Milk Allergens Research Articles

World Allergy Organization (WAO) Diagnosis and Rationale for Action against Cow's Milk Allergy (DRACMA) Guidelines update - III - Cow's milk allergens and mechanisms triggering immune activation

BackgroundThe immunopathogenesis of cow's milk protein allergy (CMPA) is based on different mechanisms related to immune recognition of protein epitopes, which are affected by industrial processing. PurposeThe purpose of this WAO DRACMA paper is to: (i) give a comprehensive overview of milk protein allergens, (ii) to review their immunogenicity and allergenicity in the context of industrial processing, and (iii) to review the milk-related immune mechanisms triggering IgE-mediated immediate type hypersensitivity reactions, mixed reactions and non-IgE mediated hypersensitivities. ResultsThe main cow’s milk allergens – α-lactalbumin, β-lactoglobulin, serum albumin, caseins, bovine serum albumins, and others – may determine allergic reactions through a range of mechanisms. All marketed milk and milk products have undergone industrial processing that involves heating, filtration, and defatting. Milk processing results in structural changes of immunomodulatory proteins, leads to a loss of lipophilic compounds in the matrix, and hence to a higher allergenicity of industrially processed milk products. Thereby, the tolerogenic capacity of raw farm milk, associated with the whey proteins α-lactalbumin and β-lactoglobulin and their lipophilic ligands, is lost. ConclusionThe spectrum of immunopathogenic mechanisms underlying cow's milk allergy (CMA) is wide. Unprocessed, fresh cow's milk, like human breast milk, contains various tolerogenic factors that are impaired by industrial processing. Further studies focusing on the immunological consequences of milk processing are warranted to understand on a molecular basis to what extent processing procedures make single milk compounds into allergens.

An innovative approach for better understanding of milk allergy

Introduction: Cow’s milk and its products are popular components of the diet all over the world. Proteins contained in it, such as casein (Bos d 8), α-lactalbumin (Bos d 4), β-lactoglobulin (Bos d 5) and bovine serum albumin (Bos d 6, BSA), can cause troublesome symptoms. Among the proteins mentioned, casein is the most frequent cause of all milk allergies. It is worth mentioning that it is a protein that does not change its properties after thermal and enzymatic treatment. Therefore, casein allergic patients must avoid dairy products in even trace amounts.
 Aim of the study: The aim of the study is to present the benefits of molecular diagnostics of allergy in cow’s milk allergy.
 Description of the state of knowledge: In order to diagnose milk allergy, a medical examination should take place with a clinician who will determine the further steps of diagnosis based on the medical interview. For a complete allergy analysis, molecular diagnostics tests can be used to determine which protein the patient is sensitised to. Such an individual patient’s allergy profile is helpful in developing an appropriate diet.
 Summary: Molecular diagnosis in cow's milk allergy allows to estimate the risk of severe reactions after contact with a specific allergen. On its basis, it is also possible to predict the result of a cow's milk allergen provocation test. By using molecular diagnosis of allergy, it is possible to manage better and control cow's milk allergy in patients.

Insight into the molecular-level details of αs1 casein interactions with IgG: Combining with LC-MS/MS and molecular modelling techniques

αs1-Casein (αs1-CN) is a major cow milk allergen, while the tertiary structure of αs1-CN and conformational epitopes of αs1-CN have not been clarified. Here, a reasonable three-dimensional structure of αs1-CN was established using ab initio methods, and hot-spot residues and epitopes were investigated by combining molecular dynamics simulation, peptides synthesis, and ELISA. Obtained results demonstrated that the binding mechanism between αs1-CN and IgG was located on three main regions: a helical structure zone (E77-Q97), the flexible loop zone (Y154-T174), and a flexible C-terminal (N190-L198), mainly connecting via hydrogen bond and ionic bonds. The hydrolysates produced by papain with lowest antigenicity (12.43%), which could considerably destroy the essential epitopes of αs1-CN confirmed by epitope synthesis, and LC-MS/MS. The results reported herein would provide novel insights into the interface interactions between αs1-CN and IgG, and prove valuable for developing hypoallergenic infant-formula and peptide vaccines for allergen-specific immunotherapy.

Long-term changes in milk component immunoglobulins reflect milk oral immunotherapy outcomes in Finnish children.

Milk oral immunotherapy (OIT) may increase the amount of milk protein that can be ingested without triggering an allergic reaction. It is important to understand why some patients benefit from the treatment while others do not. The aim was to define the differences in the milk allergen component-specific (casein, α-lactalbumin, ß-lactoglobulin) immunoglobulin (sIg [sIgE, sIgG4, and sIgA]) levels relative to the long-term outcomes of milk OIT. In this long-term, open-label follow-up study, 286 children started milk OIT between 2005 and 2015. Follow-up data were collected at two points: the post-buildup phase and long term (range 1-11 years, median 6 years). Comparisons of sIg levels were made among three outcome groups of self-reported long-term milk consumption (high-milk dose, low-milk dose, and avoidance). A total of 168 (59%) of the 286 patients on OIT participated. Most patients (57%) were in the high-dose group; here, 80% of these patients had a baseline casein sIgE value less than 28 kUA/L, they had the lowest casein sIgE levels at all time (p < .001), their casein sIgG4/IgE levels increased, and long-term casein sIgA was highest compared with the low-dose and avoidance groups (p=.02). Low-milk dose group had the highest casein sIgG4/IgE levels in long term (p=.002). The baseline Ig profiles and responses to milk OIT differed depending on long-term milk consumption. Lower casein sIgE levels were associated with better outcome. Milk casein sIgA differed in the long term among high-milk consumers.

Nasal mucosal reactivity assessment via a double-blind placebo-controlled food challenge with cow’s milk allergens

BackgroundAllergies, including food allergies, are a considerable clinical and public-health problem. The introduced preventive measures and differential diagnostics, including oral food challenges, are the gold standard for determining further treatment planning.Case presentationWe present a case of an 18-year-old girl with a cow’s milk allergy who underwent an oral food challenge (double blind oral food challenge). Such a challenge may be confounded by inducing a response from other systems and organs, which provides theoretical grounds for the use of other methods of assessing the body’s response to food allergens (response demonstrated by the upper respiratory tract). Based on this idea, in order to assess the degree of mucosal response, we used optical rhinometry as an objective method for nasal patency evaluation, as well as identification of tryptase level in nasal lavage fluid and exfoliative cytology of nasal mucosa. The results of these tests confirmed positive reaction of the nasal mucosa in the course of the oral allergen challenge.ConclusionsThe observed increase in the nasal mucosal reactivity that accompanies oral food challenges may suggest a potential for using food allergens in nasal allergen provocation testing in order to diagnose food allergies.

Breastfeeding: Maternally Transferred Allergens in Breast Milk: Protective or Sensitizing?

According to a thorough literature search, the following allergen sources have been associated with allergy symptoms in the exclusively breastfed child: hen's egg, cow's milk, peanut, trout. Subsequently, several studies use the advantage of molecular allergology and investigate the potential transfer of single allergens into breastmilk. This is shown for caseins, whey proteins, gliadin, ovalbumin, ovomucoid, the peanut allergens Ara h 2 and Ara h 6, as well as the inhalant allergens Der p 1 and Blo t 5. It is still a matter of debate whether or not food allergens transferred via breastfeeding to the baby promote allergic sensitization or induce tolerance and via which mechanisms they may shift the immune response to the one or other side. Noteworthy, some breastfed children are described to be sensitized to foods before being exposed to solid foods, and this exposure may have occurred through breastmilk. In the light of these findings the investigation of food allergens transferred from the mother's diet into breastmilk and their impact on sensitization or allergy prevention remains a current topic in research. This review describes breastmilk in its composition and provides data on the identification of food allergens therein including human and mouse studies.

Cow’s Milk Antigens Content in Human Milk: A Scoping Review

The functionality of breast milk in terms of immunity is well-known. Despite this, a significant proportion of breastfed infants exhibit sensitization to different potentially allergenic proteins and clinical reactivity (including anaphylaxis) early in life and before the introduction of complementary feeding for the first time. The potential induction of early oral tolerance to overcome early allergic sensitization through exposure to allergens in breast milk also remains controversial and not yet well-established. The objective of this scoping review is to provide a critical appraisal of knowledge about the content of cow’s milk antigens in human milk. The amount of dietary derived milk antigens found in human milk and the analytical methodologies used to detect and quantify these antigens, the allergic status of the mother, the stage of lactation, the time of sampling (before or after ingestion of food), and the impact of human milk allergen on the infant were the outcomes that were assessed. Allergy risk was explored in all reviewed studies and could help to better elucidate its role in the context of allergic disease development. According to the included literature, we can conclude that there are mainly fragments derived from bovine proteins in human milk, and the presence of potentially allergenic molecules is greater in the milk of mothers with an allergic tendency. A clear relationship between maternal diet and allergen content in breast milk could not be firmly concluded though. Also, infants receiving milk from human milk banks, where donor milk is pasteurized for preservation, may be subject to greater risk of allergy development, especially for β-lactoglobulin.

The effect of industrial processing on the allergenicity of cow´s milk allergens

1. Jensen SA, Fiocchi AG, Baars T, et al. Diagnosis and RAtionale against Cow’s Milk Allergy (DRACMA) guidelines update - III – Cow’s milk allergens and mechanisms triggering immune activation. World Allergy Organ J. Forthecoming 2022. Google Scholar

Immunomodulatory Role of BLG-Derived Peptides Based on Simulated Gastrointestinal Digestion and DC-T Cell from Mice Allergic to Cow's Milk.

Peptides, but not whole protein, elicit an allergic reaction since food allergens should be consumed by digestion. In this study, we explored the remaining peptides after simulated digestion of cow’s milk in order to search for β-lactoglobulin (BLG)-derived peptides that could play an immunomodulatory role. As a major allergen in milk, BLG-derived peptides, 109 in total, were identified both from simulated infant and adult digestion in vitro. These peptides were mainly located in four regions, and they were synthesized as five peptides, namely, BLG1–14, BLG24–35, BLG40–60, BLG82–101, and BLG123–139. Then, the effect of peptides on the Caco-2 cell’s transport absorption, the co-stimulatory molecules of DC, and the T-cell phenotype was explored. The results suggested all peptides showed better transport absorption capacity with the apparent permeability coefficient higher than 2 × 10−6 cm·s−1. The ability of BLG40–60 for promoting lamina propria-derived DC cell (LPDC) maturation was observed by the increase in MHC II. Moreover, BLG1–14 and BLG40–60 directed activation of T lymphocytes towards a Th1 phenotype. This is the first report of the immunomodulatory potential of peptides in the sensitization of allergic reaction, and one peptide, BLG40–60, was regarded as an immunomodulatory peptide, one that should be further explored in an animal model in depth.

β-Lactoglobulin versus casein indirect ELISA for the detection of cow's milk allergens in raw and processed model meat products

Two methods based on indirect enzyme-linked immunosorbent assays (ELISA) targeting β-lactoglobulin (Bos d 5) or caseins (Bos d 8) were developed for milk protein concentrate (MPC) quantification in meat products. Model mixtures simulating cooked hams and sausages with known amounts of MPC and submitted to oven-cooking (68 °C, 5 h) and autoclaving (121 °C, 1 bar for 15 min), respectively, were used. Bos d 5 ELISA enabled quantifying 100 mg/kg of MPC in raw model foods and 1000 mg/kg in cooked hams, while no quantification was possible for autoclaved sausages. Bos d 8 ELISA enabled limits of detection and quantification of 10 mg/kg for all model foods. Therefore, caseins resisted autoclaving, while β-lactoglobulin was greatly affected, highlighting the importance of selecting an appropriate marker for method development. Herein, an in-house ELISA, using a commercial anti-casein antibody, is proposed as a highly specific and sensitive method, cheaper than the available ELISA kits.

Developing a synbiotic biotherapy to prevent allergic responses to food

Abstract Over the past generation, the prevalence of food allergies has risen dramatically, but the development of treatments has lagged. The commensal gut microbiome is critically involved in regulating allergic responses to food. Our laboratory has described a humanized model of cow’s milk allergy (CMA) in which germ-free mice are colonized with feces from healthy or CMA infants and sensitized to the cow’s milk allergen b-lactoglobulin (BLG). The healthy infant microbiota and specifically a single Clostridial species, Anaerostipes caccae, protect against the development of anaphylaxis, while the CMA microbiota does not induce this protection. We isolated a novel sub-strain of A. caccae (A. caccae_lahuc) from the feces of a healthy infant, sequenced its genome, and characterized its growth on various substrates in vitro. A. caccae_lahuc produces high concentrations of butyrate, a key immunoregulatory molecule in the gut which we hypothesize is critical to its protective impact. A. caccae_lahuc engrafts readily into the dysbiotic CMA microbiota, but co-delivery with lactulose is required for measurable butyrate production in vivo. Lactulose undergoes primary degradation by bacteria in the CMA feces which release small metabolites that A. caccae_lahuc can ferment into butyrate. Preliminary data show that this novel synbiotic biotherapy (A. caccae_lahuc + lactulose) prevents allergic responses to BLG in CMA-colonized mice as measured by serum mMCPT-1 and core body temperature upon challenge, and type 2 cytokine production in BLG-stimulated splenocytes. Further exploration into the mechanisms behind this therapeutic effect will contribute toward the larger goal of developing bacterial biotherapeutics against food allergies.

Simultaneous quantification of specific food allergen proteins using a fluorescent multiplex array

The aim was to develop a fluorescent multiplex array for simultaneously measuring regulated food allergens using specific allergen protein molecules from peanut, tree nut, cow’s milk, egg, soy, fish, shellfish, sesame, mustard and celery. Microspheres coupled to specific monoclonal antibodies were used for allergen detection, with purified allergens as reference standards.Standard curves for 17 allergens covered a 5-log dynamic range. Intra- and inter-assay acceptance criteria were within 70–130% recovery and a CV of ≤15%. Food reference materials contained high levels of their respective major allergens (2000–175,000 µg/g), Similar high allergen levels were found in 10 selected foods analysed using a 9-plex array. Egg, milk, peanut, hazelnut and walnut allergens were detectable in chocolate bars with incurred allergens at 3, 10, 30, and 100 ppm. The multiplex array is an efficient tool for measuring specific food allergens, with applications for risk assessment and standardization of therapeutic products for food allergy.

Leukocyte Adherence Inhibition Test to the assessment of Immunoreactivity Against Cow’s Milk Proteins in Non—IgE-Mediated Gastrointestinal Food Allergy

Background: The non—IgE-mediated food allergy syndromes are a group of conditions diagnosed mostly by clinical criteria and Oral Food Challenge tests (OFC).&#x0D; Objective: To evaluate the feasibility of the Leukocyte Adherence Inhibition Test (LAIT) to discriminate antigen-specific immunoreactivity in a group of patients with non—IgE-mediated cow’s milk allergy diagnosed by OFC and clinical criteria.&#x0D; Methods: Ex vivo challenge tests performed with cow’s milk proteins extracts were monitored by LAIT in two groups: A) the active group consisting of 38 patients presenting non—IgE-mediated gastrointestinal lactose-free cow’s milk hypersensitivity; B) the control group consisting of 29 cow’s milk tolerant individuals&#x0D; Results: The mean Leukocyte Adherence Inhibition (LAI) of the control group was 5.48%, and the mean LAI of the active group was 54.18%. The non-parametric Wilcoxon-Mann-Whitney U test showed p &lt; α = 0.05.&#x0D; Conclusion: The Leukocyte Adherence Inhibition Test is an ex vivo immunoassay able to predict individual non—IgE-mediated immunoreactivity against cow’s milk allergens and to act as a triage test to select food allergens to be prescribed in exclusion diets to perform OFC in patients with suspected non—IgE-mediated food allergies.

Immobilization and characterization of latex cysteine peptidases on different supports and application for cow’s milk protein hydrolysis

Calotropis procera cysteine peptidases (CpCPs) have been used for reducing cow’s milk allergenicity or as rennet in cheesemaking. Due to their residual presence in food products, the present study evaluated, for the first time, different protocols for their immobilization on different supports. Although the yield of immobilization on sulfopropyl-agarose (99%, at pH 7.0) was better than when using DEAE- and MANAE-agarose (40% and 15%, respectively), the derivatives had low recovered activity (4%). On the other hand, MANAE-agarose at pH 10.0 (200 mM buffer) exhibited the highest recovered enzymatic activity (~23%). Regarding the covalent immobilization, the peptidases immobilized on glyoxyl-agarose (glyoxyl-CpCPs) showed broader pH stability (pH 3.0–10.0), 60-fold more stable at 60 °C, and retained 70% of their initial activities after five reaction cycles, even though the immobilization has induced some structural changes analyzed by Fourier-Transform Infrared (FTIR) spectroscopy as well as altered some of enzyme kinetic parameters (Vmax, Km, Kcat, and catalytic efficiency). In addition, this biocatalyst (glyoxyl-CpCPs) hydrolyzed the major cow’s milk allergens (whey proteins) to a greater extent (65%) than the soluble enzymes (8%) and a commercial hypoallergenic formula (50%).

Surface-enhanced Raman spectroscopy relying on bimetallic Au–Ag nanourchins for the detection of the food allergen β-lactoglobulin

We designed a SERS-based method to detect β-lactoglobulin (β-Lg), a major allergen of milk, utilizing aptamers to specifically anchor β-Lg. The Au–Ag nanourchins with excellent Raman enhancement effects were prepared and modified with a β-Lg aptamer as the capture substrate. By taking advantage of the hybridization of aptamer and Raman reporter molecule (6-Carboxyl-X-Rhodamine: ROX)-labeled complementary DNA (cDNA), when ROX is close to Au–Ag nanourchins, the SERS signal is enhanced. In the presence of β-Lg, aptamer binding to β-Lg induces the dissociation of ROX-cDNA from Au–Ag nanourchins, thereby decreasing the SERS signal. Under optimal conditions, the present bioassay provides a wide detection range of 10 ng/mL to 1000 ng/mL with a detection limit of 0.07 ng/mL. In addition, the established SERS sensor can selectively and accurately identify β-Lg with recovery rates of 93.25–107.0% in milk samples. It provides great potential as a sensitive and reliable detection method against the growing threat of allergens.

Suboptimal basophil activation by molecular allergens can lead to false-negative basophil activation test

Introduction ( contexte de la recherche ) Molecular allergens have improved greatly the relevance of specific IgE serologies. Data are lacking regarding the added value of molecular allergens to the basophil activation test (BAT). To compare basophil activation by similar amounts of molecular allergen and allergen extract and to assess the risk of BAT discordances between extract and related component. Systematic retrospective monocentric study of all BAT performed at the Trousseau Hospital with both molecular components and corresponding extract. Data from 164 extract/component BAT pairs (71 cow milk, 46 wheat, 28 house dust mites (HDM), 12 peanut, 5 hen egg, 1 apple, 1 peach), corresponding to 111 blood samples from 89 patients were analyzed. Basophil activation rates were positively correlated between components and corresponding extract. However, component-stimulated basophils displayed significantly lower activation rates overall and for milk, wheat, peanut, and HDM molecular allergens. In addition, higher frequency of extract+/molecular− BAT discordances were found, as compared to extract−/molecular+ cases, accounting for 25% of BAT positive cases. Higher activation rate differences between extract and component were independently associated with lower total IgE and higher fMLP positive control values, but not with other demographic, clinical, or serological factor. A single molecular allergens usually have lower capacity than extract to activate IgE-sensitized basophils. In most cases, component-based BAT did not provide added information over extract-based BAT, and can potentially lead to false-negative results. Unless proven otherwise for a particular allergen, we suggest that BAT should be preferentially performed with protein extract.

A solid-state electrochemiluminescence aptasensor for β-lactoglobulin using Ru-AuNP/GNP/Naf nanocomposite-modified printed sensor.

An electrochemiluminescence (ECL) aptasensor for the detection of the milk protein allergen β-lactoglobulin (β-LG) using nanocomposite as luminophore was fabricated. The Ru-AuNPs/GNP/Naf complex was formed by combining the Rubpy32+-AuNPs complex (Ru-AuNPs), prepared by modifying the negatively charged surface of gold nanoparticles (AuNPs) with positively charged Rubpy32+ through electrostatic interactions and the graphene nanoplatelets-Nafion (GNP/Naf) at a ratio of 2:1. The nanocomposite was coated on the surface of the screen-printed electrode (SPCE) through the film-forming properties of Nafion. A layer of chitosan (CS) was coated onto this modified electrode, and later amine-terminated β-LG aptamers were covalently attached to the CS/Ru-AuNP/GNP/Naf via glutaraldehyde (GLUT) cross-linking. When β-LG was incubated with the aptasensor, a subsequent decrease in ECL intensity was recorded. Under the optimal conditions, the ECL intensity of the aptasensor changed linearly with the logarithmic concentration of β-LG, in the range 0.1 to 1000pg/ml, and the detection limit was 0.02pg/mL (3σ/m). The constructed aptasensor displayed simple and fast determination of β-LG with excellent reproducibility, stability, and high specificity. Additionally, the proposed ECL aptasensor displayed high recoveries (92.5-112%) and low coefficients of variation (1.6-7.8%), when β-LG fortified sampleswere analyzed. Integrating Ru-AuNPs/GNP/Naf nanocomposite in the ECL aptasensor paves the way towards a cost-effective and sensitive detection of the milk allergen β-LG.

Development of a sandwich enzyme-linked immunosorbent kit for reliable detection of milk allergens in processed food

The inclusion of undeclared cow's milk proteins may cause health complications to milk-allergic consumers and is one of the leading cause of food recall in many countries all over the world. Therefore, to keep control on such incidences in processed products, we established a milk sandwich ELISA test kit by incorporating two polyclonal antibodies against milk proteins obtained from different species. Its analytical effectiveness in terms of sensitivity, specificity, accuracy, trueness, and precision were all analyzed. The limit of detection (LOD) of the test kit was 0.011 ppm, with high specificity for milk protein residues. The test kit was highly specific, apart from considerable cross-reactivity with goat milk and minor cross-reactivity with donkey and horse milk. The coefficient of variation of the test kit for intra-assay ranged from 4.02% to 14.62% and inter-assay ranged from 6.05% to 15.08% respectively. The sandwich ELISA was highly specific in detecting commercial food products. In a limited retail survey, 5/6 of the milk proteins declared on the ingredient labels tested positive for milk proteins. The study offers effective technical support for the sensitive detection of milk products both for food manufacturers and regulatory authorities.

Characteristics of Exogenous Allergen in Breast Milk and Their Impact on Oral Tolerance Induction.

Food allergy is a common health problem in childhood since its prevalence was estimated to range from 6. 5 to 24.6% in European countries. Recently, a lot of research has focused on the impact of breastfeeding on oral tolerance induction. Since it was found that breast milk contains immunologically active food antigens, it would be very helpful to clarify the factors of antigen shedding that promotes oral tolerance. This narrative review aimed to summarize the latest evidence from experimental and human studies regarding allergen characteristics in human milk that may influence oral tolerance induction. A literature search in PubMed, MEDLINE, and Google Scholar was conducted. The diet of the mother was found to have a direct impact on allergen amount in the breastmilk, while antigens had different kinetics in human milk between women and depending on the antigen. The mode of antigen consumption, such as the cooking of an antigen, may also affect the allergenicity of the antigen in human milk. The dose of the antigen in human milk is in the range of nanograms per milliliter; however, it was found to have a tolerogenic effect. Furthermore, the presence of antigen-specific immunoglobulins, forming immune complexes with antigens, was found more tolerogenic compared to free allergens in experimental studies, and this is related to the immune status of the mother. While examining available data, this review highlights gaps in knowledge regarding allergen characteristics that may influence oral tolerance.

Inorganic food additive nanomaterials alter the allergenicity of milk proteins

While inorganic nanomaterials are copiously incorporated in food products, their impact on the allergenicity food proteins is largely unknown. This study analyzed the effect of widely used food additive nanomaterials (silica and titania) on the antigenicity and allergenicity of milk proteins (β-lactoglobulin and casein) and skimmed milk. Changes in the antigenicity of milk proteins in the presence of dietary nanomaterials were identified using an indirect-ELISA assay, while the change in allergenicity was studied using mast cell (LAD2) sensitized using allergic human sera. Results showed an enhancement in the allergenicity of milk proteins/skimmed milk interacted with particles (both silica and titania). Similarly, mast cell degranulation (a proxy for allergenicity) was higher when exposed to particle interacted skim milk where nanomaterials of titania showed the highest effect, and this tendency was retained even after subjecting to simulated gut digestion. Particles induced alterations in the structure of milk proteins, as evidenced by our studies, are reasoned to expose epitopes that increase allergenicity of milk proteins.