We analyzed the total plasma IgE level by using an ImmunoCAP 250 analyzer (Phadia, Uppsala, Sweden). We added 40 µL of plasma to ImmunoCAP (Phadia) and incubated this mixture. The ImmunoCAP tube was then washed with a washing solution. Enzyme-labeled anti-IgE (conjugate against total IgE) was added to the ImmunoCAP and washed again. The bound total IgE was quantified using a fluorescent substrate. The ImmunoCAP testing system is highly automated (ImmunoCAP 250, Phadia, Uppsala, Sweden), and the process required 1.5 h in total.
Immunocap 250
Manufactured by Thermo Fisher Scientific
Sourced in Sweden, United States, Germany
The ImmunoCAP 250 is a fully automated lab equipment for the detection and quantification of specific IgE antibodies in patient samples. It utilizes fluorescence-enzyme immunoassay technology to provide reliable and standardized results for the diagnosis of allergies and related conditions.
Automatically generated - may contain errors
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38 protocols using immunocap 250
1
Automated Total Plasma IgE Quantification
2
Diagnosis of Allergic Rhinitis
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Allergic rhinitis was clinically diagnosed, based on international guidelines which recommend identifying symptoms, including sneezing, rhinorrhea, nasal obstruction, and itching with specific IgE possession confirmed by skin prick test or ImmunoCAP 250 (Phadia, Uppsala, Sweden) system14 (link). Atopy was defined as positive response to any one of the inhalant allergens (birch, oak, ragweed, mugwort, cat dander, house dust mite, etc.) by ImmunoCAP 250 (Phadia) system, as previously described15 (link).
3
ELISA and Phadia ImmunoCAP for IgG Levels
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Total IgG levels (ng/ml) and PR3-ANCA IgG levels (response units) were determined by ELISA and Phadia ImmunoCAP 250 analyser, respectively, in supernatants of anti-IgM Fab2/BAFF/IL-21-stimulated PBMCs after a 12-day culture, as described previously [21 (link)].
4
Anti-TNF Agents in Rheumatoid Arthritis
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This retrospective study involved 129 patients with established RA naïve to biological agents (98 females and 32 males, mean age 56.7±12.3 years, disease duration 6.3±1.2 years, baseline Disease Activity Score [DAS]-28 scores 3.2–5.6) who received treatment with anti-TNF agents after the failure of conventional disease-modifying antirheumatic drugs: 32 (24.8%) received IFX, 58 (44.9%) ETN, and 39 (30.3%) ADA. Table 1 shows their baseline characteristics.
After 6 months of treatment, the patients were classified as being in remission (DAS28 <2.6), having low disease activity (LDA; DAS28 2.6–3.2), or not responding (NR; DAS28 >3.2). During the 24 weeks of treatment, we also evaluated injection-site reactions in patients treated with ETN or ADA, and systemic reactions in those treated with IFX.
The patients were tested for serum ADAbs with an anti-TNFα-blocker enzyme-linked immunosorbent assay kit (Immundiagnostik, Milan, Italy) and IgG4 antibodies against TNF inhibitors (fluoroenzyme immunoassay kit for ImmunoCap® 250; Phadia, Uppsala, Sweden). ADAb (Immundiagnostik) titers higher than 0.44 OD for ETN, 0.18 OD for ADA, and 0.27 for IFX were considered positive. IgG4 antibodies against TNF inhibitors (ImmunoCap) higher than 5.9 mg arbitrary unit/liter for ETA, 19.5 mg A/L for ADA, and 8.6 mg A/L for IFX were considered positive.
After 6 months of treatment, the patients were classified as being in remission (DAS28 <2.6), having low disease activity (LDA; DAS28 2.6–3.2), or not responding (NR; DAS28 >3.2). During the 24 weeks of treatment, we also evaluated injection-site reactions in patients treated with ETN or ADA, and systemic reactions in those treated with IFX.
The patients were tested for serum ADAbs with an anti-TNFα-blocker enzyme-linked immunosorbent assay kit (Immundiagnostik, Milan, Italy) and IgG4 antibodies against TNF inhibitors (fluoroenzyme immunoassay kit for ImmunoCap® 250; Phadia, Uppsala, Sweden). ADAb (Immundiagnostik) titers higher than 0.44 OD for ETN, 0.18 OD for ADA, and 0.27 for IFX were considered positive. IgG4 antibodies against TNF inhibitors (ImmunoCap) higher than 5.9 mg arbitrary unit/liter for ETA, 19.5 mg A/L for ADA, and 8.6 mg A/L for IFX were considered positive.
5
Serological Biomarkers for Celiac Disease
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Serum IgA levels of all IgA deficient individuals with suspected CD were determined by routine turbidimetric or nephelometric methods. Serum IgG anti-tTG and anti-DGP antibodies were analyzed in the newly collected serum samples at the clinical immunology unit at Linköping University Hospital, Sweden. Commercially available immunoassays for CD diagnostic testing were used according to the manufacturer instructions: (1) IgG antibodies against tTG (EliA™ Celikey®IgG, Phadia, Freiburg, Germany) were analyzed on ImmunoCAP 250 and the recommended cut-off <7 EliA U/mL was used. (2) Two different ELISAs were performed for detection of IgG antibodies against DGP including Gliadin IgG II (Quanta Lite™, INOVA Diagnostics, San Diego, CA, USA) and GAF-3X (Euroimmun, Lübeck, Germany). The recommended cut-offs 20 AU/mL (INOVA) and 25 RU/mL (Euroimmun) were used.
6
Inhalant Allergen Identification through CRD
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Total IgE and specific IgE Assay (ImmunoCAP 250; Phadia, Sweden) were performed in patients with positive SPT to evaluate the major inhalant allergen. The level of awareness towards the main inhalant allergen was increased using the CRD. We evaluated IgE antibodies to Phl p1 (Timothy grass), Phl p5 (Timothy grass), Bet v1 and Bet v2 (Betula verrucose), Amb a1 (Ambrosia), Art v1 (Mugwort), Par j2 (Wall pellitory), Ole e1 (Olea europea), Cup a1 (Cupressus arizonica), Fel d1 (cat), Can f1 (dog), Der p1 (House dust mite), Der p2 (House dust mite), and Alt a1 (Alternaria alternata). IgE levels were considered positive at the level ≥0.35 kUA/l. Patients with IgE antibodies to Bet v2 were excluded to rule out profilin allergy [31 (link),32 (link),33 (link)].
7
IgE Antibodies Against Na-GST-1 in Helminth-Endemic Region
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Prior to initiation of the Phase 1 trials of Na-GST-1/Alhydrogel, stored serum samples collected from children and adults living in Americaninhas were tested for IgE antibodies against Na-GST-1. These sera had been collected from individuals enrolled in previously reported clinical studies of helminth epidemiology, treatment and transmission [24 (link)]. IgE antibody levels against Na-GST-1 were measured by using the fluoroenzyme immunoassay (ImmunoCAP 250; Phadia, Uppsala Sweden) at the Johns Hopkins Dermatology, Allergy and Clinical Immunology Reference Laboratory. IgE levels greater than 0.1 kU/L (0.24 ng/dL) were considered clinically significant.
8
Autoimmune Antibody Detection Protocol
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Blood samples in serum separator tubes were centrifuged for 15 min at 3000 g. An aliquot of serum was analysed for anti-double stranded DNA antibodies, C3 and C4 at the Royal Victoria Hospitals Belfast immunology labs which is a member of the UKNEQAS scheme for nuclear and related antigens. Venous blood was collected into a trisodium citrate tube for the measurement of erythrocyte sedimentation rate (ESR). ESR was measured using the Westergren method. The fluorescence enzyme linked immunoassay (Phadia, Immunocap 250) was utilised for detection of anti-dsDNA. Plasmid dsDNA was coated to a solid phase support. Serum samples were diluted 1/10 and incubated for 30 min. Secondary antibodies and washing steps were automated. C3 and C4 were quantified by rate nephleometry (Beckman Image 8000).
9
Immunoassay Techniques for RA Biomarkers
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ACPA were measured by immunofluorimetry using the Immunocap 250 system based on second generation CCP as antigen and reagents and controls provided by the manufacturer (Phadia AB, Uppsala, Sweden). According to the manufacturer the intra-run and inter-run coefficients of variation (CVs) are 5.1-10.5 % and 2.6-7.7 % respectively, with values of >10 U/ml deemed positive [26 (link), 27 (link)].
C-reactive protein (CRP) and rheumatoid factor (RF, composite IgM, IgG, IgA) were assayed by immunonephelometry (Siemens Health Care Diagnostics, BN Prospec Nephelometer, Newark, USA) using reagents and controls supplied by the manufacturer with results considered positive when they exceeded values of 5 μg/ml and 11 IU/ml respectively [26 (link), 27 (link)].
C-reactive protein (CRP) and rheumatoid factor (RF, composite IgM, IgG, IgA) were assayed by immunonephelometry (Siemens Health Care Diagnostics, BN Prospec Nephelometer, Newark, USA) using reagents and controls supplied by the manufacturer with results considered positive when they exceeded values of 5 μg/ml and 11 IU/ml respectively [26 (link), 27 (link)].
10
Fecal Calprotectin Dynamics in IBD
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FC measurements (ImmunoCAP 250; Phadia, Uppsala, Sweden, fluorescent enzyme immunoassay, values µg/g feces) were performed on stool samples before treatment with prednisolone or anti-TNF-α and six weeks later. Patients were instructed to collect stool samples from the first bowel movement of the day and deliver them to the hospital within 24 hours. Delta FC (δFC) was calculated as follows: (FC pre-treatment – FC 6 weeks post-treatment)×100/(FC pre-treatment), as a percentage.
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