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18 protocols using phadia 100

1

Biotinylation and Immobilization of Recombinant Allergen

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For custom ImmunoCAP, recombinant allergen was dialyzed against 1 L of 0.1 M NaHCO3, 1 M NaCl buffer. After dialysis, the protein concentration was measured. Biotin stock 10 mg/mL was used in 5-fold molar excess to bind the recombinant allergen (3 h, RT, mixed), followed by PBS dialysis and another measurement of the protein concentration. The biotinylated allergen was bound on Streptavidin ImmunoCAP o212 (Thermo Fisher Scientific) using a custom program (prewashed, loaded 50 µL of 100 µg/mL allergen/CAP, incubated 30’, washed) on a Thermo Scientific™ Phadia100, then detected with patient sera in a Thermo Scientific™ Phadia250 instrument according to manufacturer protocols.
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2

Multiplex Measurement of Specific IgE Antibodies

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Total serum IgE levels were measured using Phadia 100; results were reported in kIU/L. Specific serum IgE antibodies were measured with the ImmunoCAP ISAC (Thermo Fisher Scientific, Waltham, MA USA). ImmunoCAP ISAC is a miniaturized immunoassay platform that allows for multiplex measurement of specific IgE antibodies to many allergen components using 20 μL of serum or plasma. Allergens are immobilized on a microarray chip to allow simultaneous measurement of specific IgE antibodies to 112 components from 51 allergen sources.21 (link) Test results were measured with a biochip scanner and results were reported in ISAC standardized units (ISU) and categorized based on the manufacturer’s cutoff levels (< 0.3 ISU, undetectable or very low; 0.3-0.9 ISU, low; 1-14.9 ISU, moderate/high; and ≥ 15 ISU, very high). Values above 1 ISU were considered positive.
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3

Recombinant Allergen IgE Quantification

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Specific IgE levels to recombinant proteins were determined by ImmunoCAP in Phadia 100 and 250 instruments (Thermo Fisher Scientific, Uppsala, Sweden) according to the manufacturer's instructions. In the case of Bet v 1, Api g 1.01 and Cor a 1.04, commercial ImmunoCAP tests were used, whereas for Pru av 1, Dau c 1.01, NCS and its variants, experimental ImmunoCAPs were manufactured by coupling the recombinant allergens individually to the solid phase, as described elsewhere [27] , [28] (link).
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4

Cow's Milk Allergy Biomarkers Assessment

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Total IgE and specific IgE against of cow’s milk proteins (nBos d4; nBos d5; nBos d6; nBos d8) were assessed by enzymatic immunoassay (Phadia 100 ThermoFisher Scientific CAP system, Rodano Milano, Italy). Measurements were expressed as kilounits per liter (kU/L).
The concentrations of IL-4 and IL-10 were measured with a Human IL4/IL10 Enzyme immunoassay kit (Boster Biological Technology, Ltd., Fremont, CA, USA). The IL-5 and IFN- concentrations were measured using the human ELISA assay kit (BioVendor, Brno, Czech Republic). The minimum detection concentrations were 15.6 pg/mL for IL-4, 7.8 pg/mL for IL-5 and IL-10, and 0.78 pg/mL for IFN-γ.
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5

Coconut Milk IgE Reactivity Evaluation

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IgE reactivity to coconut milk was evaluated using Phadia ImmunoCAP (f36) (Thermo Fisher Scientific, Uppsala, Sweden) using the Phadia 100 (Table 1). The test was performed according to the manufacturer’s instructions. The cutoff value was set as > 0.35 kUA/L for sIgE positivity.
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6

Fecal Calprotectin Measurement Protocols

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Based on study protocol (Figure 1), extracts were analysed with fluorescence enzyme immunoassay (FEIA), EliATM Calprotectin on Phadia 100 analyser (Thermo Fisher, Uppsala, Sweden) and/or PETIA, Bühlmann fCAL® Turbo on Roche Cobas c501 analyser (Roche diagnostics, Manheim, Germany). Measuring ranges for FEIA and PETIA are 15 - 3000 mg/kg (within-laboratory coefficient of variation, CV = 4.8%) and 20 - 2000 mg/kg (within-laboratory CV = 4.9%), respectively. Cut-off value for both methods is set at 50 mg/kg (negative). Values 50 - 200 mg/kg represent the “grey zone” and > 200 mg/kg active inflammation in gastrointestinal tract (14 , 22 ).
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7

IgE Antibody Binding Assay Protocol

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Direct IgE antibody binding assays were performed by ImmunoCAP. Biotinylation of
the allergens was carried out using the EZ-Link Sulfo-NHS-LC-Biotin, No-Weigh™
Format (Thermo Fisher Scientific Inc., Pierce, Rockford, IL), with biotin added at a
10-fold molar excess to the allergens. Zeba™ Desalt Spin Columns (Thermo Fisher
Scientific Inc., Pierce, Rockford, IL) were used to remove any excess biotin from the
biotinylated allergens. A Quant*Tag Biotin Kit™ (Vector Laboratories, Inc.,
Burlingame, CA) was used to quantify the number of biotins per molecule. IgE detection and
quantification in plasma samples was determined using Specific IgE detection
streptavidin-ImmunoCAP on the Phadia® 100 (Thermo Fisher Scientific, Portage, MI).
After testing several concentrations of allergen (from 0.25 to 10 µg/ml),
streptavidin ImmunoCAPs were loaded with 5 µg/CAP of biotinylated allergen and
incubated for 30 minutes. IgE measurements were performed according to standard ImmunoCAP
procedures.
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8

Optimizing Allergen-Loaded Immunocaps

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Streptavidin ImmunoCAPs (Thermo Fisher Scientific, Portage, MI) were loaded and incubated on a Phadia 100, with the biotinylated allergen at amounts ranging from 0.5 to 10 µg/CAP. Two different human plasma samples from individuals allergic to the allergen (that had been originally tested for IgE binding to 2–3 µg/CAP) were selected for optimization experiments. Their IgE levels to the allergen-loaded CAPs were measured in a Phadia 250 following manufacturer’s instructions (Thermo Fisher Scientific, Portage, MI) to select optimal amount of biotinylated allergen to be loaded to the Streptavidin ImmunoCAPs.
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9

Measuring Serum IgE and WBC Counts

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Serum total immunoglobulin E (IgE) was measured in our research lab from plasma in duplicate on a Phadia 100 detection system (ThermoFisher Scientific, Uppsala, Sweden). If both measurements were not within 10% concordance, a 3rd measurement was assayed. White blood cell (WBC) counts were obtained from complete blood counts with differentials using commercially available and Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories through Quest Diagnostics in the GALA II dataset and through UCSF Clinical Laboratories for SAGE. Serum total IgE and WBC counts were specified as continuous predictors. However, the distributions of serum total IgE, basophils, and eosinophils were highly right-skewed and were log-transformed for analysis purposes.
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10

Murine OVA-specific IgG Isolation

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Murine OVA-specific IgG was prepared from pooled sera from mice immunized i.p. with alum (50μg protein precipitated to 1mg aluminum hydroxide gel (Sigma Aldrich, St. Louis, MO) on days 0 and 7, followed by 10μg protein on days 14 and 21). Control IgG was prepared from mock-immunized mice. Human IgG was prepared from pooled sera samples from patients who had undergone oral immunotherapy for peanut allergy or from non-allergic donors. IgG was isolated over Nab protein G spin columns (Thermo Scientific, Waltham, MA), concentrated and dialyzed with Macrosep Advance Centrifugal Devices carrying a 100 kDa cut-off (Pall Corporation, Westborough, MA) and filter-sterilized with 0.2μM syringe filters (Millex, EMD Millipore, Billerica, MA). Murine OVA-specific IgG concentrations were determined by ELISA, and mice were injected with 100μg IgG1. Peanut-specific IgG4 in human IgG preparations was quantified by ImmunoCAP on a Phadia 100 (ThermoScientific, Kalamazoo, MI).
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