similar detection sensitivity as traditional ELISA. Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool for drug and biomarker discovery.

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How it works Array support

Samples

Incubation of Sample With arrayed antibody Supports

1 hr

Cocktail of Biotin-Ab Incubation with Biotinylated Ab Labeled – streptavidin

Incubation with Cy3 equivalent dye Labeled- streptavidin Detection of signals

Data analysis and graph

Quantibody® Mouse cytokine array Q-2000

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1 hr

1 hr

II. Materials Provided Upon receipt, all the components of the Quantibody® Array kit should be stored at -200C. At -200C the kit will retain complete activity for up to 6 months. Once thawed, the glass chip, cytokine standard mix, detection antibody cocktail and Cy3 equivalent dye-conjugated Streptavidin should be kept at –200C and all other components should be stored at 40C. The entire kit should be used within 6 months of purchase. Components* Quantity

Item Description

1 2 3 4 5 6 7 8 9 10

Quantibody® Array Glass Chip Sample Diluent 20X Wash Buffer I 20X Wash Buffer II Lyophilized cytokine standard mix Detection antibody cocktail Cy3 equivalent dye-conjugated Streptavidin Slide Washer/Dryer Adhesive device sealer Manual

1+1+1 1 3 3 1+1+1 1+1+1 3 1 10 1

* There are three independent sets of reagents for Quantibody® mouse cytokine arrays 1, 2, and 3. Among all the reagents, the glass chip, lyophilized cytokine standard mix, and detection antibody cocktail are array specific, while all the other reagents are suitable for all the three arrays.

Additional Materials Required • Orbital shaker • Laser scanner for fluorescence detection • Aluminum foil • Distilled water • 1.5ml Polypropylene microcentrifuge tubes

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III. General Considerations A. Preparation of Samples • Use serum-free conditioned media if possible. • If serum-containing conditioned media is required, it is highly recommended that complete medium be used as a control since many types of sera contains cytokines. • We recommend the following parameters for your samples: 50 to 100 μl of original or diluted serum, plasma or cell culture supernatant or 20-200 μg of protein for cell lysates and tissue lysates. If you experience high background or the readings exceed the detection range, further dilution your sample is recommended. B. Handling glass chips • Do not touch the surface of the slides, as the microarray slides are very sensitive. Hold the slides by the edges only. • Handle all buffers and slides with latex free gloves. • Avoid breaking glass slide. • Handle glass chip in clean environment. C. Incubation • Completely cover array area with sample or buffer during incubation. • Avoid foaming during incubation steps. • Perform all incubation and wash steps under gentle rotation. • Cover the incubation chamber with adhesive film during incubation, particularly when incubation is more than 2 hours or <70 μl of sample or reagent is used. • Avoid cross-contamination from overflowing solution to neighboring wells. • Several incubation steps such as step 6 (blocking), step 7 (sample incubation), step 10 (Detection antibody incubation) or step 13 (Cy3 equivalent dye-streptavidin incubation) may be done at 40C for overnight. Please make sure to cover the incubation chamber tightly to prevent evaporation.

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IV. Protocol Note: There are three sets of reagents for three different arrays. Be careful to use the glass chip, lyophilized cytokine standard, and the detection antibody cocktail for the same array. Following is the procedure for processing any one of the arrays in the kit. A. Complete air dry the glass chip 1. Take out the glass chip from the box; remove it from the plastic bag; peel off the covering film, and let it air dry at room temperature for at least 1-2 hours. Note: Incomplete drying of slides before use may cause the formation of “comet tails”. B. Prepare Cytokine Standard Dilutions Note: Reconstitute the lyophilized standard within one hour of usage.

Prepare serial dilution of cytokine standards 100μl 100μl 100μl 100μl Add 500μl Sample Diluent 200μl 200μl 200μl 200μl Vial Labels

Std1

Std2

Std3

Std4

Std5

100μl CNTRL

2. Reconstitute the Cytokine Standard Mix (lyophilized) by adding 500μl Sample Diluent to the tube. Dissolve the powder thoroughly by a gentle mix. Labeled the tube as Std1. Quantibody® Mouse cytokine array Q-2000

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3. Label 4 clean microcentrifuge tubes as Std 2 to Std 5. Add 200μl Sample Diluent to each of the tubes. 4. Pipette 100μl Std1 into tube Std2 and mix gently. Perform 3 more serial dilutions by adding 100ul Std2 to tube Std3 and so on. 5. Add 100μl Sample Diluent to another tube labeled as CNTRL. Do not add standard cytokines or sample to the CNTRL tube, which will be used as negative control. C. Blocking and Incubation 6. Add 100μl Sample Diluent into each well and incubate at room temperature for 30 min to block slides. 7. Decant buffer from each well. Add 100μl standard cytokines or samples to each well. Incubate arrays at room temperature for 1 hour. (Be careful to use the corresponding cytokine standard for the matching glass slide.) Note: The sample volume can be 50-100 μl. If sample volume is less than 70 μl, cover the gasket with adhesive sealer to prevent evaporation during incubation. Incubation may be done at 40C for overnight. Note: We recommend using 50 to 100 μl of original or diluted serum, plasma or conditioned media or 20-200 μg of protein for cell lysates and tissue lysates. In order to minimize the matrix effects and to lower the background of the assay, we recommended that the samples at least diluted 2 folds with Sample Diluent. Dilute the lysate at least 5 folds with Sample Diluent to make a total volume of 50 to 100 μl. Make sure there is no bubble in the wells. Note: The amount of sample used depends on the abundance of cytokines. More samples can be used if signals are too weak. If signals are too strong, the sample can be diluted further. 8. Decant the samples from each well, and wash 5 times with 200 μl of 1x Wash Buffer I and then 2 times with 200 μl of 1x Wash Buffer II at Quantibody® Mouse cytokine array Q-2000

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room temperature with gentle shaking. Completely remove wash buffer in each wash step. Note: avoid solution flowing into neighboring wells. 9. Reconstitute the Detection Antibody by adding 1.4 ml of Sample Diluent to the tube. Spin briefly. (Be careful to use the corresponding detection antibody for the matching glass slide.) Note: the diluted Detection antibodies can be stored at 40C for 2-3 days. 10. Add 80 μl of the detection antibody cocktail to each well. Incubate at room temperature for 1 hour. Note: incubation may be done at 40C for overnight. 11. Wash as directed in step 8. 12. After briefly spinning down, add 1.4 ml of Sample Diluent to Cy3 equivalent dye-conjugated streptavidin tube. Mix gently. 13. Add 80 μl of Cy3 equivalent dye-conjugated streptavidin to each well. Cover the device with aluminum foil to avoid exposure to light or incubate in dark room. Incubate at room temperature for 1 hour. Note: incubation may be done at 40C for overnight. 14. Wash four times with 1x Wash Buffer I. D. Fluorescence Detection 15. Disassemble the device by pushing clips outward from the slide side. Carefully remove the slide from the gasket. Note: Be careful not to touch the surface of the array side Quantibody® Mouse cytokine array Q-2000

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16. Place the slide in the slide washer (50 ml centrifuge tube), add enough 1x Wash Buffer I (about 40 ml) to cover the whole slide, and then gently shake at room temperature for 15 minutes. Decant Wash Buffer I. Wash with 1x Wash Buffer II (about 40 ml) with gentle, and gently shake at room temperature for 5 minutes. Note: This step can be done using slide chamber. 17. Decant Wash Buffer II and remove water droplets by centrifuging at 1,000 rpm for 3 minutes without cap. Note: After the rinse step, proceed immediately for the drying step to prevent the deposit of the watermarks on the slide. 18. The signals can be visualized through use of a laser scanner equipped with a cy3 wavelength such as Axon GenePix. The settings should be: Excitation: 555 nm; Emission: 565 nm; Resolution: 10 um. Make sure that the signal from the standard well containing the highest concentration (Std1) receives the highest possible reading, yet remains unsaturated. Saved the image as a high resolution (16-bit) .tif file. Note: In case the signal intensity for different cytokine varies greatly in the same array, we recommend using multiple scans for the low signal ones. Note: we recommend scanning slide right after experiment. You can also store the slide at 40C in a dry dark container for several days. If you do not have a laser scanner, RayBiotech can provide service for you. Just simply send your slide to us and we will take care of it. E. Data Analysis 19. Data extraction can be done with most of the microarray analysis software (GenePix, ScanArray Express, ArrayVision, or MicroVigene). For quantitative data analysis, our RayBio® Q Analyzer software is available. It gives visual output as well as digital value. More information can be found in section VIII. Quantibody® Mouse cytokine array Q-2000

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V. Cytokine array map QAM-CYT-1 a b c d e f g h i j k

1,2,3,4

5,6,7,8

POS GM-CSF IL-1a IL-2 IL-4 IL-6 IL-10 IL-13 KC M-CSF TNFa

NEG IFNg IL-1b IL-3 IL-5 IL-9 IL-12 IL-17 MCP-1 RANTES VEGF

QAM-CYT-2 a b c d e f g h i j k

1,2,3,4

5,6,7,8

POS Axl CD30 CXCL16 Fas L IGFBP5 L-Selectin MIP-1a PF4 SDF-1a sTNFRII

NEG BLC CD40 Eotaxin-2 IGFBP3 LIX MIG MIP-1g P-Selectin TCA-3 VCAM-1

QAM-CYT-3 a b c d e f g h i j k

1,2,3,4

5,6,7,8

POS Eotaxin G-CSF ICAM-1 IGFBP-6 IL-12p40/70 MCP-5 MIP-2 Osteopontin Resistin TPO

NEG E-Selectin GITR IGFBP-2 IGF-I Leptin MDC MIP-3a OPG SCF VEGF-D

Alexa Fluor 555 dye

B

Biotin-Streptavidin complex Detect antibody Cytokine Capture antibody Glass Slide Support

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VI. Six-point cytokine standard curves and detection sensitivity Cytokines Axl BLC CD30 CD40 CXCL16 Eotaxin Eotaxin-2 E-Selectin Fas Ligand GCSF GITR GM-CSF ICAM-1 IGFBP-2 IGFBP-3 IGFBP-5 IGFBP-6 IGF-I IL-10 IL-12 IL-12 p40p70 IL-13 IL-17 IL-1α IL-1β IL-2 IL-3 IL-4 IL-5 IL-6 IL-9 INFγ KC LEPTIN(OB) LIX L-Selectin MCP-1 MCP-5 M-CSF MDC MIG MIP-1α MIP-1γ MIP2 MIP-3a

Std1 4,000 10,000 4,000 2,000 500 1000 1,000 4000 4,000 1000 4000 2000 10000 20000 20,000 20,000 20000 8000 10000 50000 400 40000 4000 20000 80000 10000 10000 1000 10000 3000 100000 80000 40000 40000 20,000 40,000 4000 1000 1000 2000 10,000 10,000 1,000 2000 8000

Std2 1333 3333 1333 667 167 333 333 1333 1333 333 1333 666 3333 6667 6667 6667 6667 2667 3333 16667 133 13333 1333 6667 26666 3333 3333 333 3333 1000 33333 26666 13333 13333 6667 13333 1333 333 333 667 3333 3333 333 667 2667

Std3 444 1111 444 222 56 111 111 444 444 111 444 222 1111 2222 2222 2222 2222 889 1111 5556 44 4444 444 2222 8889 1111 1111 111 1111 333 11111 8889 4444 4444 2222 4444 444 111 111 222 1111 1111 111 222 889

Std4 148 370 148 74 19 37 37 148 148 37 148 74 370 741 741 741 741 296 370 1852 15 1481 148 741 2963 370 370 37 370 111 3700 2963 1481 1481 741 1481 148 37 37 74 370 370 37 74 296

Std5 49 123 49 25 6 12 12 49 49 12 49 25 123 247 247 247 247 99 123 617 5 494 49 247 988 123 123 12 123 37 1230 988 494 494 247 494 49 12 12 25 123 123 12 25 99

Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Sensitivity (pg/ml) 5.7 11.2 1.0 6.1 0.3 2.7 1.2 11.3 47.8 4.5 6.2 4.0 19.3 3.8 70.3 54.0 5.5 16.3 17.0 121.0 0.3 69.0 14.0 48.0 80.0 11.0 23.0 2.0 31.0 6.0 197.0 155.0 115.0 76.4 62.6 310.1 5.0 1.1 3.0 16.1 13.0 6.2 3.7 14.8 33.3

(To be continued) Quantibody® Mouse cytokine array Q-2000

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Cytokines OPG Osteopontin PF-4 P-Selectin RANTES Resistin SCF SDF-1α sTNF RII TCA-3 TNFα TPO VCAM-1 VEGF VEGF-D

Std1 2000 10000 40,000 20,000 40000 1000 10000 40,000 1,000 1,000 40000 40000 8,000 6000 4000

Std2 667 3333 13333 6667 13333 333 3333 13333 333 333 13333 13333 2667 2000 1333

Std3 222 1111 4444 2222 4444 111 1111 4444 111 111 4444 4444 889 667 444

Std4 74 370 1481 741 1481 37 370 1481 37 37 1481 1481 296 222 148

Std5 25 123 494 247 494 12 123 494 12 12 494 494 99 74 49

Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Sensitivity (pg/ml) 16.7 37.1 91.7 11.9 33.0 1.9 32.4 117.2 1.0 1.8 74.0 71.9 7.4 13.0 8.4

VII. System Recovery The recovery of the mouse antigens by the kit was tested through spiking different levels of the recombinant proteins in both diluted mouse serum and mouse cell culture media NIH 3T3 (CM). The non-spiked serum sample and cell culture media were used as negative control. The recovery rate for each antigen was then determined by subtracting the endogenous antigen level from the observed value and divided by the spiking antigen concentration. Quantibody Mouse Cytokine Array 1 (in 2x diluted CM and Serum) Cytokine GM-CSF IFNg IL-1a IL-1b IL-2 IL-3 IL-4 IL-5 IL-6 IL-9 IL-10 IL-12 IL-13 IL-17 KC MCP-1 M-CSF RANTES TNFa VEGF

Spiking (pg/ml) 1,000 40,000 10,000 40,000 5,000 5,000 500 5,000 1,500 50,000 5,000 25,000 20,000 2,000 20,000 2,000 500 20,000 20,000 3,000

CM 47 0 0 218 0 0 0 0 34 0 0 0 0 0 0 over 17 0 0 2,787

Quantibody® Mouse cytokine array Q-2000

CM+Ag 868 52,982 12,295 49,953 3,927 3,823 482 5,102 1,453 49,153 5,448 22,182 26,127 2,265 19,984 over 459 21,114 20,043 6,147

CM% 82% 132% 123% 124% 79% 76% 96% 102% 95% 98% 109% 89% 131% 113% 100% 88% 106% 100% 112%

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Serum 62 24,109 0 6,442 0 2,685 0 67 181 0 228 0 0 57 0 0 0 138 486 332

Serum+Ag 694 57,501 8,847 46,944 5,038 8,245 448 4,525 1,436 52,989 4,471 18,772 20,982 2,088 19,948 2,016 327 21,900 20,460 2883

Serum% 63% 83% 88% 101% 101% 111% 97% 89% 84% 106% 85% 75% 105% 102% 100% 101% 65% 109% 100% 85%

Quantibody Mouse Cytokine Array 2 (in 10x diluted CM and Serum) Cytokine Axl BLC CD30 CD40 CXCL16 Eotaxin-2 Fas L IGFBP3 IGFBP5 LIX L-Selectin MIG MIP-1α MIP-1γ PF4 P-Selectin SDF-1α TCA-3 sTNFRII VCAM-1

Spiking (pg/ml) 2000 5000 2000 1000 250 500 2000 10000 10000 10000 10000 7000 5000 500 15000 8000 20000 500 500 4000

CM 11 3 0 2 0 1 1 5 2 11 9 0 0 4 0 0 13 0 8 525

CM+Ag 1861 5202 1891 1017 261 507 2076 9453 7863 10381 10352 7351 4441 464 14322 7987 17891 530 439 4515

CM% 92.5% 104.0% 94.6% 101.6% 104.4% 101.3% 103.8% 94.5% 78.6% 103.7% 103.4% 105.0% 88.8% 92.0% 95.5% 99.8% 89.4% 105.9% 86.3% 99.7%

Serum 251 135 14 8 13 5 1 over 1928 490 over 150 3 476 over over 2 5 877 over

Serum+Ag 2079 5041 2084 1000 247 433 1547 over 9646 8452 over 8054 4012 1023 over over 17695 497 1326 over

Serum% 91.4% 98.1% 103.5% 99.2% 93.7% 85.6% 77.3% 77.2% 79.6% 112.9% 80.2% 109.4% 88.5% 98.4% 89.7% -

Quantibody Mouse Cytokine Array 3 (in 10x diluted CM and Serum) Cytokine Eotaxin E-Selectin G-CSF GITR ICAM-1 IGFBP-2 IGFBP-6 IGF-I IL-12p40/70 Leptin MCP-5 MDC MIP-2 MIP-3a Osteopontin OPG Resistin SCF TPO VEGF-D

Spiking (pg/ml) 500 1600 300 1600 3500 4000 14000 3200 200 12000 500 1500 800 5000 800 1200 1600 4000 14000 1500

CM 0 0 1 0 0 0 0 0 0 10 0 0 0 0 over 0 0 0 0 0

Quantibody® Mouse cytokine array Q-2000

CM+Ag 484 1556 313 1591 3559 4641 15617 2977 145 9399 447 1201 888 5027 over 1185 1447 3426 12170 1214

CM% 96.8% 97.2% 104.0% 99.4% 101.7% 116.0% 111.5% 93.0% 72.3% 78.2% 89.5% 80.1% 111.0% 100.5% 98.7% 90.4% 85.7% 86.9% 80.9%

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Serum 24 626 21 0 175 4 0 11707 10 1119 18 0 0 18 over 2377 over 0 0 0

Serum+Ag 469 2356 266 1609 2828 3124 9815 14898 164 12764 559 1785 647 3713 over 3491 over 4340 13134 1516

Serum% 89.1% 108.1% 81.6% 100.6% 75.8% 78.0% 70.1% 99.7% 76.9% 97.0% 108.1% 119.0% 80.9% 73.9% 92.9% 108.5% 93.8% 101.1%

VIII. Troubleshooting guide Problem

Cause

Recommendation

Inadequate detection Inadequate reagent volumes or improper dilution Short incubation time Weak Signal Too low protein concentration in sample Improper storage of kit

Uneven signal

Bubble formed during incubation Arrays are not completed covered by reagent Reagent evaporation Cross-contamination from neighboring wells Comet tail formation

Poor standard Inadequate detection curve Use freeze-thawed cytokine standards

High background

Overexposure Insufficient wash Dust Slide is allowed to dry out

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Increase laser power and PMT parameters Check pipettes and ensure correct preparation Ensure sufficient incubation time and change sample incubation step to overnight Don’t make too low dilution or concentrate sample Store kit as suggested temperature. Don’t freeze/thaw the slide. Avoid bubble formation during incubation Completely cover arrays with solution Cover the incubation chamber with adhesive film during incubation Avoid overflowing wash buffer Air dry the slide for at least 1 hour before usage Increase laser power that the highest standard concentration for each cytokine receives the highest possible reading yet remains unsaturated. Always use new cytokine standard vial for new set of experiment. Discard any leftover. Lower the laser power Increase wash time and use more wash buffer Work in clean environment Don’t dry out slides during experiment.

VIII. Sample Raybio® Q Analyzer Output Raybio® Q Analyzer greatly facilitates the data analysis. Instead of tedious calculation, user can now quickly figure out the unknown sample concentration through a simple copy and paste process. The program can automatically remove the outlier spots, and users can choose either linear regression or log-log algorithms to best meet their analytical needs. 100000

Density Value (signal-BKG)

MMP-1 MMP-2

10000

MMP-3 MMP-8

1000

MMP-9 MMP-10 100

MMP-13 TIMP-1

10

TIMP-2 TIMP-4

1 10

100

1000

10000

100000

1000000

Cytokine concentration (pg/m l)

TIMP-4

TIMP-4

y = 0.0387x

10

20010

40010

60010

80010

100010 120010

Concentration (pg/ml)

4.00

Log(Signal-BKG)

Signal-BKG

TIMP-4 4500 4000 3500 3000 2500 2000 1500 1000 500 0

3.50 3.00 2.50 2.00 1.50

y = 0.9415x - 1.1507

1.00 0.50 0.00 0.00

Concentration (pg/ml)

1.00

2.00

3.00

4.00

5.00

100000 10000 1000 100

2

y = 0.0003x + 24.896x - 32.129

10 1 1

6.00

10

100

1000

Signal-BKG

Log(Concentration) (pg/ml)

Sample Cytokine Concentration (pg/ml) (Base on Linear Regression) ID MMP-1 MMP-2 MMP-3 MMP-8 MMP-9 MMP-10 MMP-13 TIMP-1 TIMP-2 TIMP-4

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

0 0 0 0 0 0 0 0 0 0

538 69 6 234 2,891 10,952 1,021 1,356 234 2,288

3,626 7,066 1,362 917 9,740 16,428 1,293 1,111 131 5,917

8,202 7,479 3,573 2,029 6,013 37,660 4,167 2,759 1,876 8,094

43,812 26,460 21,670 9,986 14,510 116,437 10,277 6,923 4,139 25,384

96,822 46,335 25,902 18,793 27,976 406,305 18,553 19,838 20,897 46,641

551 1,496 30,364 1,409 37,079 320,779 1,056 71,685 133,539 16,342

2,122 2,802 966 678 19,933 56,489 1,797 9,304 5,705 3,912

Quantibody® Mouse cytokine array Q-2000

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10000

IX. Reference List 1. Thorpe, R.C. A.R. Mire-sluis, and M. Wadhwa. 2001. Cytokine Standardization. In Cytokine Reference Volume 1: Ligands. Oppenheim, J.J., Feldmann, M., Durum, S.K., Hirano, T., Vilcek, J., and Nicola, N.A. eds. Academic Press, San Diego, CA, pp83-91

2. Biological Reference Materials 2000. National Institute for Biological Standards and Control

3. Harlow, E. and Lane, D., 1999. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory.

4. Barry, R. and Soloviev, M. 2004. Quantitative protein profiling using antibody arrays. Proteomics 4: 3717-3726

5. Huang, R.P., W. Yang, D. Yang, L. Flowers, I. R. Horowitz, X. Cao and R. Huang. 2005. The promise of cytokine antibody arrays in drug discovery process. Expert opinion on drug discovery. 9:601-615.

6. Berlier, J.E., Rothe, A., Buller, G. et al. 2003. Quantitative Comparison of Longwavelength Alexa Fluor Dyes to Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates. J. Histochem & Cytochem. 51(12): 1699-1712

RayBiotech, Inc., the protein array pioneer company, strives to research and develop new products to meet demands of the biomedical community. RayBio’s patent-pending technology allows detection of over 180 cytokines, chemokines and other proteins in a single experiment. Our format is simple, sensitive, reliable and cost effective. Products include: Cytokine Arrays, Chemokine Arrays, ELISA kits, Phosphotyrosine kits, Recombinant Proteins, Antibodies, and custom services. Quantibody® Mouse cytokine array Q-2000

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1. Antibody arrays Cytokine antibody array Human cytokine antibody arrays Mouse cytokine antibody arrays Rat cytokine antibody arrays Pathway- or disease-focused antibody arrays Inflammation antibody array Angiogensis antibody array Chemokine antibody array Growth factor antibody array MMP antibody array Atherosclerosis antibody array Antibody analysis tool, software 2. 3. 4. 5. 6. 7.

ELISA Cell-based phosphorylation assay Custom antibody arrays Antibody Recombinant protein Cytokine protein arrays

RayBiotech also provides excellent custom service: 1. Antibody arrays 2. Protein arrays 3. Peptide synthesis 4. Production of recombinant protein and antibody 5. Peptide arrays 6. Phosphorylation arrays 7. ELISA Just simply send your samples and we will do the assay for you. Technology transfer program Have you developed technologies or reagents interested to the scientific and research community? RayBiotech can help you commercialize your technologies, reagents and dream. Quantibody® Mouse cytokine array Q-2000

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X. Experiment Record Form Date: ___________________________ File Name: _______________________ Laser Power: ______________________ PMT: ____________________________ Well No. 1

Sample Name

Dilution factor

CNTRL

2

Std5

3

Std4

4

Std3

5

Std2

6

Std1

7 8 9 10 11 12 13 14 15 16

Quantibody® Mouse cytokine array Q-2000

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1

2

3

4

5

6

Note: RayBio® is the trademark of RayBiotech, Inc. Cytokine protein arrays are RayBiotech patent-pending technology. This product is intended for research only and is not to be used for clinical diagnosis. Our produces may not be resold, modified for resale, or used to manufacture commercial products without written approval by RayBiotech, Inc. Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials. Products are guaranteed for three months from the date of purchase when handled and stored properly. In the event of any defect in quality or merchantability, RayBiotech’s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase price.

This product is for research use only.

©2007 RayBiotech, Inc.

Quantibody® Mouse cytokine array Q-2000

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