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IgE-QBA™

Quantitative Binding Array for the simultaneous detection of

total and allergen-specific IgE in human samples

Storage: Microspheres and Streptavidin-PE: 2-8⁰C Coating Allergens, IgE Standard, and Detection Antibody: -20⁰C (+/-5⁰C)

For Research Use Only

© 2013, Indoor Biotechnologies, Inc. 2

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IgE-QBA

Quantitative Binding Array for the simultaneous detection of

total allergen-specific IgE in human samples

1. Intended use 2. Reagents supplied 3. Storage Conditions Upon Receipt 4. Materials Required But Not Provided 5. Technical Notes 6. Assay Workflow 7. Certificate of Analysis 8. IGE-QBA Protocol 9. IGE-QBA Data Analysis 10. Guidelines for Acceptable Data 11. Assay Performance

By opening the packaging containing this Kit (which contains fluorescently labeled microsphere beads authorized by Luminex Corporation) or using this Kit 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 Kit for a full refund prior to using it in any manner. You, the customer, acquire the right under Luminex Corporation's patent rights, if any, to use this Kit or any portion of this Kit, 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 Luminex Instrument refers to Luminex® 100, Luminex 200 and other Luminex Instruments available from Luminex Corporation and from authorized distributors including Bio-Rad Laboratories (Hercules, CA), Qiagen Corporation (Valencia, CA) and MiraiBio (South San Francisco, CA).

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1. Intended Use This is a multiplex assay kit manufactured by INDOOR Biotechnologies Inc. to be used for the simultaneous quantitative determination of total IgE and allergen-specific IgE against up to eleven common indoor and outdoor allergens: house dust mite allergens Der p 1 and 2 (Dermatophagoides pteronyssinus), Der f 1 and 2 (Dermatophagoides farinae), animal allergens Fel d 1 (cat, Felis domesticus), Can f 1 (dog, Canis familiaris), Mus m 1 (mouse, Mus musculus), Rat n 1 (rat, Rattus norwegicus), pollens Bet v 1 (birch, Betula verrucosa) and Phl p 5 (Timothy grass, Phleum pretense), and mold Alt a 1 (Alternaria alternata). This kit may be used for quantitation of total and allergen-specific IgE against the above indoor allergens in human plasma or serum samples. This product is for research use only .

2. Reagents Supplied IgE-QBA Custom kit for quantitation of total and specific human IgE Product code: Lot Number: Ship Date: Expiry is six months from ship date Kit contents: Microspheres:

□ Standard Beads Lot #: 35143

□ Total IgE Lot #: 35143

□ Der p 1 Lot #: 35116

□ Mite Group 2 Lot #: 35118 □ Der f 1 Lot #: 35117 □ Can f 1 Lot #: 35102 □ Fel d 1 Lot #: 35119 □ Mus m 1 Lot #: 35121 □ Rat n 1 Lot #: 35122 □ Bet v 1 Lot #: 35124 □ Phl p 5 Lot #: 35125 □ Alt a 1 Lot #: 32067

Coating Allergen(s):

□ CA-IGE Lot #: 35151 □ ST-DP2 Lot #:35150 □ ST-DF2 Lot #: 34059 Human IgE Standard Lot #: 35141 Detection Antibody Lot #: 35134 Streptavidin-Phycoerythrin Lot #: 35002 Standard/Sample Buffer Lot #: 35135 Multiscreen 96-well filter plate

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4. Materials Required But Not Provided 4.1 Reagents

1. Assay buffer (sterile filtered 1% BSA-PBS-0.02% Tween 20, pH 7.4). Buffer recipes can be found on Indoor Biotechnologies’ web site: www.inbio.com/MARIA.html (Multiplex Assay buffer formu-lary)

2. Luminex Sheath Fluid (Luminex Catalog #40-50000, BioRad Cat-alog# 171000055)

4.2 Instrumentation/Materials

1. Adjustable Pipettes with Tips (10 µl - 1000 µl ) 2. Multichannel Pipettes (5 µl - 50 µl and 25 µl - 200 µl) 3. Reagent Reservoirs 4. 96-well Microtiter plate (dilution of standard and samples) 5. Polypropylene Microcentrifuge Tubes 6. Aluminum Foil or Drawer (incubation in dark) 7. Absorbent Pads or Paper Towels 8. Laboratory Vortex 9. Vacuum Filtration Unit (Millipore Vacuum Manifold, Catalog #

MAVM0960R) 10. Luminex xMAP® Instrument 11. Data interpolation software* 12. Biohazard Disposal Bins

*It is recommended to use GraphPad Prism 5 software to determine IgE concentration; the data analysis instructions are based on using this pro-gram. A free two-week trial is available from the GraphPad website: http://www.graphpad.com/.

A Data Analysis Reference pdf with larger screen images of the analysis of a sample data set is available on Indoor Biotechnologies’ website: <website>

3. Storage Conditions Upon Receipt • Coating Allergens, Human IgE Standard, and Human IgE Detection

Antibody should be frozen at –20⁰C (+/- 5⁰C) upon receipt. • All other IgE-QBA reagents may be stored at 2-8ºC. • DO NOT FREEZE antibody-coupled fluorescent microspheres or

Streptavidin-Phycoerythrin.

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5. Technical Notes The IgE-QBA kit operator should carefully read the entire product insert before performing the assay and be sure to follow the recommended protocol in order to collect reliable and reproducible results.

• The Assay Buffer requires sterile filtration. Unfiltered assay buffer has a high particle load that will interfere with measurement in the xMAP® system. It will cause high bead aggregation ratios and may increase the time it takes to read the plate by 3 to 4-fold.

• DO NOT INVERT PLATE at any time throughout the assay. • The IgE-QBA plate should be protected from light during all incubation

steps to prevent photo-bleaching of the antibody-coupled florescent microspheres.

• Always ensure that the instrument needle is routinely cleaned to prevent clogs during plate reading.

• It is recommended that the IgE-QBA plate be read on the instrument on the same day the assay is performed. Note: Microspheres should be re-suspended immediately before read.

• Instrument settings: calibrate on low PMT, sample size 50µl, read 100 beads per analyte.

• Vacuum manifold settings: 5-7 in Hg suction. • When working with human sera or plasma, care must be taken to

avoid contact with human fluids. BSL-II level precautions must be taken when handling plasma or sera samples.

• At the time of print, Indoor Biotechnologies is not aware of assay interference caused by typically-used anticoagulants.

• To mix plasma samples, invert tubes gently 3-5 times. Indoor Biotechnologies does not recommend mixing plasma samples by vortexing.

• In the case of clogged plate wells, use a pipet positioned at the bottom of the plate to manually suction the fluid from the well. Wells that do not unclog should be disregarded during analysis.

• The same Mite Group 2 microsphere set is used in the analysis of specific IgE to Mite Group 2 allergens Der p 2 and Der f 2. Therefore, if quantitative analysis of specific IgE against both Der p 2 and Der f 2 is desired, the analyses must be performed separately. Quantitation of specific IgE against Der p 2 OR Der f 2 OR combined Mite Group 2 allergens is possible using a single kit, but additional kits are required if quantitative results for specific IgE against Der p 2 AND Der f 2 are desired.

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6. Assay Workflow

Estimated Assay Time Required: Sample Preparation: 0.5 hours; Incubation: 3.5 hours; Plate Reading: 1.5 hours

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7. Certificate of Analysis • Microsphere Details: Standard Microspheres: Anti-IgE Antibody-coupled Fluorescent Microspheres Product Code: MS-HIGE Specificity: Mouse monoclonal anti-human IgE

Sample Microspheres: Antibody-coupled fluorescent microspheres are supplied individually * Polyclonal antibody Store Microspheres at 2-8⁰C. • Human IgE Standard Details: Standard: Chimeric mouse monoclonal anti-Der p 2/human IgEε Product Code: ST-HIGE Concentration: 2500 IU/mL by ImmunoCAP® Freeze Human IgE Standard at –20ºC (+/- 5⁰C) upon receipt.

Analyte Bead Region Ab

Anti-IgE 42 α-Human IgE

Analyte/Specificity

Product Code Bead Region Antibody

Der p 1 MS-DP1 33 10B9 Der f 1 MS-DF1 51 6A8 Der p 2 MS-MG2 53 1D8 Der f 2 MS-MG2 53 1D8 Fel d 1 MS-FD1 58 6F9 Can f 1 MS-CF1 20 10D4

Mus m 1 MS-MM1 62 pAb α-Mus m 1* Rat n 1 MS-RN1 69 RUP-6 Bet v 1 MS-BV1 39 3B4 Phl p 5 MS-PP5 43 1D11 Alt a 1 MS-AA1 28 121

Total IgE MS-HIGE 42 α-Human IgE

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7. Certificate of Analysis (cont.) • Coating Allergens Details: Coating Allergen: 9-Allergen Mix Product Code: CA-IGE Composition: A premixed formulation of nine purified natural and recombinant allergens prepared in 5% BSA/50% glycerol/PBS, pH 7.4 Allergens Included: nDer p 1, nDer f 1, nCan f 1, nFel d 1, nMus m 1, nRat n 1, rBet v 1, rPhl p 5, rAlt a 1 Coating Allergen: Natural Der p 2 Product Code: ST-DP2 Composition: Purified natural Der p 2 prepared in 5% BSA/50% glycerol/PBS, pH 7.4 Coating Allergen: Natural Der f 2 Product Code: ST-DF2 Composition: Purified natural Der f 2 prepared in 1% BSA/50% glycerol/PBS, pH 7.4 Freeze Coating Allergens at –20ºC (+/- 5⁰C) upon receipt. • Biotinylated Detector Antibody Details: Detection Antibody: Biotinylated Anti-human IgE Product Code: BI-HIGE Specificity: Goat polyclonal anti-Human IgEε Freeze Human IgE Detection Antibody at –20ºC (+/- 5⁰C) upon receipt. • Streptavidin-Phycoerythrin Details: Formulation: Streptavidin-R-Phycoerythrin solution in 0.1M sodium phosphate, 0.1M NaCl with 5mM NaN3, pH 7.5 Product Code: SAP-MRA Concentration: 1 mg/mL Store Streptavidin-PE in the dark at 2-8⁰C.

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8. IgE-QBA Protocol 8.1 Retrieve samples from freezer and allow to reach room temperature. 8.2 Pre-wet each well of the 96 well filter plate with 100 μL of assay buffer (sterile filtered 1% BSA-PBS-0.02% Tween 20, pH 7.4). *Tip: When pipetting into the 96 well filter plate, insert the pipette tip at an angle into the bottom corner of the well. This will help ensure that the tip does not puncture the filter. Suggested Plate Layout

Preparation of Microsphere Solutions and Coating Allergens 8.3 Prepare the microsphere solution for the standard curve by first vortexing the Standard Bead stock (Product Code: MS-HIGE) for one minute and then adding 6 µL of the bead stock to a total of 1.5 mL of assay buffer. Mix by vortexing. 8.4 Prepare the microsphere solution for samples by first vortexing the tube(s) of Sample Bead stock (Product Codes will vary) for one minute and then adding 15 µL bead stock from each tube to 5.5 mL of assay buffer. Mix by vortexing. 8.5 Bring Coating Allergens (Product Code: CA-IGE and/or ST-DP2 and/or ST-DF2) to room temperature and add 600 µL stock from each tube to 5.5 mL assay buffer. Mix by vortexing.

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8. IgE-QBA Protocol (cont.) The Assay 8.6 Remove buffer from the 96 well filter-bottom plate by vacuum filtration. Tap the plate on paper towels to remove excess buffer from the plate bottom. Repeat vacuum filtration. Tap plate again on paper towels. *Do Not Invert Plate* 8.7 Pour the microsphere solution for the standard into a pipette basin and use a multichannel pipette to add 50 μL of microsphere solution to duplicate standard wells in columns 1-3. 8.8 Pour the microsphere solution for the sample beads into a pipette basin and use a multichannel pipette to add 50 μL of microsphere solution to appropriate wells. 8.9 Add 50 μL of diluted Coating Allergen solution to all wells. Set a multichannel pipette to 50 μL and mix all wells vigorously (5-10 repetitions).* *Note: foaming may occur when mixing 8.10 Incubate for one hour at room temperature in the dark. During this incubation, move on to step 8.11. 8.11 Prepare a standard curve using tripling dilutions of the Human IgE Standard (Product Code: ST-HIGE) starting at 300 IU/mL concentration by first pipetting 133 uL Standard/Sample Buffer (Product Code: SB-IGE) into columns 1-3 on a microtiter plate, then add an additional 43 uL Standard/Sample Buffer to wells A1 and B1 only. Pipette 24 μL Human IgE Standard into wells A1 and B1 and mix well. Transfer 67 µL from A1 and B1 (Standard 1) to wells C1 and D1 (Standard 2). Continue to make a total of ten 3-fold duplicate dilutions according to the suggested plate layout on page 10. Use Standard/Sample Buffer only in blank wells. 8.12 Invert samples 3-5 times to mix, then add 30 µL of sample to 120 µL Standard/Sample buffer for a dilution of 1:5. Mix thoroughly and transfer 30µL of the 1:5 dilution to 120µL Standard/Sample Buffer twice more to create 1:25 and 1:125 dilutions. Use Standard/Sample Buffer only in blank wells. 8.13 Remove Coating Allergens from assay plate by vacuum filtration and filter-wash wells 2x with 100 µL assay buffer.* *Note: Filter-wash wells by adding 100 µL assay buffer, then removing buffer by vacuum filtration. No mixing is required. 8.14 Transfer 100 µL diluted Standard or Sample from the microtiter plate to appropriate wells of assay plate and mix vigorously by pipetting. 8.15 Incubate for one hour at room temperature in the dark. 8.16 Dilute the Biotinylated Detection Antibody (Product Code: BI-HIGE) stock solution by adding 6 µL to 12 mL assay buffer.

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8. IgE-QBA Protocol (cont.) 8.17 Remove samples and standards by vacuum filtration and filter-wash wells 2x with 100 µL assay buffer. 8.18 Add 100 µL diluted Biotinylated Detection Antibody to each well and mix vigorously by pipetting. 8.19 Incubate for 45-60 minutes at room temperature in the dark.* *Note: Increasing incubation time past 60 minutes may result in very high background fluorescence. 8.20 Dilute Streptavidin-Phycoerythrin (Product Code: SAP-MRA) by adding 50 µL stock to 12mL assay buffer. 8.21 Remove biotinylated antibody solution by vacuum filtration and filter-wash wells 2x with 100 µL assay buffer. 8.22 Add 100 µL diluted Streptavidin-Phycoerythrin and mix vigorously by pipetting. 8.23 Incubate for 30 minutes at room temperature in the dark.* *Note: During this incubation period, prepare the instrument for plate reading according to the manufacturer’s instructions and according to the Instrument Settings detailed in Section 5: Technical Notes. 8.24 Remove Streptavidin-Phycoerythrin solution by vacuum filtration and filter-wash wells 2x with 100 μL of assay buffer. Add 100 μL of assay buffer to all wells and resuspend the microspheres by pipetting repeatedly, taking care not to create bubbles. Wipe the bottom of the filter plate with a paper towel and read the plate on the xMAP Instrument. Plate Reading Protocol Set-Up Note: IgE analysis requires running two separate protocols on the xMAP instrument: one for the standard curve and one for samples. 8.25 Create a protocol for the Standard Curve. • From the menu bar, click File > New. Enter a IgE-QBA in the author box and a

description of the protocol. • From the menu box on the left side of the screen, click ‘Select Analytes’. Select

‘Add Panel’ and enter a panel IgE-QBA (example: “Human IgE Standard Beads”).

a) Click ‘Add’. Enter the bead region and analyte IgE-QBAs for the standard beads: Region Analyte IgE-QBA 42 Anti-IgE

b) Click ‘OK’. All available analytes are now shown on the screen. c) Click ‘Add’ to select Anti-IgE microspheres.

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8. IgE-QBA Protocol (cont.) • From the menu box, select ‘Format Plate’. Select duplicate wells for the

standard curve according to the plate layout, including blanks for the standard curve.

Note: There are no samples analyzed during the standard curve analysis, so do not enter any sample information.

• Select ‘Enter Standards Info’ from the menu box. Enter the value for the most concentrated standard (300) and the dilution factor (3) to calculate values for the standard curve. The concentration units are IU/mL.

• Start the protocol and save the results file as the standard curve for that plate. The analysis of the standard curve takes approximately 20 minutes. When analysis is complete, perform a Wash Between Plates on the xMAP instrument. Export the data to an Excel file in ‘Single Analyte Layout’ and save the file as that plate’s Standard Curve data. 8.26 Create a protocol for the Sample Analysis. • From the menu bar, click File > New. Enter a IgE-QBA in the author box and a

description of the protocol. • From the menu box on the left side of the screen, click ‘Select Analytes’. Select

Add Panel and enter a panel IgE-QBA (example: “Human IgE Detection”). a) Click ‘Add’. Enter the number region for the microsphere sets used and the analyte IgE-QBAs according to the analytes included in the kit:

Region Analyte IgE-QBA 42 Total IgE 33 Der p 1 51 Der f 1 53 Mite Group 2 (Der p 2 and/or Der f 2) 58 Fel d 1 20 Can f 1 39 Bet v 1 43 Phl p 5 28 Alt a 1 69 Rat n 1 62 Mus m 1

b) Click ‘OK’. All available analytes are now shown on the screen. c) Click ‘Add’ to select microshpere sets. • Format the plate according to the plate layout, including blanks. Type in a

description and appropriate dilution factors (5, 25, 125) for the samples. • Start the protocol and save the results file as the sample data for that plate. Note: There are no standards for the sample run, so do not enter any standard information. The analysis of 22 samples generally takes 45-60 minutes. When analysis is complete, export the data to an Excel file and save the file as that plate’s Sample Analysis. Shut Down the xMAP instrument after completion of analyses.

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9. IgE-QBA Data Analysis and Handling Note: Larger images of the screen shots presented here are available in the Data Analysis Reference pdf that can be found on Indoor Biotechnologies’ website: <Site>. 9.1 If not done so upon completion of sample analysis, export the Bio-Plex Manager data file into Excel using the program icon on the menu bar. Export using the “Single Analyte Layout” format. 9.2 Open the Excel files for the Standard Curve and Sample data. Create a new worksheet in the Excel file for the Sample Analysis and label it “Analysis.” 9.3 Copy all of the standard curve data, including the column headings, into the newly-created Analysis worksheet for the Sample Analysis file. Copy and paste all information for the sample microsphere sets, excluding column headings, below the data for the standard curve. Be sure to copy over all information from every microsphere set analyzed. 9.4 At the top of the page, add new columns labeled “Interpolated X,” “High cutoff,” “Low cutoff,” and “Data within range” to the right of the column for dilution factor. These will be filled in as the analysis progresses. A sample set of data is shown in Figure 9.4a below and Figure 9.4b on the following page. Figure 9.4a: Standard curve and sample data pasted into Analysis worksheet in Excel. Four new columns are listed to the right of “Dilution” column.

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9. IgE-QBA Data Analysis and Handling (cont’d) Figure 9.4b: Aspects of new Analysis worksheet: Standard Curve data (blue rectangle) has been copied and pasted into the newly-created Analysis worksheet, followed by the data for each Sample data (green rectangle). New columns (red circle) “Interpolated X,” “High cutoff,” “Low cutoff,” and “Data within range” have been added to the right of the Dilution column.

Transfer data from Excel to GraphPad 9.5 Open GraphPad and set up a new graph in the “XY” format on the left menu. Be sure that the “Start with an empty data table” option is selected. • Choose a “Points only” graph and “Enter and plot a single Y value for each

point.” Click Create to start a new data table. • IgE-QBA the table in the left-hand menu by double-clicking “Data 1” and

inserting the file IgE-QBA and date. 9.6 Copy and paste the standard curve concentrations from the “Exp Conc” column from the Excel Analysis sheet into the X column on the GraphPad data table. 9.7 Copy and paste the “FI-Bkgd” values for the standards and samples from the Excel Analysis sheet into the Y column on the GraphPad data table. Only the points on the standard curve will have both X and Y values, points for the samples will only have Y values. 9.8 In GraphPad, choose the graph for the data table from the left-hand menu under the Graphs icon. There will be several points on the graph to correspond with the standard curve. • Click on the graph’s axes to IgE-QBA them—”IgE concentration” for X axis,

“MFI” for Y axis.

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9. IgE-QBA Data Analysis and Handling (cont’d) • Double-click on the X axis values to bring up the Format Axes menu. Select

“Log 10” for axis scale. Leave the Y axis as linear. Click OK. • Choose “Fit a curve with nonlinear regression” from the Analysis panel at

the top of the page. A pop-up dialog box stating the data will be fit to the actual data, not the log values of the X axis. Select OK.

• In the Parameters box, click the “+” next to “Binding-Saturation” to reveal the drop-down options. Choose “One Site—Specific Binding.” • Be sure the “Least squares” fitting method is selected. • Check the box by “Interpolate unknowns from standard curve.” • Leave the confidence interval selection at “None.” Click OK. • A sigmoidal line for the standard curve on the graph should now be

visible. 9.9 GraphPad will create a Results sheet with Interpolated X Values. Select this sheet from the left-hand menu and select the values in the left-most column (the Interpolated data).

• Copy the Interpolated X values for all your data and paste into the “Interpolated X” column of the Excel Analysis worksheet.

• Align the first Interpolated X data point with the first FI-Bkgd value below the standard curve. There will be no Interpolated X data for the standard curve.

• See Figure 9.9 below for a sample data set showing the alignment of Interpolated X values with FI-Bkgd values (red circles).

Figure 9.9: Interpolated X values copied from GraphPad and pasted into “Interp X” column in the Excel Analysis worksheet. Ensure the first Interp X value is in the same row as the first data point (red circles).

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9. IgE-QBA Data Analysis and Handling (cont’d) Analyze data in Excel *Note: Check that each sample’s dilution factor listed in the Analysis worksheet is correct before proceeding. 9.10 Determine the usable range of the standard curve. • On the Analysis sheet, look at the “(Obs/Exp)*100” column for the standard

curve and determine the highest point on the curve that is usable. Usable data fall within and include 85-115%. See the close-up image of the (Obs/Exp)*100 column in Figure 9.10 below.

Figure 9.10: Close-up view of (Obs/Exp)*100 column (blue rectangle). 9.11 Determine the “High Cutoff” value. • Assign the FI-Bkgd value of the topmost usable standard as the “High cutoff”

and enter that value at the top of the High Cutoff column. • A screen shot of a sample data set is shown in Figure 9.11 below:

Figure 9.11: Assigning a High Cutoff. In this sample standard curve, all points have (Obs/Exp)*100 value between 85-115%, therefore the topmost usable point is the 300 IU/mL standard (S1). The FI-Bkgd value corresponding with the 300 IU/mL standard is 10715.3. This is the High Cutoff. Enter this value at the top of the High Cutoff column.

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9. IgE-QBA Data Analysis and Handling (cont’d) 9.12 Determine the “Low Cutoff” value. • Multiply the standard deviation of the standard curve’s blank FI by 3 and add it

to the blank value for the standard curve. Enter this value at the top of the column for Low cutoff.

• A screen shot of a sample Low Cutoff calculation is shown in Figure 9.12 below:

Figure 9.12: Calculation of Low Cutoff. The Low Cutoff is the standard curve of the blank + 3SD of the blank.

9.13 Determine which data points fall within the usable range of the Standard Curve. • Select the box directly to the right of the first Interpolated X value in the High

cutoff column. • Select “fx” button to bring up the Insert Function dialog box. • Choose the “If” function and click OK.

9.13.1 Determine if the sample’s FI-Bkgd value is less than the High Cutoff value.

• In the Function Arguments, formulate Logical_test field to test if the High cutoff value is greater than the FI-Bkgd of the sample.

*Note: Type out the numeric value of High Cutoff, then use the “>” sign, then highlight the cell containing the FI-Bkgd of the sample being analyzed for this formula to execute properly.

• In the Value_if_true field, multiply the Interpolated X value by the dilution factor for that sample.

• In the Value_if_false field, enter “OOR HI”. Click OK. • An example of how to structure the arguments is presented in Figure

9.13.1 on the following page.

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9. IgE-QBA Data Analysis and Handling (cont’d) Figure 9.13.1: Determine if the sample’s FI-Bkgd is less than the High Cutoff value. Top: Select the cell directly to the right of the first interpolated X value in the High Cutoff column. Then click the “fx” button (red circle) to open Insert Function dialog box. Select an If function and click OK. Bottom: Formulation of the Function Arguments for calculating the values in the High cutoff column for each sample. Type out the numeric value of High Cutoff, then use the “>” sign, then select the cell containing the FI-Bkgd of the sample being analyzed in the Logical_test field. To fill out the Value_if_true field, select the cell including the Interp X value for that data point, then type “ * ”, then select the cell including the Dilution for that data point. Enter “OOR HI” in the field for Value_if_false. Click “OK.”

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9. IgE-QBA Data Analysis and Handling (cont’d)

9.13.2 Determine if the sample’s FI-Bkgd is greater than the Low Cutoff value. • Select the box directly to the right of the first High cutoff value in the Low

cutoff column. • Formulate Function Arguments to test if the Low Cutoff value is less than

the FI-Bkgd for the sample.

*Note: Type out the numeric value of Low Cutoff, then use the “>” sign, then select the cell containing the FI-Bkgd of the sample you are analyzing in the Logical_test field for this formula to execute properly.

• If true, enter “OOR LO,” if false, multiply the Interpolated X value by the dilution factor for that sample.

• A sample data set and Function Arguments is shown in Figure 9.13.2 below:

Figure 9.13.2: Determine if the sample’s FI-Bkgd is greater than the Low Cutoff. Top: Select the cell directly to the right of the previously-calculted value in the Low Cutoff column. Then click the “fx” button (red circle) to open Insert Function dialog box. Select an If function and click OK. Bottom: Formulation of the Function Arguments for calculating the values in the Low cutoff column for each sample. Enter “OOR LO” in the field for Value_if_true. To fill out the Value_if_false field, select the cell including the Interp X value for that data point, then type “ * ”, then select the cell including the Dilution factor for that data point. Click “OK.”

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9. IgE-QBA Data Analysis and Handling (cont’d)

9.13.3 Determine if the FI-Bkgd value for the sample falls between the High Cutoff and Low Cutoff. A sample Function Arguments is shown in Figure 9.13.3 below.

• Select the box directly to the right of the value just computed in the Data within range column.

• Set up an If function to test if the values in the High cutoff and Low cutoff columns are equal.

*Note: Select the value in the Low Cutoff column, then type “=”, then select the value in the High Cutoff column for this formula to execute properly.

• If true, multiply the Interpolated X value by the dilution factor for that sample. If false, enter “OOR.”

Figure 9.13.3: Determine if the sample’s FI-Bkgd falls between the High and Low cutoff values. Top: Select the cell directly to the right of the previously-calculted value in the Data Within Range column. Then click the “fx” button (red circle) to open Insert Function dialog box. Select an If function and click OK. Bottom: Formulation of the Function Arguments for calculating the values in the Data Within Range column for each sample. Select the newly-calculated values in the Low Cutoff and High Cutoff columns, separated by the “=” sign for the Logical_test field. To fill out the Value_if_true field, select the cell including the Interp X value for that data point, then type “ * ”, then select the cell including the Dilution for that data point. Enter “OOR” in the field for Value_if_false. Click “OK.”

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9. IgE-QBA Data Analysis and Handling (cont’d)

9.14 Repeat these steps for all samples on the plate until all samples are analyzed. A screen shot of a completed sample data set is shown in Figure 9.14 below: Figure 9.14: Screen shot of a completed Analysis page.

9.15 Create a second new worksheet in Excel and IgE-QBA it “Concentration Data”. Select all the data in columns for Analyte, Description, Dilution, and Data Within Range. Copy all of this data and paste into the new worksheet. Figure 9.15: Screen shot of new Concentration Data page with information pasted from “Analyte,” “Description,” “Dilution,” and “Data within range” columns.

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9. IgE-QBA Data Analysis and Handling (cont’d) 9.16 Make a separate table containing the sample ID and IgE concentration for each dilution. 9.17 Calculate the average IgE concentration using the three dilutions for a final concentration. Calculate the CV% by dividing the Standard Deviation of the three concentrations by the Average of the three concentrations, then multiply by 100%. A screen shot of a sample data set is shown in Figure 9.17 below. Refer to Section 10: Guidelines for Acceptable Data on the following pages.

Figure 9.17: Screen shot of new table with the average of the three dilutions’ IgE concentration and the CV% for each sample ID per analyte.

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10. Guidelines for Acceptable Data • The Lower Limit of Detection is 0.70 IU/mL. Any samples that are OOR

low for all three dilutions or have a calculated IgE concentration below 0.70 IU/mL should be assigned <0.70 IU/mL.

• The Upper Limit of Detection for this assay is >37,500 IU/mL. • Three serial five-fold dilutions are recommended for samples: 1:5, 1:25,

and 1:125. Accept the average of the IgE concentration of all three dilu-tions if the CV% between the concentrations is <30%.

• If the CV >30%, refer to the raw data (MFI) for that sample’s three dilutions. • Samples that are OOR low for the 1:5 dilution but within range for one

or more of the higher dilutions are suspected of showing a background amplification effect or contamination and should be assigned <0.70 IU/mL.

• In general, allergen-specific IgE concentration of less than 200 IU/mL tend to have similar IgE concentration at the 1:5 and 1:25 dilutions while the IgE concentration at the 1:125 dilution is much higher. This is a mathematical amplification and does not accurately reflect the IgE concentration of the sample. In this case, use only the 1:5 and 1:25 concentrations to generate an average IgE concentration for that sam-ple.

• For samples with low Total IgE concentration (<10 IU/mL), use only the concentration for the lowest dilution factor (1:5), unless the CV of the average IgE concentrations is within 30%.

• Samples with very high IgE concentration (Total or allergen-specific) tend to be above the saturation point of the assay at the 1:5 dilution. Therefore, the 1:5 dilution may be too concentrated to yield accurate data. For samples with Total IgE concentration of above 1000 IU/mL or allergen-specific IgE concentration of above 200 IU/mL, generate an average IgE concentration using the values for the 1:25 and 1:125 dilutions.

• A sample data set is shown in Figures 10.1a and Figure 10.1b on the fol-

lowing page.

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10. Guidelines for Acceptable Data (cont’d) Figure 10.1a: Bold red values should be rejected based on the Guidelines for Accepta-ble Data that are described on the preceding page. Samples that are “OOR” (below the detection limit) for all three dilutions have been replaced with <0.70 IU/mL.

Figure 10.1b: Screen shot of final data set with all unacceptable data points removed.

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11. Assay Performance

Data were generated from a panel of 69 allergic and 10 non-allergic patient plasma by INDOOR Biotechnologies, Inc. * Mean Intra-assay CV%—mean coefficient of variance of fluorescence intensity values of intra-assay triplicates. ** Mean Inter-assay CV%—mean coefficient of variance of IgE concentrations from thrice-repeated samples.

Assay

Reproducibility

Mean Intra-Assay CV% * Mean Inter-Assay CV% **

Total IgE 4.41 16.0 Der p 1 19.0 11.9 Der f 1 22.6 15.5 Der p 2 26.7 10.9

Fel d 1 7.9 10.5 Can f 1 11.3 10.1 Mus m 1 13.8 15.9 Rat n 1 33.4 9.6 Bet v 1 21.8 12.5 Phl p 5 11.0 11.1 Alt a 1 15.5 15.6

Der f 2 5.2 12.1

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