Major Allergen Research Articles

Epitope profiling of cow's milk allergen-specific antibodies with determining IgE content in epitopes-ALL, a 14-epitopes mixture.

Allergen-specific antibodies (Abs), IgE, and IgG4 increase during the early phase of oral immunotherapy (OIT) of allergen food in patients; subsequently, IgE levels decrease and specific IgG4 levels increase after successful OIT treatment. The detailed profile of these Abs during OIT remains largely unclear. We developed a diagnostic tool to assess the OIT efficacy and extent of responsiveness based on a profiling method by identifying epitopes recognized by the Ab classes of IgE or IgG4. A peptide microarray followed by microplate analysis using synthetic peptides was used to identify 14 epitopes widely recognized by IgE and/or IgG4 in the serum samples of patients with OIT among the amino acid sequences of five major cow's milk allergens. The set of defined 14 epitopes clarified different epitope profiles of allergen-specific IgE and IgG4 in each patient's serum samples. Moreover, the total signal of Abs recognizing all 14 epitopes was equal to the sum of all individual epitope-specific Abs. It was further observed that the quantitative value of IgE concentrations of 14 epitopes-ALL correlated with the ImmunoCAP IgE value. These findings strongly imply that the quantity of IgE and IgG4 recognizing epitopes-ALL may easily be used to measure allergy severity. To investigate this potential, we developed an immunochromatographic method that can detect IgE and IgG4 levels in patient samples. This study clearly demonstrated the usefulness of the defined 14 epitopes and their mixture, "epitopes-ALL," and that the simple and reliable methods of immunochromatography and microplate analyses demonstrating the epitope profile of allergen-specific Abs are applicable for diagnostic use at multiple disease stages and the OIT-treatment course in patients with cow's milk allergy.

Total protein concentration and stability of Amb a 1 in glycerinated ragweed sublingual immunotherapy stored at room temperature and refrigerated cold temperature.

Few studies have investigated optimal storage conditions or expiration dates for sublingual immunotherapy (SLIT) formulations prepared from glycerinated allergen extracts. The objective of this study was to compare concentrations of short ragweed major allergen (Amb a 1) and total protein in SLIT formulations stored at two different temperatures. It was hypothesised that protein concentrations would show greater decline over time in a formulation stored at room temperature (RT) than in one stored under refrigeration. Two SLIT samples containing equal volumes of 20,000 PNU Amb a 1 extract were prepared and stored at refrigerated cold (CT) (2-8°C) or RT (20-24°C) for 140 days. Changes in total protein and major allergen concentration and composition were measured by Bradford assay, two-site enzyme-linked immunosorbent assay and SDS-PAGE. Presence of Amb a 1 was confirmed with Western immunoblot. Data were analysed using an analysis of covariance, with p < 0.05 considered significant. SDS-PAGE showed compositional changes in a ~26-30 kDa protein band under RT and not CT storage. The Amb a 1 concentration of the RT SLIT sample declined significantly over time, compared to that of the CT SLIT sample (F(1,8) = 47.69, p < 0.0001). There was no significant difference in total protein concentration over time between groups (F(1,8) = 1.79, p = 0.22). These results demonstrate that storage of glycerinated SLIT formulations in refrigerated CT preserved the highest concentration of the specific allergen Amb a 1, suggesting that SLIT formulations containing short ragweed should be stored under refrigeration.

Allergenic Potential of Common Hops (Humulus lupulus L.) in the Context of Cross-Reactions with Pollen Allergens

Background: Common hops (Humulus lupulus L.) play a key role in brewing, providing the bitterness, flavor, and aroma of beer, and are widely used in supplements for their antibacterial, anti-inflammatory, and antioxidant properties. However, despite their broad applications, the allergenic potential of common hops remains underexplored, particularly when compared to the closely related Humulus japonicus. This preliminary study aimed to investigate the allergenic potential of common hops and their potential cross-reactivity with common pollen allergens. Methods: The immunoreactivity of hop stalks, leaves, and cones was assessed using antibodies against major allergens from birch (Bet v1a), mugwort (Art v1), and timothy grass (Phl p5b), as well as three sera from pollen-allergic patients. Slot Blot analysis was performed using phosphate-buffered saline extracts from the stalks, leaves, and cones of three hop cultivars, while Western Blotting followed SDS-PAGE protein separation. Results: The results revealed significant immunoreactivity in native hop proteins, with diminished reactivity observed in denatured proteins. Cross-reactivity between hop proteins and major pollen allergens was confirmed, indicating that hop proteins may contribute to allergic sensitization in pollen-sensitive individuals. Conclusions: These findings underscore the potential allergenic risks associated with the consumption or exposure to hop-containing products.

Improved quality control of allergen products: Assessing the molecular allergen composition by mass spectrometry.

Natural allergen sources contain a variety of allergens, against which allergic subjects have developed individual sensitization profiles. Ideal allergen products for skin prick testing (SPT) and allergen immunotherapy (AIT) should contain the complete set of allergens of the respective allergen sources to cover all sensitization profiles. However, commercially available allergen products were shown to vary regarding their allergen composition. The qualitative allergen composition of different SPT and AIT products produced from pollen of grasses, birch, mugwort and from house dust mites was assessed by a consistent high-resolution liquid chromatography-coupled tandem mass spectrometry method (LC-MS/MS). All major, mid-tier and most minor allergens were detected in each of the investigated three batches of SPT and AIT products, demonstrating the completeness of the allergen composition and a high degree of batch-to-batch consistency. This is the first study using a single consistent high-resolution LC-MS/MS method to provide solid data on the qualitative allergen composition of SPT and AIT products manufactured from various common allergen sources. The applied method showed high reliability in qualitative batch-to-batch consistency testing and can be performed fast and with high throughput. High-resolution LC-MS/MS is applicable for process development and quality control to ensure market availability of allergen products corresponding to the composition of the respective natural allergen sources.

First Report of Alternaria alternata Causing Alternaria Leaf Spot of Black Nightshade in China.

Black nightshade (Solanum nigrum L.) belongs to Solanaceae family, which is distributed all over China and has become one of the malignant weeds in cotton field in Xinjiang (Zhang et al., 2020; Zhang et al., 2021). During July 2016 to August 2023, leaf spot was observed on black nightshade in Hami and Shihezi city, Xinjiang, China. The spots appeared as brown circular or irregular, with some small black dots surrounded with yellow halos. Twenty five black nightshade plants were collected from a cotton field (100 m2) for pathogen isolation in Shihezi, July 2023. Leaf samples (2×2 mm) from the boundary between diseased tissue and healthy tissue, sterilized in 70% ethanol for 45 s, dipped in 2% NaClO for 30 s, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA) after dried and cultivated at 25°C in the dark for 4 days. Twenty-five fungal isolates were obtained and purified by using the single-spore isolation method, and all isolates were found to be similar in morphology, appeared as dark brown and produced dark brown pigmentation on PDA. Conidiophores were black brown, 8 to 58 × 10 to 20 μm, with 1 to 5 transverse septa and 0 to 3 longitudinal septa, the results similar to those of Alternaria alternata isolated from buckwheat (Li et al., 2021). To confirm this identification, total genomic DNA of the isolates were extracted by CTAB method (Doyle et al., 1987). The internal transcribed spacer (rDNA-ITS), Alternaria major allergen gene (Alt α1) and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) of the A. alternata 26 were amplified using the primers ITS1/ITS4 (Glass et al., 1995), Altα1-F/Altα1-R (Li et al., 2024) and gpd-F/gpd-R (5'-CAACGGCTTCGGTCGCAT- TG-3' / 5'-GCCAAGCAGTTGGTTGTG-3'), respectively. The rDNA-ITS, Altα1 and GAPDH gene sequences were deposited in GenBank (KX904867, PP263361, PP263360), and showed 100% identity (rDNA-ITS: 572 out of 609 bp; Alt α1: 514 out of 516 bp; GAPDH: 619 out of 619 bp) to A. alternata (KU179665; MW522975; MK451977), respectively. The multi-gene phylogenetic tree showed that the representative isolate was grouped with A. alternata and identified as A. alternata. Black nightshade was used for pathogenicity assay. Fifteen plants at 3 to 4-leaf stage were selected and sprayed with the conidial suspension (1.0×106 spores/mL) of A. alternata 26 after wound the underside of leaves (2 mm length) with inoculating needle. Five plants were sprayed with sterilized water as control. After inoculation, placed the plants in a plastic box and covered with plastic wrap to keep 80% relative humidity for 3 days at 25℃ in the greenhouse, then removed the plastic box. Symptoms of leaf spots appeared within 12 - 15 days that were similar to the symptoms observed in the field, and the fungal pathogen that was re-isolated from symptomatic leaves was identical to original pathogen on the basis of morphological and molecular analysis to fulfill Koch's postulates. This experiment was repeated three times, and the disease incidence was above 80% after inoculation at each time. To our knowledge, this is the first report of A. alternata causing leaf spot of black nightshade in China. This report will help us to monitoring distribution of the disease and providing theoretical basis for disease prevention and control.

First Report of Alternaria alternata Causing Leaf Spot of Magnolia delavayi in China

Magnolia delavayi, known as Delavay's magnolia or Chinese evergreen magnolia, is an enchanting deciduous tree native to southwestern China (Xu et al. 2020). In March 2024, leaf spots were observed on M. delavayi plants at Qujing Normal University (25.527°N, 103.744°E), Qujing, Yunnan, China. About 80% of the trees (n=92) on campus were infected, with infected leaves accounting for about 10 to 30% of all leaves. The leaf spots exhibited a round or oval shape, with a yellowish-brown center and a brown outer ring. As the spots expanded, the leaves gradually dried up and fell off. Five symptomatic leaves were collected from the middle sections of five diseased plants. Tissue samples (5 × 5 mm) were excised from the lesion margins and subjected to surface sterilization in 75% ethanol for 30 seconds and 1% sodium hypochlorite for 60 seconds. They were then rinsed three times with sterile water and dried on sterilized paper. The samples were plated on potato dextrose agar (PDA) and incubated at 28 °C for 3 days. Developing colonies were retransferred to new PDA and purified by the hyphal tip technique (Senanayake et al. 2020). Two representative isolates (SYL1 and SYL2) were selected and confirmed to be the same species based on morphological characteristics and molecular identification. On PDA, the fungal colonies initially appeared grayish-white and turned dark gray over time with cotton-like aerial hyphae on the surface. The conidia are light brown, pear-shaped or rod-shaped, with 1 to 4 transverse septa and 0 to 1 oblique septa, and measured 5.4 to 12.7 × 10.4 to 25.7 μm (n = 50). Molecular identification was performed by partial sequencing of the internal transcribed spacer (ITS: ITS4/ITS5) region, glyceraldehyde 3-phosphate dehydrogenase (GAPDH:gpd1/gpd2), translation elongation factor 1-alpha (TEF1:EF1/EF2), and Alternaria major allergen (Alt a 1:Alt-for/Alt-rev) (Woudenberg et al. 2015). The resulting sequences of ITS (PP951882, PP951883), GAPDH (PP968942, PP968943), Alt a 1(PP968940, PP968941) and TEF1 (PP968938, PP968939) were deposited in GenBank. BLAST analyses revealed that the sequences from these two isolates showed 100% identity with those from Alternaria alternata strains for each gene. Additionally, a multigene phylogenetic analysis revealed a distinct group containing SYL1, SYL2 and other A. alternata strains with a posterior probability of 100%. The morphological and phylogenetic evidence collectively suggested that the two isolates belonged to A. alternata. Strain SYL1 was used for the pathogenicity test on three 5-year-old M. delavayi plants on campus. Three healthy leaves of each plant were punctured with a sterile needle and sprayed with 20 μl of spore suspension (1 × 106 conidia/ml). Similarly, another nine wounded leaves were sprayed with 20 μl of sterile water. All inoculated leaves were enclosed in plastic bags for 48 hours. Disease symptoms were observed on leaves inoculated with spore suspension after 7 days, while control plants remained healthy. This experiment was repeated three times with same results. To fulfill Koch’s postulates, A. alternata was reisolated from the inoculated leaves and identified based on morphological characteristics and ITS sequencing. To our knowledge, this is the first report of A. alternata causing leaf spot on M. delavayi in China. This research expands the known host range of this pathogen and provides relevant information to the design of disease management strategies.

A new leaf spot caused by Alternaria alternata on Atractylodes lancea in Jiangsu, China.

Atractylodes lancea is an important Chinese herbal medicine, which is mainly produced in Jiangsu province, China. In June 2022, leaf spots symptom were observed on some A. lancea seedlingsgrowing in a Chinese herbal medicine resource garden of NanjingBotanical Garden, Jiangsu. Approximately 75% of 100 A. lancea seedlings suffered from the disease. Initially, gray to black spots appeared at the tip of the blade, then spread deep into the petiole, finally causing the blade to wither and fall. To isolate the pathogen, five diseased leaves were collected from five different seedlings. Leaf sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% alcohol for 30 s, then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃in darkness. Pure cultures were obtained by monosporic isolation. Eighteen isolates were obtained, and 77.8% of isolates was identified as Alternaria spp. A representative isolate, CS4-1 was used for further investigation. The colony of CS4-1, growing on PDA was cotton-like and black to brown with gray-white aerial hyphae on their surfaces, and dark gray on the back. The conidia were solitary on conidiophores and were oval to pear-shaped, brown in color, with 1 to 4 transverse septa and 0 to 1 oblique septa, parietal cells extending into the beak, and measured 8.9 to 39.5×6.0 to 13.5 µm (n=35). These characteristics were consistent with the description of Alternaria spp. (Simmons 2007). Six DNA regions, i.e.,internal transcribed spacer (ITS),large subunit ribosomal RNA (LSU), small subunit ribosomal RNA (SSU), anonymous region OPA10-2, Alt a 1 major allergen (Alta1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH)and translation elongation factor 1-alpha (TEF1)with the respective GenBank Accession No. OP836052, OP836054, OP836051, OP851487, OP851488, OP851489, and OP851490, were amplified and sequenced with the primer pairs described by White et al. (1990) and Woudenberg et al. (2015). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7, and CS4-1 clustered in the A. alternata clade. To test pathogenicity, 12 leaves, on three one-month-old A. lancea seedlings (four leaves from each seedling) were wounded with a sterile needle and inoculated with 20 μL of conidial suspension (1×106 spores/mL) on the left sides of leaves. The right sides of the leaves were inoculated with 20 µL sterile water and used as the control. All inoculated detached leaves and seedlings were covered with clear polyethylene bags to keep moisture and were incubated in a greenhouse at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 4 days, typical gray to black spots were visible on the left sides of all inoculated leaves and the inoculated seedlings, and the right sides remained asymptomatic. Subsequently, the same fungus was reisolated and identified based on morphological and molecular traits. A. alternata has a very wide host range but has never been reported on A. lancea worldwide (nt.ars-grin.gov). Because of the medicinal value of A. lancea, further studies should be directed toward control of this disease.

Parent-reported offering of allergen foods to infants during complementary feeding: An observational study of New Zealand infants

The prevalence of food allergies in New Zealand infants is uncertain but is believed to be similar to Australia, exceeding 10%. Current recommendations for reducing food allergy risk are to offer all major food allergens to infants from as early as six months of age (start of complementary feeding), and before 12 months of age. However, little is known regarding parental practices around introducing major food allergens. This study aimed to explore parental offering of major food allergens to infants during complementary feeding, and parent-reported food allergies. The cross-sectional study is a secondary analysis of the multi-centre (Auckland and Dunedin) First Foods New Zealand study of 625 parent-infant dyads. Participants were recruited in 2020–2022 when infants were 7–10 months of age. Questionnaires assessed sociodemographic characteristics, complementary feeding approach, infant pouch use and parental responses to five food allergy questions. All major food allergens had been offered to only 17% of infants by 9–10 months of age. Having offered egg, peanut, tree nuts, sesame, soy and seafood was more commonly associated with using a baby-led complementary feeding approach than a parent-led approach (p < 0.001). Frequent baby food pouch use was associated with a lower likelihood of offering egg and peanut (both p < 0.001). Overall, 12.6% of infants had a reported food allergy, with symptomatic response after exposure being the most common diagnostic tool. Most infants are not offered all major food allergens during early complementary feeding, with some parents actively avoiding major food allergens in the first year of life. These results provide up-to-date knowledge of parental practices, highlighting the need for more targeted advice and strategies to improve parental engagement with allergy prevention and diagnosis.

Identification, Purification, and Characterization of Kunitz-Type Protease Inhibitors from White-Fleshed Dragon Fruit (Selenicereus undatus) and Red-Fleshed Dragon Fruit (Selenicereus costaricensis) Seeds as Novel Food Allergens.

There have been clinical reports of allergies to dragon fruits. However, little is known about the allergens that trigger these reactions. This study aimed to investigate novel allergens in dragon fruits. The peptide mass fingerprints of two purified natural allergens were identified by LC-MS/MS with chymotrypsin proteolysis. The complete amino acid sequences of the allergens were deduced from cDNA amplicon sequences. The coding sequences of two allergens were inserted in the expression vector pColdI, and recombinant allergens were produced in Escherichia coli. Circular dichroism was performed to analyze the secondary structures of natural and recombinant allergens. Our results identified two novel allergens as Kunitz-type protease inhibitors. Recombinant allergens exhibited well-folded structures, predominantly composed of β-sheets, similar to their natural counterparts. IgE reactivity analysis with sera from ten patients primarily sensitized to dragon fruit indicated that Kunitz-type protease inhibitors are major allergens in dragon fruits. These results improve our understanding of allergen sources and provide important insights into the allergenicity of proteins from different species.

Effect of enzymatic hydrolysis and ultrasound pretreatment on allergenicity, functional properties and bioactivity of whey protein isolates

Whey protein isolate (WPI) is an important food ingredient and a major allergen in food. Enzymatic hydrolysis is an important method for WPI modification, but there is limited information on the effect of ultrasound pretreatment on WPI hydrolysates (WPH). The aim of this study was to analyze the effect of enzymatic hydrolysis and ultrasound pretreatment on the allergenicity, functional properties and bioactivity of WPH by multispectroscopy and peptidomics combined with bioinformatics. The results showed that ultrasound pretreatment contributed to the production of WPH with low allergenicity (IgE binding capacity is 55.59%) and good functional properties (emulsifying activity index is 27.02%, foaming characteristic is 50.00%) and bioactivity (total antioxidant capacity is 65.94%). This was attributed to the synergistic hydrolysis of WPI by endopeptidases and exopeptidases, and ultrasound pretreatment that altered the accessibility of WPI to proteases and the peptide profiles and main proteins of WPH, affecting the changes in their spatial and primary structure. This research will advance the application of WPI in hypoallergenic foods.

Ecological and Allergenic Significance of Atmospheric Pollen Spectra from a Grassland-Savanna Ecotone in North-West Province (South Africa)

ABSTRACT Southern Africa's floral, topographic and climatic diversity manifests in distinctive bioregions where aerobiology studies are needed to manage allergenic health problems, especially in urban areas. Previous studies underline different seasonal pollen fluctuations for the biomes, encouraging regional studies. These studies have been previously restricted to large cities such as Cape Town, Johannesburg and Pretoria whereas small cities such as Potchefstroom in the North-West province are understudied. The South African Pollen Monitoring Network (SAPNET) seeks to fill this lacuna, enhancing ecological and health-related knowledge, with regards to the allergenicity of the recorded pollen. Here we investigate plant phenology and resulting pollen production within the Grassland and Savanna biomes around Potchefstroom, a fast-growing city in the North West province, comparing it with other SAPNET sites. As a SAPNET initiative, pollen monitoring in Potchefstroom involved monthly collections using a Lanzoni 7-day spore trap over 13 months from December 2022 to 2023. This facilitated the development of a preliminary pollen calendar and a pollen atlas, detailing pollen types' morphology, ecology, pollination modes, and allergenicity. Initial findings revealed Potchefstroom's pollen calendar, with tree pollen (60% of the Annual Pollen Index (API)) mainly from northern hemisphere species such as Cupressus (c. 11% of API) and Platanus (c. 18%) which released pollen in spring. Grass pollen was predominant from November to April (c. 18% of API). Potchefstroom recorded South Africa's highest levels of Ambrosia pollen (c. 7% of API), a major allergen, in March and April. Noteworthy is the unusual abundance of pollen of the neophyte Ulmus (c. 14%) in December. This study highlights the pollen patterns of the Grassland and Savanna biomes around Potchefstroom. The region, with its mosaic of urban spaces, natural grass-/woodland, and agricultural land, exhibits distinct characteristics when contrasted with major, more densely populated cities such as Bloemfontein, Pretoria, and Johannesburg.

Optimizing β-Lactoglobulin antigenicity through single enzyme hydrolysis: Exploring structural changes and effects on linear epitopes

β-lactoglobulin (β-LG) is the major allergen in dairy products, but research on the optimal conditions for antigen reduction in β-LG using different enzymes remains limited. Therefore, this study aims to investigate the antigenicity, structural characteristics, and peptide distribution of advantageous protease hydrolysates capable of eliminating the allergenic epitopes of β-LG selected via bioinformatics tools. The results showed that under optimal enzymatic hydrolysis conditions, the antigen reduction rates for the four advantageous proteases acting on β-LG were 47.37 % (pepsin), 33.54 % (chymotrypsin A), 38.71 % (papain), and 45.91 % (stem bromelain), respectively. The four proteases effectively degraded β-LG, causing shorter peptide chain formation, reduced content of highly ordered α-helix, decreased fluorescence intensity, and lower surface hydrophobicity. Furthermore, they cleaved the linear epitopes of β-LG into peptides of varying sizes, leading to different antigen reduction rates among the hydrolysates. These findings provide a theoretical basis for developing targeted enzymatic hydrolysis technologies and low-allergenicity dairy-based materials.

Sensitisation to Lep d 2- Lepidoglyphus destructor allergy in asthma and rhinitis.

Background. Mites are responsible for allergic diseases, such as asthma and rhinitis, in nearly 1.5% of the world population. It is known that nowadays, besides House Dust Mites (HDM), Storage Mites (SM) are important sensitisers in urban non-occupational dwellings, predominantly in the Mediterranean area. The main objective of our study was to analyse the clinical relevance of the most prevalent SM, Lepidoglyphus destructor, by assessing the relationship between sensitisation to the major molecular allergen Lep d 2 and allergic respiratory disease phenotype in an urban population monosensitised to this molecular allergen. Methods. Cross-sectional study which included consecutive patients evaluated in our Allergy Department between 2012 and 2018, from urban non-occupational setting, with rhinitis and/or asthma, perennial symptoms and proven sensitisation to SM Lep d 2, tested using ImmunoCAP Immuno Solid-phase Allergen Chip (ISAC) technology, which detects 112 allergens. Results. A total of 37 allergic patients were included (23 female), aged 20.1 ± 12.8, min-max 5-58 years-old. The molecular sensitisation profile showed 18.9% of L. destructor mite monosensitised patients, having only sIgE to Lep d 2. This subset of patients mainly had allergic rhinitis, with 28.6% also being asthmatic. Regarding severity, most patients showed a persistent moderate phenotype of respiratory disease. The mean value of Lep d 2 sIgE was 8.3 ± 9.8 ISU-E, lower than in mite polysensitised patients (21.7 ± 21.5, p = 0.049). Conclusions. Our proof-of-concept study focused on Lep d 2 monosensitisation, showed that SM may have clinical relevance in perennial allergic asthma and rhinitis, and should be taken into account when assessing and treating allergic respiratory disease. Conclusions. The present survey demonstrated that Italian primary care pediatricians accomplish ARIA guidelines and adapt treatment on the basis of the intensity of symptoms. Corticosteroids and antihistamines are the most common prescribed medications. Nasal lavages are also popular.

Recombinant Gad c 1 improves diagnostic efficacy of cod allergy but not horse mackerel allergy.

Parvalbumin Gad c1 is a major cod allergen used as a follow-up marker of fish-allergic children. However, the diagnostic efficacy of recombinant Gad c 1 (rGad c 1) for fish allergy diagnosis remains controversial. This study aimed to evaluate the efficacy of rGad c1 for diagnosing cod and horse mackerel allergy. This single-centered, retrospective study obtained oral food challenges (OFCs) information performed for cod and horse mackerel. Cod-, horse mackerel-, and rGad c1-specific immunoglobulins (sIgEs) were investigated. Diagnostic performances of these parameters were compared using areas under the curve (AUC). We enrolled 45 and 38 children with suspected cod and horse mackerel allergies, respectively. The median age (interquartile range) of children with suspected cod allergy was 5.7 (0.7-11.7) years and that of children with suspected horse mackerel allergy was 6.0 (1.0-12.3) years. Fourteen and 22 children reacted to OFCs with 25 (10-40) g of cooked pacific cod and 40 (10-40) g of cooked horse mackerel, respectively. The cod sIgE and rGad c 1 sIgE AUCs for cod allergy diagnosis were 0.85 and 0.90, respectively. For horse mackerel allergy diagnosis, AUCs of horse mackerel and rGad c 1 sIgE were 0.76 and 0.72, respectively. Both AUCs for cod and mackerel allergy were significantly different. rGad c 1 sIgE is more effective than cod sIgE as a diagnostic marker of cod allergy, but less effective than horse mackerel sIgE as a diagnostic marker of horse mackerel allergy. Further studies are warranted to explore the potential applications of rGad c 1 sIgE in the diagnosis of various fish allergies.

Development of a graphene field effect transistor-based immersible biosensor for immunodetection of the birch pollen allergen Bet v 1 in air samples

Pollen traps, the current gold standard to determine pollen load and thereby the allergy season, are not sufficient to determine the allergenic risk. Therefore, the establishment of highly sensitive assays for allergen measurement is of highest interest. Herein, a graphene field-effect transistor (GFET) was constructed on an interdigitated electrodes chip to develop an immersible biosensor, which was used to detect the major birch pollen allergen Bet v 1. Graphene was wet-transferred on interdigitated electrodes that contain a reference electrode used as a liquid gate in the GFET. Using a standard ELISA protocol, two different anti-Bet v 1 antibodies were chosen and immobilized on graphene for the specific capture of the target allergen. The sensitivity of the GFET biosensor was evaluated using a standard Ag/AgCl liquid gate electrode and a reference electrode when the chip was immersed in Bet v 1-containing solutions. The results showed a higher performance and sensitivity for Bet v 1 detection compared to a mediator release method, one of the most sensitive assays for allergen detection. Compared with conventional methods of allergen detection, these immersible biosensors significantly improved the speed and level of detection providing the foundation of a point-of-need platform for in-field application. Furthermore, the proposed technique provides both a new biosensor for allergen detection and a strategy for designing low-cost integrated biosensors.

NMR residual dipolar couplings investigation in the topology of house dust mite Group V allergens

Blo t 5 is an important major allergen protein from Blomia tropicalis mites, which are prevalent in tropical and subtropical regions, including Taiwan. It is a coiled-coil triple helical bundle, but there currently is ambiguity around its structural fold and packing of the three helices. We have relied on NMR residual dipolar coupling data collected from four different alignment media to confirm that Blo t 5 has left-handed helical topology and further used that data to refine its solution structure. Earlier we had described conformational epitope for a detection monoclonal antibody by exclusive use of TROSY NMR experiments that studied Blo t 5 binding with the antibody FAB’ fragment. Here, we confirm those findings with an extensive mutagenesis and biophysical study to validate the NMR epitope mapping approach proposed by us.

Allergen immunotherapy using recombinant Culicoides allergens improves clinical signs of equine insect bite hypersensitivity.

Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis of horses caused by bites of Culicoides spp., sharing some common features with human atopic dermatitis. Allergen immunotherapy (AIT) using Culicoides whole-body extracts has limited efficacy. This study aimed to evaluate AIT with a pool of major Culicoides recombinant allergens in a prospective, double-blinded, placebo-controlled study. The IBH lesion score was assessed during a pre-treatment year and first treatment year (May-October) in 17 horses and in May and July of a second treatment year. Nine horses were immunized subcutaneously 3× with a combination of nine r-allergens (20 μg each/injection) in alum and monophosphoryl lipid A (MPLA). Eight horses received a placebo. The immunization was repeated twice the following year. The specific antibody response to one of the AIT Culicoides r-allergens was assessed. In the first treatment year, the decrease in average IBH lesion score was significantly larger in the AIT compared to the placebo group, with 67% of the AIT group and 25% of the placebo horses reaching >50% improvement of the average IBH lesion score. The response to the AIT was enhanced in the 2nd treatment year when 89% of the AIT vs. 14% of the placebo horses showed an improvement (p ≤ 0.01). IgG antibodies of all subclasses were induced, with IgG4/7 showing the most significant differences between groups. The post-AIT sera showed IgE blocking activity. AIT using only a few injections of small amounts of r-allergens in alum and MPLA as immunomodulators seems a promising approach for the treatment of insect bite allergy.

Allergy mapping in children with allergic rhinitis in different subjects of the Russian Federation

Given the climatic and geographical location and differences in the change of seasons in the Russian Federation, there is a marked heterogeneity in the representation of respiratory allergens. Obviously, in the case of double or triple sensitization, the diagnosis of its true picture is a difficult task. In such cases, allergy-component analysis is an extremely necessary stage of allergological examination. Purpose of the study: to assess the sensitization spectrum of AR patients in the age group from 3 to 17 years inclusive, living in different regions of the Russian Federation. Comparative analysis of differences in the sensitization spectrum of AR patients in the Republic of Ingushetia in different age groups. The study involved 47 patients, 36 of them were children under 18 years of age: Moscow n = 9 (Central Federal District), Samara n = 8 (Volga Federal District), Magas n = 19 (North Caucasus Federal District, Republic of Ingushetia). Also, 11 patients aged 18 years and older from Magas city took part in the study. The results obtained by ImmunoCAP ISAC and ALEX2 immune solid-phase allergy test were analyzed. The numerical values of the main respiratory allergens were evaluated, in patients with clinical signs of AR in the period from 01.01.2023 to 27.01.2024. The diagnosis of AR was made according to generally accepted criteria for diagnosis. The main causative factor of AR formation is the major allergen of birch pollen (Bet v1). However, for the Central Federal District, heterogeneity of representation in the group of buciferous trees was noted, which should be taken into account when planning allergen-specific immunotherapy. The spectrum of sensitization of children with AR in the North Caucasian Federal District (Magas) differs significantly from the Central and Volga Federal Districts, where the main major allergen of Amb a 1 ragweed pollen forms the etiological basis of AR formation. When comparing patients with AR of different age groups from Magas city, no differences in the sensitization spectrum were found. The results indicate a high degree of importance of allergy-component diagnostics to improve the effectiveness of allergen-specific immunotherapy.

Silencing of RDR1 and RDR6 genes by a single RNAi enhances lettuce's capacity to express recombinant proteins in transient assays.

Enhanced recombinant protein expression was achieved in Salinas lettuce and commercial lettuce by designing a unique RNAi that knockdown the gene-silencing mechanism in transient assays. Improved yields of recombinant proteins (RP) are necessary for protein-production efficiency and ease of purification. Achieving high yield in non-tobacco plants will enable diverse plants to be used as hosts in transient protein-expression systems. With improved protein yield, lettuce (Lactuca sativa) could take the lead as a plant host for RP production. Therefore, this study aimed to improve RP production in lettuce var. Salinas by designing a single RNA interference (RNAi) construct targeting LsRDR1 and LsRDR6 using the Tsukuba system vector. Two RNAi constructs, RNAi-1 and RNAi-2, targeting common regions of LsRDR1 and LsRDR6 with 75% and 76% similarity, respectively, were employed to evaluate simultaneous gene silencing. Quantitative transcription analysis demonstrated that both RNAi constructs effectively knocked down LsRDR6 and LsRDR1, but not LsRDR2, at both 3 and 5days post-infiltration (dpi), with RNAi-1 exhibited slightly higher efficiency. Based on the protein yield, co-expression of RNAi-1 with enhanced green fluorescent protein (EGFP) increased EGFP expression by approximately 4.9-fold and 3.7-fold at 3dpi and 5dpi, respectively, compared to control. A similar but slightly lower increase (2.4-fold and 2.33-fold) was observed in commercial lettuce at 3and 5 dpi, respectively. To confirm these results, co-infiltration with Bet v 1, a major allergen from birch pollen, resulted in a 2.5-fold increase in expression in Salinas lettuce at 5 dpi. This study marks a significant advancement in enhancing transient protein production in lettuce, elevating its potential as a host for recombinant protein production.

Pollen Cross-Reactivity: A Primer for Allergy Specialists

There are over 400,000 land-based plant species that comprise our biodiverse habitat. Only a small subset of those plant species satisfies Thommen’s postulates, classifying them as allergens. The number of allergenic species, however, remains so vast that it is prohibitive to both test and treat for all relevant species within a geographical region. Allergy specialists have a valuable tool that can be used to help simplify the management of allergic patients: cross-reactivity. Cross-reactivity is the ability for an allergen to induce an IgE-mediated response, regardless of previous exposure. Allergens are a complex milieu of proteins, some of which have allergic potential while others do not. A number of proteins are conserved across allergen species. When exposure to these conserved proteins occurs, the immune system recognizes them in a similar manner. For homologous or cross-reactive allergenic proteins, this conserved molecular recognition initiates the allergic cascade. Allergen characterization is critical to the understanding of cross-reactivity. Characterization, in this context, refers to the protein make-up of a particular allergen. The process of allergen characterization began in 1962 with the discovery of antigen E, the first identified allergenic protein. Antigen E, commonly known as Amb a 1, is an allergenic protein in ragweed, and is the primary sensitizing protein for ragweed allergy sufferers. These primary sensitizers are referred to as major allergens and can be defined as such when &gt;50% of the allergic patient population is sensitized to them. An entire branch of research arose from this discovery, which has allowed for major advances to be made in the understanding of cross‑reactive relationships among allergen species. Cross‑reactivity is not limited to homologous major allergen expression. Rather, minor allergens and panallergens, though less clinically relevant, play a similar role in cross‑reactive relationships. This primer will explore the science behind cross‑reactivity, as well as provide a general overview of the cross‑reactive relationships that have been defined for plant allergens found across North America.