Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms of respiratory allergy (e.g., rhinitis, asthma) to ragweed pollen. Among the eleven characterized allergens, Amb a 6 is a non-specific lipid transfer protein (nsLTP). nsLTPs are structurally stable proteins in pollen and food from different unrelated plants capable of inducing severe reactions. The goal of this study was to produce Amb a 6 as a recombinant and structurally folded protein (rAmb a 6) and to characterize its physicochemical and immunological features. rAmb a 6 was expressed in Spodoptera frugiperda Sf9 cells as a secreted protein and characterized by mass spectrometry and circular dichroism (CD) spectroscopy regarding molecular mass and fold, respectively. The IgE-binding frequency towards the purified protein was evaluated using sera from 150 clinically well-characterized ragweed-allergic patients. The allergenic activities of rAmb a 6 and the nsLTP from the weed Parietaria judaica (Par j 2) were evaluated in basophil activation assays. rAmb a 6-specific IgE reactivity was associated with clinical features. Pure rAmb a 6 was obtained by insect cell expression. Its deduced molecular weight corresponded to that determined by mass spectrometry (i.e., 10,963 Da). rAmb a 6 formed oligomers as determined by SDS-PAGE under non-reducing conditions. According to multiple sequence comparisons, Amb a 6 was a distinct nsLTP with less than 40% sequence identity to currently known plant nsLTP allergens, except for nsLTP from Helianthus (i.e., 52%). rAmb a 6 is an important ragweed allergen recognized by 30% of ragweed pollen allergic patients. For certain patients, rAmb a 6-specific IgE levels were higher than those specific for the major ragweed allergen Amb a 1 and analysis also showed a higher allergenic activity in the basophil activation test. rAmb a 6-positive patients suffered mainly from respiratory symptoms. The assumption that Amb a 6 is a source-specific ragweed allergen is supported by the finding that none of the patients showing rAmb a 6-induced basophil activation reacted with Par j 2 and only one rAmb a 6-sensitized patient had a history of plant food allergy. Immunization of rabbits with rAmb a 6 induced IgG antibodies which strongly inhibited IgE binding to rAmb a 6. Our results demonstrate that Amb a 6 is an important source-specific ragweed pollen allergen that should be considered for diagnosis and allergen-specific immunotherapy of ragweed pollen allergy.

BACKGROUND: German cockroach (GCr) aeroallergens are associated with allergic rhinitis and asthma. Vitellogenin (Vg) and vitellin (Vn) are abundant proteins in GCr blood and eggs (including egg cases), respectively, and are possible high molecular mass allergens. Prior efforts to purify Vg/Vn yielded amounts too small for subsequent studies. In this study, we report the affinity purification of Vg/Vn from whole-body defatted GCr powder and determination of the binding of Vg/Vn to anti-GCr IgE.
METHODS: New Zealand white rabbits were immunized with pure Vg/Vn in Freund\'s adjuvant, and IgG was purified from the rabbit sera and conjugated to cyanogen bromide (CNBr)-activated Sepharose. Aqueous extracts from GCr powder were passed over the column. After extensive washing, putative Vg/Vn was eluted in low-pH buffer, neutralized, and analyzed by SDS-PAGE and liquid chromatography high-resolution mass spectrometry (LC-HRMS). IgE binding of Vg/Vn was evaluated by inhibition of IgE binding to GCr-ImmunoCAP(I6) in sera from 10 GCr-allergic individuals. In addition, Vg/Vn was biotinylated and bound to ImmunoCAP-streptavidin, and direct IgE antibody binding to the immobilized Vg/Vn was determined in sera from 26 GCr-allergic individuals.
RESULTS: Vg/Vn isolated by affinity chromatography was 91% pure by LC-HRMS; contaminants included Bla g 3 (0.9%), human keratin (6%), and rabbit IgG. Vg/Vn inhibited IgE binding to GCr-ImmunoCAP(I6) in 8 of 10 sera. In direct-binding experiments, 21/26 (80%) sera had anti-Vg/Vn IgE at >0.10 kUA/L, while 11/26 (42%) sera were >0.35 kUA/L.
CONCLUSIONS: We affinity-purified Vg/Vn and demonstrated that Vg/Vn-specific IgE antibody is a major component of GCr-specific IgE.

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UNASSIGNED: Imported fire ant (IFA) venom immunotherapy (VIT) is the only disease-modifying treatment reported to be effective at decreasing the risk of systemic reactions (SRs) to IFA stings.
UNASSIGNED: Our aims were to determine the baseline rates of IFA sensitization in subjects, describe IFA VIT prescribing patterns across the military health system (MHS), and retrospectively evaluate the safety and efficacy of IFA VIT.
UNASSIGNED: We prospectively compared IFA sensitization in participants with and without an SR to flying Hymenoptera venom. Separately, IFA VIT prescription records were extracted from a centralized repository, and rates were described across the MHS. Additionally, we retrospectively reviewed the clinical course of patients being treated with IFA VIT at 11 military treatment facilities.
UNASSIGNED: The in vitro IFA sensitization rates in our prospective cohort ranged from 19.1% to 24.1%. Sensitization rates did not differ statistically between the subjects with or without an SR to flying Hymenoptera venom. We found that 60.9% of all MHS IFA VIT prescriptions (491 of 806) were from the 11 facilities in this study. We retrospectively identified 137 subjects actively undergoing IFA VIT. Among the subjects actively undergoing IFA VIT, 28 reported an SR to IFA venom and repeat stings by IFAs after reaching VIT maintenance, and 85.7% (24 of 28) of the subjects noted symptoms no worse than a large swelling reaction after a repeat IFA sting. Notably, only 2.9% of the subjects (4 of 137) had an SR due to VIT.
UNASSIGNED: This study\'s results align with those of prior IFA sensitization reports. A substantial proportion of patients undergoing IFA VIT experienced protection against anaphylaxis with reexposure, with relatively few adverse events.

A precise diagnosis of peanut allergy is extremely important. We identified 4 Ara h 2 peptides that improved Ara h 2-specific IgE (sIgE) diagnostic accuracy.
To assess the diagnostic utility of sIgE to the mixture of these peptides and their role in mast cell response to peanut allergens.
sIgE to the peptide mix was determined using ImmunoCAP. Its diagnostic utility was compared with Ara h 2-sIgE and sIgE to the individual peptides. The functional relevance of the peptides was tested on the mast cell activation test using laboratory of allergic diseases 2 cell line and flow cytometry.
A total of 52 peanut-allergic (PA), 36 peanut-sensitized but tolerant, and 9 nonsensitized nonallergic children were studied. Peptide mix-sIgE improved the diagnostic performance of Ara h 2-sIgE compared with Ara h 2-sIgE alone (area under the receiver operating characteristic curve .92 vs .89, respectively; P = .056). The sensitivity and specificity of Ara h 2-sIgE combined with the peptide mix were 85% and 96%, respectively. sIgE to individual peptides had the highest specificity (91%-96%) but the lowest sensitivity (10%-52%) compared with Ara h 2-sIgE (69% specificity and 87% sensitivity) or with peptide mix-sIgE (82% specificity and 63% sensitivity). Peptide 3 directly induced mast cell activation, and the peptide mix inhibited Ara h 2-induced activation of mast cells sensitized with plasma from Ara h 2-positive PA patients.
sIgE to the peptide mix improved the diagnostic performance of Ara h 2-sIgE similarly to sIgE to individual peptides. The peptides interfered with Ara h 2-induced mast cell activation, confirming its relevance in peanut allergy.

Serum immunoglobulin E (IgE) assays to food specific IgE (s-IgE) are useful tools for the confirmation of clinical suspicion of food allergy. However, the specificity of these assays is poor given that sensitization is much more common than clinical food allergy. Therefore, the use of broad panels to assess sensitization to multiple foods often leads to overdiagnosis and unnecessary food avoidance. Unintended consequences that may occur as a result include physical harm, psychological harm, financial cost, opportunity cost, and even worsening of existing health care disparities. Although current guidelines recommend against the use of s-IgE food panel testing, these tests are widely available and frequently used. To limit the negative impacts of s-IgE food panel testing, further work is needed to effectively spread the message that these food panels may cause unintended harm to patients and families.

Cross-reactivity of allergens is the cause of various, sometimes unexpected, clinical reactions. There are no standard methods to investigate cross-reactivity. We present an experimental model of a two-sided inhibition test (IT) on ImmunoCAP membranes (CAP). We constructed the described model based on the known cross-allergy syndrome to red meat developing in people bitten by ticks (α-Gal syndrome; AGS). Some individuals who are bitten by ticks develop IgE antibodies specific to the carbohydrate determinant, galactose-α-1,3-galactose (α-Gal), present in the tick\'s saliva. These antibodies can cross-react with α-Gal molecules expressed on mammalian meat proteins. The well-known property of anti-α-Gal IgE antibodies binding by various sources of this allergen was used by us in the proposed model of the two-sided inhibition test on ImmunoCAP membranes. We expected that anti-α-Gal IgE antibodies bind allergens from mammalian meat and blocking them abolishes this reactivity, and the two-sided inhibition test model we proposed on ImmunoCAP membranes allowed us to observe such a relationship. We conducted the experiment three times on biological material from people with different clinical manifestations of allergy to α-Gal, each time obtaining similar results. In conclusion, the model of bilateral inhibition on ImmunoCAP membranes proposed by us seems to be an attractive, simple tool for direct testing of allergic cross-reactivity.

UNASSIGNED: Aspergillus diseases are frequently encountered in patients who are immunocompromised. Without a prompt diagnosis, the clinical consequences may be lethal. Aspergillus-specific antibodies have been widely used to facilitate the diagnosis of Aspergillus diseases. To date, universally standardized cut-off values have not been established. This study aimed to investigate the cut-off values of Aspergillus-specific antibodies and perform a narrative review to depict the geographic differences in the Taiwanese population.
UNASSIGNED: We analyzed enrolled 118 healthy controls, 29 patients with invasive aspergillosis (IA), chronic pulmonary aspergillosis (CPA), and allergic bronchopulmonary aspergillosis (ABPA) and 99 with disease control, who were tested for Aspergillus fumigatus and Aspergillus niger-specific IgG and IgE using ImmunoCAP. 99 participants not fulfilling the diagnosis of IA, CPA, and ABPA were enrolled in the disease control group. The duration of retrieval of medical records from June 2018 to September 2021. Optimal cut-offs and association were determined using receiver operating characteristic curve (ROC) analysis.
UNASSIGNED: We found that patients with CPA had the highest A. fumigatus-specific IgG levels while patients with ABPA had the highest A. fumigatus-specific IgE, and A. niger-specific IgG and IgE levels. In patients with CPA and ABPA, the optimal cut-offs of A. fumigatus-specific IgG and A. niger-specific IgG levels were 41.6, 40.8, 38.1, and 69.9 mgA/l, respectively. Geographic differences in the cut-off values of A. fumigatus-specific IgG were also noted. Specifically, the levels were different in eco-climatic zones.
UNASSIGNED: We identified the optimal cut-offs of Aspergillus-specific antibodies to facilitate a precise diagnosis of aspergillosis. The observed geographic differences of the antibody levels suggest that an eco-climatic-specific reference is needed to facilitate a prompt and accurate diagnosis of aspergillosis.

BACKGROUND: ALEX multiplex platform has been recently commercialized but its clinical utility as quantitative technique respect to ImmunoCAP-singleplex as the reference method has not yet been confirmed on patients suffering from nut allergy and co-sensitization to different nuts.
METHODS: 58 serum samples from patients with nut allergy from a Mediterranean population were assayed in parallel by ALEX-multiplex and ImmunoCAP-singleplex techniques. Patients were diagnosed based on clinical symptoms and positive skin prick tests (SPTs). The following whole extracts were compared between both techniques: walnut, hazelnut, peanut, almond, pistachio and sunflower seed; besides the recombinant Pru p 3. A qualitative and quantitative study was carried out.
RESULTS: Both techniques had similar sensitivities respect to whole extracts from walnut, hazelnut and peanut as well as to Pru p 3 (p > 0.05). However for whole extracts from almond, pistachio and sunflower seed the sensitivity obtained by ALEX was much lower than ImmunoCAP (9.09 % vs 88.63 %; 14.81 vs 70.37 %; and 8.51 % vs 88.88 %; respectively). The concordance between both techniques showed only a substantial agreement for Pru p 3 (k = 0.791); moderate agreement for hazelnut and peanut (k = 0.550 and k = 0.544, respectively); fair agreement for walnut (k = 0.386) and poor agreement for almond, pistachio and sunflower seed (k < 0.2). Quantitative analysis showed that ImmunoCAP for walnut, peanut and sunflower seed had higher mean values than ALEX. Relationships were significant for all specific IgE levels except to for almond, pistachio and sunflower seed.
CONCLUSIONS: ALEX platform is a suitable technique to patients with nut allergy from the Mediterranean area except to for those suffering from allergy to almond, pistachio and sunflower seed.

BACKGROUND: The reagent which is available for single allergenic tests is Oriton IgE, ImmnoCAP, Alastat in Japan. No study has investigated the correlations of Oriton IgE and ImmnoCAP or Alastat, and, used for specific IgE antibody testing.
METHODS: Six frequently tested allergens (dust mite, cedar pollen, dog dander, egg white, milk, and candida) were measured by three methods, and Spearman rank correlation coefficient and class-judged agreement were evaluated. Furthermore, we did the evaluation like other 2 methods when we made small short sample volumes of Oriton IgE.
RESULTS: As for the examination result of Oriton IgE and ImmnoCAP or Alastat, constant correlation was confirmed. However, the tendency was a different result by assay method and an allergenic item. No significant differences were observed in the results of the Oriton IgE test when standard sample volumes and small short sample volumes were used.
CONCLUSIONS: These comparison results help us to understand each characteristic and select an optimal test method. In addition, it can be inferred that it is beneficial to choose tests requiring small sample volumes in pediatric patients.