Phadia 250

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.

All participants who consented to biosample collection provided a blood sample immediately prior to the OFC and another sample either at the end of the observation period (in the case of no reaction) or as soon as possible after a reaction was identified, generally within 5 minutes, without interfering with clinical care (in the case of a reaction). Blood samples were collected into K2 EDTA vacutainers and immediately placed in 4°C and promptly separated into plasma by centrifugation. Plasma samples were aliquoted and immediately snap-frozen in liquid nitrogen until the time of analysis. Samples were not subjected to multiple freeze-thaw cycles.
The following analyses were performed using both pre- and post-OFC plasma samples. All kits were used according to the manufacturer’s instructions. The ImmunoCAP Tryptase fluoroenzyme immunoassay, analyzed on a Phadia 250 instrument (Thermo Fisher Scientific), was performed in the University of Michigan’s Clinical Laboratory Improvement Amendments–certified (CLIA-certified) clinical laboratory. Cytokines including TNF-α, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, and VEGF were analyzed using a customized cytokine/chemokine/growth factor immunology multiplex assay kit (MILLIPLEX, MilliporeSigma).

Levels of RF were measured using EliA test on Phadia 250 (Thermo Fisher, Waltham, USA). Anti-CCP IgG (ACPA) levels were measured using chemiluminescent microparticle immunoassay on the Alinity i-platform (Abbott Laboratories, Chicago, USA). CRP was analysed using Turbidimetry Alinity and ESR was analysed according to standard lab protocols. Serum calprotectin (S100A8/A9) levels were analysed using particle-enhanced turbidimetric immunoassay (Gentian Diagnostics, Moss, Norway) on an Optilite system (Binding Site, Birmingham, England).

Total IgE was measured using the Immage 800 (Beckman Coulter, Mississauga, ON, Canada) and peanut-specific IgE antibodies were measured using the Phadia 250 (Thermo Scientific, Waltham, MA, USA).

A panel of circulating proteins reflecting immune activation, especially associated with B cells, was selected based on data from the literature. Serum concentrations of β2-microglobulin were measured using commercially available assays on the fully automated Optilite^® turbidimetric analyzer (The Binding Site Group Ltd). Serum concentrations of rheumatoid factor (RF) were measured using commercially available fluorimetric assays (Phadia 250^®, Thermo Fischer Scientific). Serum concentrations of IgA, IgG and IgM were measured using commercially available nephelemetric assays (Siemens). Normal range values were defined according to the manufacturer’s specifications. Serum concentrations of sBCMA and sCD21 were measured in duplicate using commercial ELISA assays (Human BCMA/TNFRSF17 DuoSet ELISA, cat. #DY193; Human CD21 DuoSet ELISA, cat. #DY4909-05). Serum concentrations of APRIL, BAFF, sTACI, CXCL13, sCD23, sCD25 and sCD27 were measured in duplicate using commercial customized pre-mixed multiplex assays (Luminex Assay, R&D Systems). All experiments were conducted according to the manufacturer’s protocol.

The primary outcome variable was major relapse rate after two and five years. Secondary outcome variables were clinical data including infections requiring treatment, ESRD, malignancy, and mortality and laboratory parameters including renal function, MPO- or PR3-ANCA and immunoglobulin levels, and circulating CD19⁺ B cells.
MPO- and PR3-ANCA levels were measured with ImmunoCAP using Phadia 250 (Thermo Fisher Scientific, Waltham, MA) [16 (link)]. The number of CD19⁺ B cells was quantified by flowcytometry [17 ].