beadplex human cancer panel 2 (growth factors) · bphcp002-tgf 20x tgf-alpha capture beads 1.1 ml...

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User Protocol TB538 Rev. B 0710JN of 21

BeadPlex® Human Cancer Panel 2 (Growth Factors)

1) Cat. No. BPHCP002-12 (Complete Kit)

2) Cat. No. BPHCP002-BASE, with any combination of Panel-Specific Capture Bead(s) (Mix and Match Format)


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Table of Contents

I. Assay Principle ................................................................................................. 3 1. BeadPlex ® System Overview 3 2. Panel Overview 4 3. Assay FlowChart 5

II. Materials and Storage ..................................................................................... 5

II. Materials and Storage ..................................................................................... 6 1. Panel Components 6 2. Materials Required But Not Supplied 7

III. Assay Preparation .......................................................................................... 8 1. Considerations Before You Begin 8 2. Resuspension of Individual Reagents 8 3. Sample Preparation 9 4. Capture Beads Mix Preparation 9 5. Standard Curve Preparation 10 6. Streptavidin-PE Preparation 10

IV. Assay Protocol ............................................................................................. 11

V. Performance Characteristics ....................................................................... 13

VI. References ................................................................................................... 18

VII. Troubleshooting ......................................................................................... 19

VIII. Assay Plate Design .................................................................................... 21

© 2010 EMD Chemicals, Inc., an Affiliate of Merck KGaA, Darmstadt, Germany. All rights reserved. WideScreen® and BeadPlex® are registered trademarks of EMD Chemicals, Inc. in the United States and in certain other jurisdictions. Bio-Plex® is a registered trademark and Bio-Plex® Manager™ is a trademark of Bio-Rad Laboratories, Inc. Luminex® and xMAP® are registered trademarks and Luminex® 100™ IS is a trademark of Luminex Corporation. ProClin® is a registered trademark of Rohm and Haas Co. TWEEN® is a registered trademark of ICI Americas Inc.

Manufactured by Rules Based Medicine, Inc. (RBM)

By opening the packaging containing this product (which contains fluorescently labeled microsphere beads authorized by Luminex Corporation) or using this product in any manner, you are consenting and agreeing to be bound by the following terms and conditions. You are also agreeing that the following terms and conditions constitute a legally valid and binding contract that is enforceable against you. If you do not agree to all of the terms and conditions set forth below, you must promptly return this product for a full refund prior to using it in any manner. You, the buyer, acquire the right under Luminex Corporation’s patent rights, if any, to use the product or any portion of this product, including without limitation the microsphere beads contained herein, only with Luminex Corporation’s laser-based fluorescent analytical test instrumentation marketed under the name Luminex Instrument.

The terms and conditions governing EMD Chemicals’ sale of this product are as indicated on our website (www.emdbiosciences.com).






I. Assay Principle

1. BeadPlex ® System Overview

Figure 5

Figure 4

Figure 3

Figure 1

Figure 2

BeadPlex® assays are bead-based flow cytometric assays that utilize xMAP® technology. This system allows for sensitive and precise quantification of multiple analytes within a single sample. Such assays are essentially ELISAs on a bead. This is accomplished through the use of Luminex® detection system (e.g. Luminex 100, 200, or Bio-Rad® BioPlex™ analyzer).

Figure 1. The BeadPlex Kits include analyte-specific antibody-conjugated differentially-dyed capture beads and biotinylated detection antibodies (Figure 2). Each of the differential-coded capture beads allow for the capture and detection of analytes from various samples (e.g. standards of known concentration, control, and test samples). The biotinylated detection antibodies, which bind to different epitopes on the same protein or complex, allow for the use of immuno-fluorescent molecules.

Figure 3. The samples are mixed with the antibody capture beads and the analytes of interests are captured. The beads are washed to remove any unbound material.

Figure 4. The biotinylated detector antibodies are added and incubated with the analyte-specific captured beads. During this incubation step, the biotinylated detector antibodies bind to the appropriate immobilized analytes, and form a Bead-Immuno-Sandwich Complex.

Figure 5. An immuno-fluorescent molecule, Streptavidin R-Phycoerythrin (RPE), is added and allowed to incubate for 30 minutes. During this incubation step, the Streptavidin-RPE binds to the biotinylated detector antibodies, which allows for the quantification and detection of bound analytes.

After washing to remove unbound Streptavidin-RPE and subsequent re-suspension in wash buffer, the beads are analyzed with the Luminex® detection system (e.g. Luminex 100, 200, or Bio-Rad® BioPlex™ analyzer). The simultaneous detection and quantification of multiple analytes is made possible by measuring the spectral properties of the beads and the amount of associated (RPE) fluorescence.






2. Panel Overview Many growth factors are thought to play a role in cancer progression (1). Levels of EGFR and related growth factors are often elevated or dysregulated in tumor cells and can be detected in blood samples from cancer patients. EGFR activation is initiated through the autocrine or paracrine binding of EGF and EGF-like growth factors to the extracellular domain of EGFR or other members of the ErbB family, resulting in receptor dimerization and initiation of signal transduction cascades. Amphiregulin, EGF, TGF-α, betacellulin, epiregulin, and HB-EGF all bind to EGFR, and in some cases ErbB-4. Normal and malignant cells also produce soluble forms of EGFR through alternate mRNA splicing or proteolytic cleavage of the transmembrane receptor (2). Many therapeutic approaches targeting EGFR have been developed; however, combinational therapy directed at cancer-related growth factors is still an emerging area of research that may prove effective in overcoming tumor resistance (3).

Tumor cells induce new blood vessel formation by secreting a variety of growth factors that act on endothelial cells. VEGF-A is the major stimulator of angiogenesis; nevertheless, the coordination of multiple cytokines is required including: EGF, FGF-basic, PDGF-BB, PlGF, Tenascin C, and TGF-α. In turn, angiogenesis promotes tumor metastasis, providing a means for malignant cells to establish at distal sites. Cancerous tumors frequently express elevated levels of angiogenic growth factors, and therefore can be useful as diagnostic and prognostic markers. For instance, tenascin C expression correlates with angiogenesis and carcinoma metastasis (4).

This novel multi-analyte profiling kit provides a deeper view of the following individual growth factors and soluble EGFR receptor levels in normal and disease processes.

Analyte Alternative/Full Name Bead Region AREG Amphiregulin 35

BTC Betacellulin 20

EGF Epidermal Growth Factor 04

EGFR Epidermal Growth Factor Receptor 06

EREG Epiregulin 22

FGF-basic Fibroblast Growth Factor-basic, FGF-2 08

HB-EGF Heparin-Binding Epidermal Growth Factor 07

PDGF-BB Platelet-Derived Growth Factor-BB 63

PlGF Placenta Growth Factor 33

TnC Tenascin C 39

TGF-α Transforming Growth Factor 19

VEGF-A Vascular Endothelial Growth Factor-Alpha 15






3. Assay FlowChart

Add the Reaction Mix

10 µL of blocking buffer 30 µL of standard / control or samples 10 µL of capture bead mix 50 µL of total reaction mix

Standard and Control Preparation

Capture Bead Mix Preparation

Streptavidin – PE Preparation

Filter

Pre Wet Filter Plate 50 µL of Assay Buffer

Shake and Incubate 1 hour at room temperature in the dark. Plate Shaker: 750 rpm.

Resuspend Beads

100 µL of Assay Buffer

Acquire Data with Luminex

Add Detection Antibody

40 µL of 1X Detection Antibodies

Add Streptavidin - PE 20 µL of 1X Streptavidin - PE

Filter & Wash 2X

Do NOT Filter & Wash

Filter & Wash 2X

Shake and Incubate 1 hour at room temperature in the dark. Plate Shaker: 750 rpm.

Shake and Incubate 30 minutes at room temperature. Plate Shaker: 750 rpm.






II. Materials and Storage

1. Panel Components Storage : All components of the BeadPlex® Human Cancer Panel 2 (Growth Factors) are shipped at 2 to 8°C. Upon receipt, store all kit components at 2 to 8°C. Do not freeze.

The BeadPlex Human Cancer Panel 2 is available in two kit configurations: 1. Mix and Match Format 2. Complete Kit

For maximum flexibility, end-users may configure their own customized multiplex assay through the Mix and Match Format. This format consists of a BeadPlex Human Cancer Panel 2 - Base Kit (Cat. No. BPHCP002-BASE) and a customized choice of any or all analyte-specific capture beads from the following table. The Base Kit and selected components contain enough reagents and buffers to assay one 96-well plate.

Alternatively, end-users may order the BeadPlex Human Cancer Panel 2 - Complete Kit (Cat. No. BPHCP002-12). This kit contains all respective capture beads and all components in the BeadPlex Human Cancer Panel 2 - Base Kit. Desired capture beads will need to be pre-mixed prior to running the assay. The Base Kit contains enough components to assay one 96-well plate.

Cat. No. Description Amount BPHCP002-12 BeadPlex® Human Cancer Panel 2 - Complete Kit

(The Complete Kit includes all of the capture beads listed below and all of the components in the BeadPlex Human Cancer Panel 2 - Base Kit)

1 Kit

BPHCP002-AMP 20X Amphiregulin Capture Beads 1.1 mL BPHCP002-EGF 20X EGF Capture Beads 1.1 mL BPHCP002-EGR 20X sEGF-R Capture Beads 1.1 mL BPHCP002-BTC 20X Betacellulin Capture Beads 1.1 mL BPHCP002-EPI 20X Epiregulin Capture Beads 1.1 mL BPHCP002-FGF 20X FGF basic Capture Beads 1.1 mL BPHCP002-HBE 20X HB-EGF Capture Beads 1.1 mL BPHCP002-PDG 20X PDGF-BB Capture Beads 1.1 mL BPHCP002-PIG 20X PIGF Capture Beads 1.1 mL BPHCP002-TEN 20X Tenascin C Capture Beads 1.1 mL BPHCP002-TGF 20X TGF-alpha Capture Beads 1.1 mL BPHCP002-VEG 20X VEGF-A Capture Beads 1.1 mL BPHCP002-BASE

72005-4.4ML 71996-150UL 71997-100UL 71998-100UL 71999-60ML 72000-1.5ML 71990-7.5ML 72473-1.1ML 72474-1EA 72002-1ML 72003-450UL 71786-EA

BeadPlex Human Cancer Panel 2 - Base Kit (The Base Kit includes only those components which are listed below)

Human Cancer Panel 2 Detection Antibodies Human Cancer Panel 2 Standards Mix Human Cancer Panel 2 Control 1 Human Cancer Panel 2 Control 2 1X Assay Buffer Type 3 Blocking Buffer Type 2 1X Sample Dilution Buffer Type 1 Bead Diluent Bead Mixing Vial Standard Curve Diluent Type 2 5X Streptavidin-Phycoerythrin 96-well Filter Plate and Sealer

1 Kit

4.4 mL 150 µL 100 µL 100 µL 60 mL 1.5 mL 7.5 mL 1.1 mL 1 ea 1 mL 450 µL

1 ea






*Following reconstitution of lyophilized reagents, store any unused reagent at –70°C. See Reagent Preparation section (p 4).

Note: BeadPlex® Human Cancer Panel 2 is not compatible with other bead kits or buffers sold by EMD Chemicals, Inc. or other vendors.

Note: Grossly hemolyzed samples or whole blood cannot be used with this kit.

Caution: Human Cancer Panel 2 Standards Mix, Control 1, and Control 2 contain materials derived from human sources and mammalian cell lines. All human source materials have tested negative for HIV-1, HIV-2, HCV antibodies, HIV Ag and HBsAg. However, all materials derived from human fluids or mammalian cell lines should be considered potentially biohazardous and handled accordingly. Refer to MSDS for additional information.

2. Materials Required But Not Supplied � Luminex® xMAP® System (or equivalent) (Cat. No. 72490) � Vacuum manifold for filter plates (Pall 5017 or Millipore MSVMHTS00) � 96-well plate platform shaker � Polypropylene microcentrifuge tubes � 15 mL polypropylene centrifuge tubes � Vortex mixer � Ultrasonic bath, such as Cole Parmer EW-08849 (optional) � Multichannel pipet � Distilled or deionized water






III. Assay Preparation

1. Considerations Before You Begin � Excessive vacuum will cause the filter plate membrane to perforate. Adjust the pressure

using a non-filter (ELISA or tissue culture) plate. Ensure that vacuum does not exceed 5 in. (127 mm) Hg. − After adjusting the vacuum with a non-filter plate, place filter plate on the manifold. Add

liquid to the wells. Use fingertips to apply pressure evenly across the plate. The liquid should drain in 2–5 seconds.

− To avoid drying out the beads, vacuum only long enough to drain all wells. Do not allow drained beads to sit for > 5 minutes before rehydrating with buffer.

− It is critical to remove excess buffer from the underside of the filter plate by tapping it on a paper towel several times before adding samples or reagents. This prevents samples from wicking out of the wells during incubation steps. For the same reason, avoid placing filter plate on an absorbent surface during incubations.

− To avoid perforating the filter plate membrane, make certain that the probe height on the xMAP® system is adjusted correctly. Do not touch the membrane with pipet tips. For accurate pipetting, touch tips to the sides of the filter plate wells. Change tips as necessary to prevent cross-contamination.

� Capture Beads and Streptavidin-PE are light-sensitive. To avoid photobleaching, protect beads and Streptavidin-PE in microcentrifuge tubes from light. Cover filter plates with aluminum foil during incubation steps.

� To prevent fluorescent dye loss, do not use organic solvents with capture beads. Beads are incompatible with DMSO concentrations > 1%.

� Many of the washing and reagent dispensing steps may be done with an 8-channel or 12-channel pipette (manual or automatic). For accurate results, use calibrated single-channel pipettes for manipulation of standards and test samples.

� Store test samples (cell lysate, tissue homogenate, serum, plasma, tissue culture supernatant) at -70°C prior to use.

2. Resuspension of Individual Reagents Both the Complete Kit and the Mix and Match Kit are supplied with select lyophilized reagents, which are described in the following table.

1. Resuspend each of the following reagents as indicated in the following table.

Reagent Volume of dH 20 Human Cancer Panel Standards Mix 150 µL

Human Cancer Panel 2 Control 1 100 µL

Human Cancer Panel 2 Control 2 100 µL

Blocking Buffer Type 2 1.5 mL

Standard Curve Diluent Type 2 1.0 mL

Human Cancer Panel 2 Detection Antibodies 4.4 mL






2. Mix by vortexing at medium speed and allow to sit at room temperature for a minimum of 5 minutes and no longer than 1 hour, prior to use.

Note: Following reconstitution, store any unused reagents at –70°C. Unused reagents can be stored at –70°C for up to one month. Avoid multiple freeze-thaw cycles.

3. Sample Preparation 1. Thaw and dilute samples within 1 hour of use. Remove any particulates by centrifugation or

filtration. Avoid multiple freeze/thaw cycles.

2. Dilute serum or plasma samples 5-fold in Sample Dilution Buffer Type 2 (Duplicate Samples: 15 µL sample + 60 µL Sample Dilution Buffer Type 2), mix well and store on ice. If desired, further dilutions of serum or plasma samples can also be performed in Sample Dilution Buffer Type 2 to ensure reading within the range of the assay standards.

Note: PDGF-BB is found at much higher levels in serum than in plasma due to release from platelets during serum preparation. Serum dilutions greater than 5-fold may be necessary for accurately quantifying PDGF-BB if levels are elevated above the normal range.

3. Dilute tissue culture supernatants 5-fold in PBS (Duplicate Samples: 15 µL sample + 60 µL PBS, mix well and store on ice). If desired, further dilutions of tissue culture supernatants can also be performed in Sample Dilution Buffer Type 2 to ensure reading within the range of the assay standards.

4. Capture Beads Mix Preparation Note: Capture beads are light-sensitive. Prepare 1X diluted capture bead mix within 1 hour of use. Protect from light.

Both the Complete Kit and the Mix and Match Kit are supplied with individual vials of analyte-specific Capture Beads at 20X concentrate that must be custom mixed and dilut ed prior to use .

1. Add the following volume of 1X bead diluent = [1.1 mL - (number of desired Panel-Specific Capture Beads X 0.055 mL)] to the Bead Mixing Vial.

2. Sonicate and vortex gently for 15–30 seconds each desired vial of 20X Capture Beads. Centrifuge the contents to the bottom of the vial (100 rcf for 5 seconds).

3. Resuspend the beads by gently pipetting 25 µL up and down 10 times, taking care not to create bubbles.

4. Transfer 55 µL of each desired Panel-Specific Capture Bead (20X) to the Bead Mixing Vial. Protect from light and store at 4°C until use.






5. Standard Curve Preparation Label 8 polypropylene tubes S8 through S1. Alternatively, prepare standard dilutions in a 96-well plate. Pipet Standard Curve Diluent Type 9 into labeled tubes as described below. Transfer the reconstituted Human Kidney Toxicity Panel 1 Standards Mix to the S8-labeled tube. Prepare 3-fold serial dilutions of S8 following the table below. Ensure that each new standard is mixed well by vortexing before proceeding to the next dilution. Change tips between each dilution.

Note: The kit contains sufficient standards for a dilution series performed in duplicate. Standard concentrations are lot-specific. Refer to Certificate of Analysis of appropriate lot for specific standard concentrations.

6. Streptavidin-PE Preparation Note: Streptavidin-PE is light-sensitive. Use 1X Streptavidin-PE within 15 minutes after preparation. Protect from light.

Both the Complete Kit and the Mix and Match Kit are supplied with a 5X concentrate of Streptavidin-PE that must be diluted prior to use.

1. Quickly spin down the vial of Streptavidin-PE (5X).

2. Determine the number of test wells required for the entire assay. Each test well requires a total volume of 20 µL of 1X Streptavidin-PE.

3. Determine the total volume of 1X Streptavidin-PE required for the entire assay by multiplying the number of test wells by the total volume of 1X Streptavidin-PE per well (20 µL). Account for 10% extra.

4. Divide total volume of 1X Streptavidin-PE required for the entire assay by the concentration of Streptavidin-PE (5X) to determine the necessary amount of 5X Streptavidin-PE. Subtract this calculated amount from the total volume of 1X Streptavidin-PE required for the entire assay to determine the necessary amount of 1X Assay Buffer.

5. In an appropriately sized bottle, dilute the calculated volume of 5X Streptavidin-PE into the calculated volume of 1X Assay Buffer Type 2. Cap the bottle and vortex for 3 to 5 seconds. Protect from light and store at 4°C until use.

Standard Volume of Standard Curve Diluent Type 7

Volume of Standards Mix

S8 0 µL 150 µL from vial

S7 80 µL 40 µL of S8












IV. Assay Protocol

1. Start up the Analyzer and warm up lasers and perform daily Start-up procedure on Luminex® machine, unless samples are to be incubated overnight.

2. Seal any unused wells of 96-well filter plate with plate sealer or lab tape for future use.

3. Pre-wet 96-well filter plate wells with 50 µL Assay Buffer Type 3 and incubate for a minimum of 5 minutes. Immediately prior to Step 3, remove liquid from filter plate by vacuum filtration. Do not exceed 5 in Hg or 127 mm Hg vacuum; liquid should drain in 2–5 seconds. Tap filter plate on a paper towel to remove any buffer remaining on the underside.

Note: It is critical to remove excess buffer from the underside of the filter plate before adding samples or reagents. Otherwise, samples may wick out of the wells during incubation steps. For the same reason, avoid placing filter plate on an absorbent surface during incubations. If a well does not drain, use the non-tip end of a 200 µL pipet tip to flick the center of the plastic support on the underside of the well, then reapply vacuum.

4. Add 10 µL Blocking Buffer Type 2 to each filter plate well that will be used.

5. Add 30 µL each standard, sample, or control to appropriate well of the 96-well filter plate.

Note: Human Cancer Panel 2 Control 1 and Control 2 do not need to be diluted.

6. Sonicate 10 seconds (optional) and vortex the bead mixing vial of 1X Human Cancer Panel 2 mixed capture beads for 10 seconds. Add 10 µL to each well.

7. Vortex the plate by gently gliding the plate over the vortex mixer.

Note: Gradually increase vortex speed from low to medium. Hold plate with a loose grip. Mix thoroughly for 10 seconds. Avoid splashing. Alternatively, use a multi-channel pipet to mix the contents of each well by pipetting 25 µL up and down 5 to 7 times.

8. Cover plate with aluminum foil to protect from light and incubate 1 h at room temperature on a plate shaker (750 rpm).

9. Remove liquid from filter plate by vacuum filtration (5 in Hg or 127 mm Hg maximum).

10. Wash the capture beads by adding 100 µL Assay Buffer Type 3 to each well and applying vacuum to remove buffer. Repeat wash step for total of two washes in Assay Buffer Type 3. After second wash and vacuum, tap filter plate on paper towels to remove any buffer remaining on the underside.

Note: Do not resuspend beads in Assay Buffer Type 3 after second wash.

11. Add 40 µL Human Cancer Panel 2 Detection Antibodies to each well. Mix the contents of each well as described in Step 6.

12. Cover plate with aluminum foil to protect from light and incubate 1 h at room temperature on a plate shaker (750 rpm)

Note: Do not wash beads after Detection Antibody incubation.

Note: Wrapping the entire plate, including the bottom side, will prevent wells from “clogging” due to salt drying over plate gasket(s).

13. Add 20 µL 1X Streptavidin-PE to each well.

14. Cover plate with aluminum foil to protect from light and incubate 30 minutes at room temperature on a plate shaker (750 rpm).


15. Remove liquid from filter plate by vacuum filtration.

16. Wash beads by adding 100 µL Assay Buffer Type 3 to each well and applying vacuum to remove buffer. Repeat wash step for total of two washes in Assay Buffer Type 3. After second wash and vacuum, tap filter plate on paper towels to remove any buffer remaining on the underside.






17. Add 100 µL Assay Buffer Type 3 to each well.

18. Cover plate to protect from light and incubate 3–5 minutes at room temperature on a plate shaker (750 rpm).


19. Analyze using a Luminex instrument.

20. Read on Luminex Machine.

For detailed instructions on the operation of Luminex® systems, refer to the user guide for your specific instrument and software. General recommendations are given below.

a. Using your Luminex system software, prepare a protocol for the assay you will run. b. Enter information for each bead target, and for the standards, samples, and controls. c. Select the following bead regions as indicated in Section I or in the Certificate of Analysis. d. Acquire data using the system settings shown in the following table:

*

*If the time spent acquiring samples needs to be reduced, collect as few as 50 events per bead region.

Generation of Standard Curves and Quantification of Experimental Samples

� Protein standards are supplied in the BeadPlex® Mouse CVD Panel 1 Base Kit, allowing for accurate quantification using a titrated standard curve. Representative standard curves and assay performance information can be found in the Certificate of Analysis for the specific lot.

� Refer to the Certificate of Analysis for expected control ranges. � The eight data points obtained with the concentration standards are plotted using Median

Fluorescent Intensity (MFI) as the signal readout (Y-axis), against concentration of standard dilutions (X-axis).

� Five-parameter logistic (5PL) curve fitting is recommended for modeling data obtained from bead-based immunoassays. Most ranges of standard concentrations are too wide for accurate linear regression analysis. Four-parameter logistic (4PL) equations will often give a good fit, but are not ideal because they assume the standard curve is symmetrical.

� If the signal from an unknown sample exceeds the highest point of the standard curve, the concentration of the unknown should not be calculated by extrapolation, because the non-linear shape of the standard curve cannot be accurately modeled past the last measured point. In this case, dilute the samples and test again.

� When concentrations of unknowns have been determined by reading off of the standard curve, remember to multiply this value by the dilution factor to obtain the concentration of the target in the original sample.

Software Sample Size

Events per Bead Region

Timeout Doublet Discriminator

CAL2 Gain Setting

Luminex® IS™ or equivalent

50 µL 50–100* 60 seconds 7,500–15,500 default

Bio-Plex® Manager™

default (50 µL)

50–100* default default (4335–10000)

RP1 low






V. Performance Characteristics

Note: All data and results shown below are representative and not lot specific. Please refer to certificate of analysis for lot specific testing data. Data were generated using the complete kit configuration with all analytes multiplexed together.

1. Representative Data

a. Growth Factor Levels in Human Sera The BeadPlex® Human Cancer Panel 2 was used to quantify growth factor and soluble receptor levels in randomly selected serum samples from normal and pregnant patients: Normal (non-pregnant, light bars, n = 5) and Pregnancy (dark bars, n = 4). Elevated levels of several growth factors and soluble receptor were observed in the sera from pregnant patients. The values denoted in the bar graph represent pg/mL, with the exception of Tenascin C (ng/mL). Values that fall at or below the corrected least detectable dose (LDD) are marked. Note: PDGF-BB is found at much higher levels in serum (compared to plasma) due to platelet degranulation during serum preparation. The VEGF indicated in the graph is representative of VEGF-A.






b. Growth Factor Levels in Human Sera (Normal vs. D isease State) Examples of BeadPlex® Human Cancer Panel 2 analyte measurements in normal and disease state sera: Normal (N, n = 6), Colorectal cancer (CR, n = 4), Ovarian cancer (OV, n = 3), Gastrointestinal cancer (GI, n = 2). Individual points are the average of duplicate wells; results from two separate experiments have been compiled. Data points at or below the corrected least detectable dose (LDD) are plotted on the x-axis. Sera from normal individuals have growth factor concentrations grouped in the normal range, whereas high concentrations of growth factors are found in some disease state patients.

Note: The VEGF indicated in the graph is representative of VEGF-A.

c. Growth Factor Levels in Human Cell Lines

The BeadPlex Human Cancer Panel 2 was used to quantify growth factors or soluble receptor levels released into tissue culture supernatant by human cell lines and peripheral blood mononuclear cells (PBMC). Cell lines were grown to confluence, and cells were stimulated with PMA (50ng/mL, Cat. No. 524400), ConA (10mg/mL, Cat. No. 234567), or unstimulated (-) for 24 hours. Supernatants were clarified and diluted five-fold prior to testing (Note: MCF-7 Amphiregulin assay was diluted two hundred-fold). The values in the table have been multiplied by the dilution factor in order to signify the level in undiluted supernatants. Targets that were below the assay level of detection are denoted as <LOW>. All targets were undetectable tissue culture media alone. Results showing high level biomarker expression are consistent with reports from the literature.

Note: The VEGF indicated in the table is representative of VEGF-A.

A-549 MDA-MB-468 HT-1080 PBMC MCF-7 HUVEC Analyte - + PMA - + PMA - + PMA - +ConA - + PMA - + PMA

Amphiregulin (pg/mL) 381 3100 145 172 <low> 435 <low> 583 15600 169000 <low> <low> Betacellulin (pg/mL) <low> <low> <low> <low> <low> <low> <low> <low> <low> <low> <low> <low> EGF (pg/mL) <low> <low> 9 6 <low> <low> 53 64 <low> <low> <low> <low> EGFR (pg/mL) 22 16 18500 39000 <low> 36 22 31 <low> 19 <low> <low> Epiregulin (pg/mL) <low> 54 68 52 <low> <low> <low> 22 <low> <low> <low> <low> FGF-basic (pg/mL) <low> <low> 13 20 <low> <low> <low> <low> <low> <low> <low> <low> HB-EGF (pg/mL) <low> <low> 55 69 150 331 26 104 19 379 42 599 PDGF-BB (pg/mL) <low> <low> 195 180 <low> <low> 260 329 2000 3030 428 2170 PIGF (pg/mL) 11 8 9 11 690 483 <low> <low> 17 44 281 1190 Tenascin C (pg/mL) <low> <low> 214000 543000 360000 752000 <low> <low> <low> <low> <low> <low> TGF-α (pg/mL) <low> 4 5 5 4 17 <low> <low> 53 236 <low> <low> VEGF-A (pg/mL) 1810 3530 35700 36800 29700 33400 15 41 10900 41600 <low> 26






2. Sensitivity The least detectable dose (LDD) in biological fluids was determined by adding two standard deviations to the average of the signal for 20 replicate determinations of the Standard Curve Diluent. This value was quantified by interpolation from the standard curve. Corrected LDD is the LDD multiplied by the dilution factor of 5 used for testing plasma/serum sample types.

The lower limit of quantification (LLOQ) in biological fluids was determined using a 2-fold dilution series of the standards in Standard Curve Diluent assayed in triplicate over three different runs, and is defined as the point at which the Coefficient of Variation (CV) for the measurements was 30%. The CV was calculated and plotted against concentration, and the LLOQ was interpolated from this plot. Corrected LLOQ is the LLOQ multiplied by the dilution factor of 5 used for testing plasma/serum sample types.


3. Precision Reproducibility within an assay (intra-assay precision) and between assays (inter-assay precision) was determined by performing quantitative measurements of Human Cancer Panel 2 Control 2 in duplicate over 5 runs on multiple days, operators and instruments. The Total %CV is the inter-assay precision across all five runs.


Amphiregulin (pg/mL)

Betacellulin (pg/mL)

EGF (pg/mL)

EGFR (pg/mL)

Epiregulin (pg/mL)

FGF-basic (pg/mL)

LDD 25 17 5.7 23 17 5.5

LDD (corrected for dilution) 127 84 29 117 85 28

LLOQ 17 12.7 7.7 19.8 8.0 11.8

LLOQ (corrected for dilution) 85 64 39 99 40 59

HB-EGF (pg/mL)

PDGF-BB (pg/mL)

PIGF (pg/mL)

Tenascin C (pg/mL

TGF-α (pg/mL)

VEGF-A (pg/mL)

LDD 3.0 31 5.1 12 2.5 16

LDD (corrected for dilution) 15 154 25 58 12 81

LLOQ 4.1 17.3 3.3 7.0 1.4 11.8

LLOQ (corrected for dilution) 21 87 17 35 7.0 59

Percent Coefficient of Variation (%CV) Analyte Run 1 Run 2 Run 3 Run 4 Run 5 Total

Amphiregulin 1% 19% 2% 6% 13% 18%

Betacellulin 6% 7% 2% 2% 6% 10%

EGF 1% 31% 13% 5% 9% 18%

EGFR 0% 19% 6% 3% 7% 16%

Epiregulin 15% 10% 7% 12% 15% 21%

FGF-basic 0% 9% 3% 1% 3% 6%

HB-EGF 1% 10% 7% 5% 19% 22%

PDGF-BB 6% 7% 3% 3% 9% 11%

PIGF 2% 5% 14% 0% 6% 6%

Tenascin C 0% 1% 6% 6% 3% 5%

TGF-α 5% 15% 2% 1% 7% 4%

VEGF-A 9% 12% 3% 3% 7% 10%






4. Recovery and Linearity To assess recovery, two concentrations of purified standards were spiked into six human serum or plasma (EDTA) samples. Measurements from the spiked samples (minus the measured levels in unspiked samples) were compared to the expected values.

Linearity was measured by spiking physiological levels of purified standards into serum, and then serially diluting the sample. Back-calculated concentrations at each dilution were compared to the measurement from the 5-fold dilution (defined as 100%).


5. Matrix Interference Matrix factors that can interfere with immunoassays were spiked into Human Cancer Panel 2 Control 2, and matrix interference was measured by comparing observed (spiked control sample) to expected (unspiked control sample).


Recovery Serum (n = 6) Plasma (n = 6)

Linearity (% of expected)

Analyte Average Range Average Range 1/10 1/20 1/40 Amphiregulin 76 (49–106)% 90 (77–100)% 117 119 140 Betacellulin 70 (52–91)% 100 (85–114)% 107 100 97

EGF 60 (40–101)% 86 (74–97)% 118 113 111

EGFR 52 (11–93)% 78 (49–103)% 106 100 97 Epiregulin 50 (25–80)% 69 (49–108)% 127 122 120 FGF-basic 109 (85–147)% 119 (109–131)% 79 69 77 HB-EGF 93 (63–135)% 59 (49–69)% 109 111 137

PDGF-BB 80 (39–107)% 111 (99–128)% 102 100 94

PIGF 97 (70–120)% 108 (89–120)% 91 89 93

Tenascin C 99 (63–155)% 70 (56–80)% 117 112 120

TGF-α 59 (33–89)% 80 (63–106)% 116 105 110

VEGF-A 97 (43–148)% 128 (101–169)% 103 98 89

Recovery from matrix (as % of expected) Matrix Amphiregulin Betacellulin EGF EGFR Epiregulin FGF-basic

Hemoglobin (5 mg/ml) 163% 125% 98% 133% 107% 118% Bilirubin (0.2 mg/ml) 130% 115% 98% 115% 96% 113%

Triglycerides (5 mg/ml) 153% 129% 124% 156% 134% 126%

Recovery from matrix (as % of expected) Matrix HB-EGF PDGF-BB PIGF Tenascin C TGF-αααα VEGF-A

Hemoglobin (5 mg/ml) 195% 143% 118% 118% 101% 114% Bilirubin (0.2 mg/ml) 143% 90% 119% 106% 99% 102%

Triglycerides (5 mg/ml) 229% 146% 149% 147% 114% 118%






6. Specificity Cross-reactivity of the BeadPlex® Human Cancer Panel 2 was tested by using the multiplex panel beads and each individual detection antibody to assay S5 of the standards dilution series. Cross-reactivity between all assays is < 1%, with the exception of the prolactin assay, which shows approximately 5% cross-recognition of AFP.

Correlations with validated assays were performed for the BeadPlex Human Cancer Panel 2 assays by quantifying and comparing biomarker measurements from 80 individual serum samples. Strong correlations with DPC IMMULITE® clinical tests were seen for the CA 15-3 (R2 = 0.78; slope = 0.91) and prolactin (R2 = 0.91; slope = 0.86) assays. Similar results were seen correlating the remaining four assays with the RBM HumanMAP® analysis. Data are available upon request.






VI. References 1. Miyamoto, S., et al. 2006. Cancer Sci. 97, 341.

2. Lafky, J.M., et al. 2008. Biochim. Biophys. Acta. 1785, 232.

3. Tabernero, J., 2007. Mol Cancer Res. 5, 203.

4. Chiquet-Ehrismann, R., 2004. Int. J. Biochem. Cell Bio. 36, 986.






VII. Troubleshooting

Problem Probable Cause

Solution

Leaking wells in filter plate � Wicking due to adherent drops

− Tap filter plate several times on paper towel before adding samples or reagents. Do not place filter plate on an absorbent surface during incubations.

− If wells leaked during data acquisition, it is possible to reacquire data from these wells. Blot underside of wells and add 100 µL/well Assay Buffer Type 3.

� Perforation of filter plate membranes

− Adjust the vacuum setting to <5 in (127 mm) Hg.

− Do not touch membranes with pipet tips.

Filter plate wells not draining under vacuum

� Vacuum is too low − Adjust vacuum setting to 3–5 in (76–127 mm) Hg.

− Clean rubber seals. Apply fingertip pressure to filter plate to ensure formation of a good seal. Use the plate sealer to cover wells not in use.

� Clogged membranes − Clarify samples by centrifugation or filtration. If samples are viscous, dilute further before assaying.

− Use the non-tip end of a 200 µL pipette to flick the center support on the underside of the well, then reapply vacuum.

Low bead counts during data acquisition

� No beads in the wells − See “Leaking wells in filter plate” solutions above.

− Verify that beads were added at the correct concentration, and that correct bead regions and wells were selected during acquisition setup.

� xMAP® fluidics system is clogged

− Clear system of clogs or air using maintenance steps described in the instrument user manual (sanitize, alcohol flush, probe sonication, etc.).

− Make sure that the probe height is set correctly.

− Make sure that beads are in suspension by incubating plate for 3–5 minutes on plate shaker (750 rpm) immediately before analysis.

− Microbial growth in buffers can cause beads to stick to the filter plate membrane. Do not use contaminated reagents.

� Timeout limit is set too low − 50–100 events per bead region should be acquired within the 60 second timeout limit. If necessary, the timeout limit can be set higher, e.g. 75 seconds.

Beads are not falling into the gates properly

� Beads were not resuspended in Assay Buffer Type 2 before analysis

− The setting of the Doublet Discriminator (DD) gate is buffer-specific. This gate can be adjusted, but Assay Buffer Type 2 is the recommended buffer for running samples. Other buffers may cause bead aggregation.

� DD gate setting not optimal

− Use the DD gate setting recommended in the Data Acquisition Section. If necessary, raw data results can be reanalyzed with different DD gate settings; see software user manual.

� Beads were exposed to organic solvents

− Do not use organic solvents in the immunoassay - they will damage beads irreversibly.

� Beads are falling outside the bead region gates due to photo bleaching

− Do not use expired beads.

− Do not expose the beads to ambient light for >10 minutes. Avoid intense light.

� Fluidics system is not running properly

− Confirm that the waste container is not full, the sheath fluid is not empty, and the SD fluidics module is turned on.

− Check system calibration using approved calibration beads.

− Verify correct system pressure. Confirm that the system is free of air or particulate buildup. Follow maintenance steps described in the instrument user manual.






Insufficient volume of an immunoassay reagent

� Solutions were not prepared or used as described in protocol

− Confirm correct buffer dilutions and use.

− If additional Assay Buffer Type 2 is needed, PBS can be used for the final bead resuspension step.

− If additional 96-well filter plates are required, we recommend Millipore Cat. No. MSBVN1210.

Sample measurements not falling on the standard curve

� Dilution of sample is too low or too high

− If values are higher than the standard curve, dilute samples further in Sample Dilution Buffer Type 2.

� Target concentration is below detection

− Verify that curve fitting at the lower end of the standard curve is accurate.

− Not all serum/plasma samples contain detectable levels of all analytes.






VIII. Assay Plate Design

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beadplex human cancer panel 2 (growth factors) · bphcp002-tgf 20x tgf-alpha capture beads 1.1 ml...

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