Allergen-specific B cell responses in oral immunotherapy-induced desensitization, remission, and natural outgrowth in cow's milk allergy.

BACKGROUND: Antigen-specific memory B cells play a key role in the induction of desensitization and remission to food allergens in oral immunotherapy and in the development of natural tolerance (NT). Here, we characterized milk allergen Bos d 9-specific B cells in oral allergen-specific immunotherapy (OIT) and in children spontaneously outgrowing cow's milk allergy (CMA) due to NT. METHODS: Samples from children with CMA who received oral OIT (before, during, and after), children who naturally outgrew CMA (NT), and healthy individuals were received from Stanford biobank. Bos d 9-specific B cells were isolated by flow cytometry and RNA-sequencing was performed. Protein profile of Bos d 9-specific B cells was analyzed by proximity extension assay. RESULTS: Increased frequencies of circulating milk allergen Bos d 9-specific B cells were observed after OIT and NT. Milk-desensitized subjects showed the partial acquisition of phenotypic features of remission, suggesting that desensitization is an earlier stage of remission. Within these most significantly expressed genes, IL10RA and TGFB3 were highly expressed in desensitized OIT patients. In both the remission and desensitized groups, B cell activation-, Breg cells-, BCR-signaling-, and differentiation-related genes were upregulated. In NT, pathways associated with innate immunity characteristics, development of marginal zone B cells, and a more established suppressor function of B cells prevail that may play a role in long-term tolerance. The analyses of immunoglobulin heavy chain genes in specific B cells demonstrated that IgG2 in desensitization, IgG1, IgA1, IgA2, IgG4, and IgD in remission, and IgD in NT were predominating. Secreted proteins from allergen-specific B cells revealed higher levels of regulatory cytokines, IL-10, and TGF-ß after OIT and NT. CONCLUSION: Allergen-specific B cells are essential elements in regulating food allergy towards remission in OIT-received and naturally resolved individuals.

Legume Allergens Pea, Chickpea, Lentil, Lupine and Beyond.

PURPOSE OF THE REVIEW: In the last decade, an increasing trend towards a supposedly healthier vegan diet could be observed. However, recently, more cases of allergic reactions to plants and plant-based products such as meat-substitution products, which are often prepared with legumes, were reported. Here, we provide the current knowledge on legume allergen sources and the respective single allergens. We answer the question of which legumes beside the well-known food allergen sources peanut and soybean should be considered for diagnostic and therapeutic measures. RECENT FINDINGS: These "non-priority" legumes, including beans, pea, lentils, chickpea, lupine, cowpea, pigeon pea, and fenugreek, are potentially new important allergen sources, causing mild-to-severe allergic reactions. Severe reactions have been described particularly for peas and lupine. An interesting aspect is the connection between anaphylactic reactions and exercise (food-dependent exercise-induced anaphylaxis), which has only recently been highlighted for legumes such as soybean, lentils and chickpea. Most allergic reactions derive from IgE cross-reactions to homologous proteins, for example between peanut and lupine, which is of particular importance for peanut-allergic individuals ignorant to these cross-reactions. From our findings we conclude that there is a need for large-scale studies that are geographically distinctive because most studies are case reports, and geographic differences of allergic diseases towards these legumes have already been discovered for well-known "Big 9" allergen sources such as peanut and soybean. Furthermore, the review illustrates the need for a better molecular diagnostic for these emerging non-priority allergen sources to evaluate IgE cross-reactivities to known allergens and identify true allergic reactions.

The shape of and applied weight on skin prick lancets critically affect the wheal size in the skin prick test.

The global application of the skin prick test (SPT) is attributed to the low costs, easy execution, and in vivo approach. Still, the healthcare professionals' technique and the lancet shape may challenge the standardization of the method. Thus, we investigated the influence of the shape of the lancet and the applied weight on the wheal size of SPT. Two allergic and one non-allergic individual were tested with allergens (Dermatophagoides pteronyssinus and Phleum pratense) and histamine solution (positive control), respectively. Horizontally (HS) and diagonally (DS) shouldered lancets with the same tip length (1 mm) were tested under two different conditions: either 60 g or 120 g weight pressure. The wheal size induced by the 4 different combinations was measured. The higher-weight device (120 g) induced a significantly larger and less variable wheal response with the tested allergens and histamine. However, the shape of the lancet affected the wheal size more than the applied weight. The least variable response was measured to histamine for the horizontal-shouldered lancet combined with the higher weight, whereas the same lancet with the lower weight resulted in a significant number of false negative results.

Transition between subcutaneous and sublingual allergen immunotherapy: Recommendations of the Brazilian Association of Allergy and Immunology (ASBAI).

The use of allergen immunotherapy (AIT) in Brazil has specific regional conditions owing to the pattern of allergen sensitization, as well as to genetic, socioeconomic, and cultural characteristics. This review article aims to discuss the clinical practice of AIT by the subcutaneous or sublingual route in Brazil, addressing the possibilities of transition between these forms of administration. A systematic review using the PubMed and Cochrane databases was performed, and the websites of major allergy and immunology organizations were consulted. Knowledge of the mechanism of action of subcutaneous immunotherapy and sublingual immunotherapy, together with Brazilian real-life experience, allowed us to establish recommendations regarding switching routes of AIT administration in selected cases. Careful analysis of each clinical situation is necessary to perform the transition between subcutaneous and sublingual allergen immunotherapy.

Development of a rapid and reliable method to simultaneously detect seven food allergens in processed foods using LC-MS/MS.

Rapid analysis of multiple food allergens is required to confirm the appropriateness of food allergen labelling in processed foods. This study aimed to develop a rapid and reliable method to simultaneously detect trace amounts of seven food allergenic proteins (wheat, buckwheat, milk, egg, crustacean, peanut, and walnut) in processed foods using LC-MS/MS. Suspension-trapping (S-Trap) columns and on-line automated solid-phase extraction were used to improve the complex and time-consuming pretreatment process previously required for allergen analysis using LC-MS/MS. The developed method enabled the simultaneous detection of selected marker peptides for specific proteins derived from seven food ingredients in five types of incurred samples amended with trace amounts of allergenic proteins. The limit of detection values of the method for each protein were estimated to be <1 mg/kg. The developed analytical approach is considered an effective screening method for confirming food allergen labelling on a wide range of processed foods.

A composite enzyme derived from papain and chymotrypsin reduces the Allergenicity of Cow's Milk allergen casein by targeting T and B cell epitopes.

Casein, the major allergen in cow's milk, presents a significant challenge in providing nutritional support for children with allergies. To address this issue, we investigated a composite enzyme, comprising papain and chymotrypsin, to reduce the allergenicity of casein. Enzymatic hydrolysis induced substantial structural changes in casein, diminishing its affinity for specific IgE and IgG antibodies. Additionally, in a BALB/c mouse model, casein hydrolysate alleviated allergic symptoms, evidenced by lower serum IgE and IgG levels, reduced plasma histamine, and decreased Th2 cytokine release during cell co-culture. Peptidomic analysis revealed a 52.38% and 60% reduction in peptides containing IgE epitopes in casein hydrolyzed by the composite enzyme compared to papain and chymotrypsin, respectively, along with a notable absence of previously reported T cell epitopes. These results demonstrate the potential of enzyme combinations to enhance the efficiency of epitope destruction in allergenic proteins, providing valuable insights into the development of hypoallergenic dairy products.

Mechanisms for initiation of food allergy by skin pre-disposed to atopic dermatitis.

Food allergy can be life-threatening and often develops early in life. In infants and children, loss-of-function mutations in skin barrier genes associate with food allergy. In a mouse model with skin barrier mutations (Flakey Tail, FT+/- mice), topical epicutaneous sensitization to a food allergen peanut extract (PNE), an environmental allergen Alternaria alternata (Alt) and a detergent induce food allergy and then an oral PNE-challenge induces anaphylaxis. Exposures to these allergens and detergents can occur for infants and children in a household setting. From the clinical and preclinical studies of neonates and children with skin barrier mutations, early oral exposure to allergenic foods before skin sensitization may induce tolerance to food allergens and thus protect against development of food allergy. In the FT+/- mice, oral food allergen prior to skin sensitization induce tolerance to food allergens. However, when the skin of FT+/- pups are exposed to a ubiquitous environmental allergen at the time of oral consumption of food allergens, this blocks the induction of tolerance to the food allergen and the mice can then be skin sensitized with the food allergen. The development of food allergy in neonatal FT+/- mice is mediated by altered skin responses to allergens with increases in skin expression of interleukin 33, oncostatin M and amphiregulin. The development of neonate food allergy is enhanced when born to an allergic mother, but it is inhibited by maternal supplementation with α-tocopherol. Moreover, preclinical studies suggest that food allergen skin sensitization can occur before manifestation of clinical features of atopic dermatitis. Thus, these parameters may impact design of clinical studies for food allergy, when stratifying individuals by loss of skin barrier function or maternal atopy before offspring development of atopic dermatitis.

House dust mite immunotherapy: A real-world, prescription data-based analysis.

BACKGROUND: House dust mite (HDM) sensitisation can contribute to the development of allergic rhinoconjunctivitis (AR) or allergic asthma (AA). As treatment, allergen immunotherapy (AIT) is a promising approach, since it aims building immunotolerance against allergens, therewith establishing long-term efficacy. The evaluation of AIT has been investigated in many randomised controlled trials, whereas few real-world evidence studies are available. METHODS: We used data from the longitudinal prescription data base IQVIA™ LRx. Data on initial AIT prescriptions against HDM from January 2009 to December 2013 was analysed regarding treatment (subcutaneous AIT with either depigmented polymerised allergen extract [dSCIT] or other allergens [oSCIT], or sublingual immunotherapy [SLIT]) and treatment duration. Treatment groups were compared with a control group of AR patients not receiving AIT. Data on symptomatic medication was collected until February 2017 and progression of AR and AA was compared. RESULTS: Data of 7260 patients with AIT prescriptions and of 21,780 control patients was analysed. AIT was associated with a significant decrease of AR medication intake compared with control (dSCIT: -34.0%, p < 0.0001; oSCIT: -25.7%, p < 0.0001; SLIT: -37.7%, p = 0.0026). In asthmatics, SCIT was associated with a significant decrease of asthma medication compared with control (dSCIT: -45.2%, p < 0.0001; oSCIT: -32.9%, p < 0.0001). Further, a significantly reduced likelihood for onset of asthma medication was demonstrated in patients treated with SCIT compared with controls (dSCIT OR: 0.759, p = 0.0476; oSCIT OR: 0.815, p = 0.0339). CONCLUSION: Real-world data analyses indicate that AIT, particularly given via a subcutaneous route, reduces the need of medication against AR and AA and might delay the onset of asthma medication in patients with AR.

Prevalence and determinants of food allergy in the era of early allergen introduction: the EarlyNuts population-based study.

BACKGROUND: Infant feeding guidelines in Australia changed in 2016 to recommend introduction of common allergy causing foods by age 1 year to prevent food allergy. Although the majority of Australian infants now eat peanut and egg by age 6-months, some still develop food allergy despite introducing allergens early. OBJECTIVE: We aimed to describe the prevalence of food allergy in a cohort recruited after introducing the nation-wide allergy prevention recommendations; identify characteristics of infants who developed allergy despite early introduction of allergens; and estimate the causal effect of modifiable exposures on food allergy prevalence and whether this differed between infants who were introduced to allergen before or after age 6 months. METHODS: We recruited a population-based sample of 12-month-old infants in Melbourne, Australia. Infants had skin prick tests to 4 foods and parents completed questionnaires. Infants with evidence of sensitisation were offered oral food challenges. Prevalence estimates were adjusted using inverse probability weighting. RESULTS: In a cohort of infants (n=1420) where nearly all infants had been introduced to common allergens such as egg, milk and peanut by one-year-of age, the prevalence of food allergy remained high at 11.3% (95% CI 9.6-13.4%). Infants who developed food allergy despite introducing the allergen by age 6-months were more likely to have Asian-born parents. Early-onset moderate/severe eczema was associated with an increased odds of food allergy, irrespective of whether allergens were introduced before or after age 6 months. Among infants who were introduced to peanut ≤6m, antibiotic use by age 6 months was associated with an increased odds of peanut allergy (aOR 6.03 (95%CI 1.15-31.60). CONCLUSION: In a cohort where early allergen introduction was common, the prevalence of food allergy remained high. Infants who developed food allergy despite introducing the respective allergen by 6 months were more likely to have Asian parents and early-onset eczema. New interventions are needed for infants with a phenotype of food allergy that is not amenable to early allergen introduction.

Treatment with the SQ tree sublingual immunotherapy tablet is safe and well tolerated in real-life.

BACKGROUND: The SQ tree sublingual immunotherapy (SLIT)-tablet is authorised for treatment of allergic rhinoconjunctivitis with or without asthma in trees of the birch homologous group in 21 European countries. The primary objective of this study was to explore the safety in real-life. METHODS: In a prospective, non-interventional post-authorisation safety study (EUPAS31470), adverse events (AEs) and adverse drug reactions (ADRs) at first administration and follow-up visits, symptoms, medication use, and pollen food syndrome were recorded by physicians in 6 European countries during the first 4-6 months of treatment. RESULTS: ADRs with the SQ tree SLIT-tablet were reported in 57.7% of 1069 total patients (median age 36.0 years, 53.7% female) during the entire observation period (severity, mild-to-moderate: 70.1%, severe: 4.7%, serious: 0.7%) and in 45.9% after first administration. ADRs were not increased with pollen exposure at first administration. With coadministration of the SQ tree and grass SLIT-tablet AEs were reported in 73.8% of patients and in 52.8% with the SQ tree SLIT-tablet alone. Nasal and eye symptoms improved in 86.9% and 80.9% of patients and use of symptomatic medication in 76.0%. PFS with symptoms was reported in 43.0% of patients at baseline and in 4.3% at the individual last visit. CONCLUSIONS: The results of this non-interventional safety study with the SQ tree SLIT-tablet confirm the safety profile from placebo-controlled clinical trials and support effectiveness in real-life according to the published efficacy data. Safety was not impaired by pollen exposure at first administration or co-administration with other SLIT-tablets.

[Molecular allergen IgE tests show neosensitizaions occurring during house dust mite specific immunotherapy].

Objective:Neosensitizations may be occur during the allergen specific immunotherapy（AIT） due to the differences between allergen vaccine's content and a patient's molecular sensitization profile. This study investigates whether AIT with HDM extract changes the sensitization profile, whether de novo sensitization occurs, and the clinical importance of the neosensitization. Methods:Fifty-three patients with HDM allergic rhinitis ,with/without asthma, patients were received one year HDM subcutaneous AIT . Fourteen patients were recruited as control group and received only necessary medications. Serum samples were collected at baseline, 6thmoths and 12thof AIT, respectively. Serum samples were tested specific IgE against Der p, Der p 1/2/3 and Der f, Der f 1/2/3, as well as IgG4 against Der p, Der p 1/2 and Der f, Der f 1/2. VAS were collected at the time-points as well. Results:In AIT group, Der p, Der p 1/3, and Der f 1/3 specific IgE levels were significantly higher after one-year treatment, especially for Der p 3. There were 69.2%（18/26） patients whose Der p 3 specific IgE below 0.35 kU/L at baseline but became positive（>0.35 kU/L） after treatment, that is, neosensitization occurred. All tested allergen specific IgG4 level significantly increased after one year AIT treatment and the VAS declined dramatically. However, for patients with neosensitization and without neosensitization, there were no significantly changes concerning to IgG4 level and VAS. Conclusion:Patients undergoing AIT might have a risk of neosensitization to the allergen components in the vaccines. However, the clinical importance of the neosensitization remains unclear and warrants further studies.

Vitamin D and allergic diseases.

In recent years, the relationship between vitamin D and allergic diseases has received widespread attention. As a fat-soluble vitamin, vitamin D plays a crucial role in regulating the immune system and may influence the onset and progression of diseases such as atopic dermatitis, allergic rhinitis, and asthma. To understand the underlying mechanisms, we have summarized the current research on the association between vitamin D and allergic diseases. We also discuss the impact of vitamin D on the immune system and its role in the course of allergic diseases, particularly focusing on how vitamin D supplementation affects the treatment outcomes of these conditions. We aim to provide a theoretical basis and practical guidance for optimizing the management and treatment of allergic diseases by modulating vitamin D levels.

Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma.

Introduction: Equine asthma (EA) is a common lower airway disease in horses, but whether its pathogenesis is allergic is ambiguous. Extrinsic stimuli like hay dust induce acute exacerbation of clinical signs and sustained local neutrophilic inflammation in susceptible horses. Aspergillus fumigatus is an EA stimulus, but it is unclear if it merely acts as an IgE-provoking allergen. We aimed to comprehensively analyze immunoglobulin (Ig) isotypes in EA, elucidating their binding to different A. fumigatus antigens, and their quantities systemically in serum and locally in bronchoalveolar lavage fluid (BALF). Methods: Serum and BALF from healthy horses (HE, n = 18) and horses with mild-moderate asthma (MEA, n = 20) or severe asthma (SEA, n = 24) were compared. Ig isotype (IgG1, IgG3/5, IgG4/7, IgG6, IgA, and IgE) binding to nine antigens (A. fumigatus lysate, and recombinant Asp f 1, Asp f 7, Asp f 8, dipeptidyl-peptidase 5, class II aldolase/adducin domain protein, glucoamylase, beta-hexosaminidase, and peptide hydrolase) was compared by enzyme-linked immunosorbent assays. Total Ig isotype contents were determined by bead-based assays. Results: MEA and SEA differed from HE but hardly from each other. Compared to HE, asthmatic horses showed increased anti-A. fumigatus binding of IgG (BALF and serum) and IgA (BALF). Serum and BALF IgE binding and total IgE contents were similar between HE and EA. Single antigens, as well as A. fumigatus lysate, yielded similar Ig binding patterns. Serum and BALF IgG1 binding to all antigens was increased in SEA and to several antigens in MEA. Serum IgG4/7 binding to two antigens was increased in SEA. BALF IgA binding to all antigens was increased in SEA and MEA. Total BALF IgG1 and IgG4/7 contents were increased in SEA, and serum IgG4/7 content was increased in MEA compared to HE. Yet, total isotype contents differentiated EA and HE less clearly than antigen-binding Ig. Discussion: A. fumigatus immunogenicity was confirmed without identification of single dominant antigens here. A. fumigatus provoked elevated BALF IgG1 and IgA binding, and these isotypes appear relevant for neutrophilic EA, which does not support allergy. BALF Ig isotype differentiation beyond IgE is crucial for a comprehensive analysis of immune responses to fungi in EA pathogenesis.

Allergen immunotherapy in asthma.

Allergen immunotherapy (AIT), including SCIT and SLIT, is a treatment that involves the administration of allergens to which patients with allergic diseases have been sensitized. HDM-SCIT for asthma is indicated in cases of HDM-sensitized allergic asthma with normal lung function. HDM-SCIT improves asthma symptoms and AHR, and decreases the medication dose. Importantly, AIT can improve other allergic diseases complicated by asthma, such as allergic rhinitis, which can also contribute to the improvement of asthma symptoms. Several studies have suggested that HDM-SLIT also attenuates the risk of asthma exacerbations, and improves lung function in asthma cases with allergic rhinitis. Furthermore, AIT can modify the natural course of allergic diseases, including asthma. For example, the effects of AIT are maintained for at least several years after treatment discontinuation. AIT can prevent the onset of asthma when introduced in allergic rhinitis, and can also inhibit or reduce new allergen sensitizations. Recent data have suggested that AIT may suppress non-targeted allergen-induced immune responses in addition to targeted allergen-induced responses, and suppress infections of the lower respiratory tract by enhancing IFN responses.

Sex differences in sociodemographic, clinical, and laboratory variables in childhood asthma: A birth cohort study.

BACKGROUND: There are marked sex differences in the prevalence and severity of asthma, both during childhood and adulthood. There is a relative lack of comprehensive studies exploring sex differences in pediatric asthma cohorts. OBJECTIVE: To identify the most relevant sex differences in sociodemographic, clinical, and laboratory variables in a well-characterized large pediatric asthma cohort. METHODS: We performed a cross-sectional analysis of the Mayo Clinic Olmsted County Birth Cohort. In the full birth cohort, we employed a natural language processing algorithm based on the Predetermined Asthma Criteria for asthma ascertainment. In a stratified random sample of 300 children, we obtained additional pulmonary function tests and laboratory data. We identified the significant sex differences among available sociodemographic, clinical, and laboratory variables. RESULTS: Boys were more commonly diagnosed with asthma than girls and were younger at the time of asthma diagnosis. There were no sex differences in relation to socioeconomic status. We identified a male predominance in the presence of a tympanostomy tube and a female predominance in the history of pneumonia. A higher percentage of boys had an FEV1/FVC ratio <0.85. Blood eosinophilia and atopic sensitization were also more common in boys. Finally, boys had higher levels of serum periostin than girls. CONCLUSION: This study describes significant sex differences in a large pediatric asthma cohort. Overall, boys had earlier and more severe asthma than girls. Differences in blood eosinophilia and serum periostin provide insights into possible mechanisms of the sex bias in childhood asthma.

The Availability of Allergen-Friendly Food for College Students Experiencing Food Insecurity: Exploring Current Campus Practices.

BACKGROUND: The burden of dietary restrictions includes the high cost and limited availability of allergen-friendly products which disproportionately impacts people with food insecurity. OBJECTIVE: We aimed to better understand access to allergen-friendly food college students experiencing food-insecurity with food allergies or other diet-treated conditions by surveying college campus food pantries. METHODS: 120 randomly selected United States colleges/universities were recruited to complete FOODiversity's Food Insecurity Questionnaire about campus food pantry operations, dietary restrictions, and food-insecurity initiatives, and provide details about their resources and training dedicated to supporting food-insecure young adults who avoid a specific food(s). RESULTS: Forty-eight percent of respondents ask students about dietary restrictions at food pantry intake, 37% track the number of patrons with restrictions, 30% process allergen-friendly product requests, 17% train staff about dietary restrictions, and 4% modify processes to accommodate dietary restrictions. Of the 48% who inquire about dietary restrictions, follow-up interventions vary in levels of accommodation. Dairy-free and gluten-free products are most commonly requested, and gluten-free products are the most donated/stocked and most difficult to accommodate. CONCLUSION: Food pantries play a critical role in reducing the burden experienced by food-insecure individuals, however, most pantries are unable to provide sufficient accommodations for clients with food allergies or other diet-treated conditions, including staff training, provision of allergen-friendly products, or identifying food-insecure college students with dietary restrictions; and promoting food allergy safety and nutritional impacts.

The Allergen Profile of Two Edible Insect Species-Acheta domesticus and Hermetia illucens.

SCOPE: Edible insect proteins are increasingly introduced as an alternative sustainable food source to address the world's need to feed the growing population. Tropomyosin is the main insect allergen; however, additional potential allergens are not well characterized and the impact of extraction procedures on immunological reactivity is unknown. METHODS AND RESULTS: Proteins from different commercial food products derived from cricket (Acheta domesticus) and black soldier fly (BSF) (Hermetia illucens) are extracted using five different extraction buffers. The proteins are analyzed by SDS-PAGE and immunoblotting using allergen-specific antibodies and crustacean allergic patient sera. IgE binding bands are analyzed by mass spectrometry as well as the complete allergen profile of all 30 extracts. Urea-based buffers are most efficient in extracting insect allergens. Shrimp-specific antibody cross-reactivity to tropomyosin from cricket and BSF indicates high sequence and structural similarity between shrimp and insects. Additional unique allergens are identified in both species, including hemocyanin, vitellogenin, HSP20, apolipophorin-III, and chitin-binding protein. CONCLUSIONS: Identifying potential allergenic proteins and their isoforms in cricket and BSF requires specific extraction approaches using urea-based methods. While tropomyosin is the most abundant and immunoreactive allergen, seven unique allergens are identified, highlighting the need for insect species-specific allergen detection in food products.

Hand and Foot Dermatitis Secondary to Zucchini Exposure.

Contact dermatitis is an inflammatory condition mediated by allergens and irritants, including food. There have been few reports of zucchini causing contact dermatitis outside of ingestion. We report a case of allergic contact dermatitis to zucchini secondary to sensitization by a past squash exposure. The patient was treated with both systemic and topical corticosteroids.

Establishment of monoclonal antibodies of BW10kDa distinguish it from Fag e 2 related to anaphylaxis.

BW10kDa, which is a buckwheat (BW) allergen, belongs to the 2S-albumin protein family, akin to Fag e 2. Detailed analyses of BW10kDa were lacking until this study. Herein, we conducted these analyses using monoclonal antibodies (mAbs) to recombinant BW10kDa (rBW10kDa). We successfully generated anti-rBW10kDa mAbs capable of distinguishing between Fag e 2 and BW10kDa. These mAbs were categorised into two types (type 1 and type 2) based on their reactivity to BW plant seed extracts in western blot analyses. Type 1 mAbs revealed two bands (15 kDa and 10 kDa), while type 2 mAbs showed a single band (15 kDa). Spot analyses using these mAbs confirmed that type 1 mAbs recognised epitopes near the C-terminal region, with the 10 kDa band representing the C-terminal subunit cleaved by protease. The mAbs targeting rBW10kDa enabled to assess the concentration of BW10kDa in wild type and also in diagnostic buckwheat extracts.