Beef Allergens Research Articles

Bos d 13, A Novel Heat-Stable Beef Allergen.

Red meat, a staple food of Western diets, can also induce IgE-mediated allergic reactions. Yet, apart from the heat-labile protein serum albumin and the carbohydrate α-Gal, the molecules causing allergic reactions to red meat remain unknown. IgE reactivity profiles of beef-sensitized individuals are analyzed by IgE-immunoblotting with protein extracts from raw and cooked beef. Two IgE-reactive proteins are identified by peptide mass fingerprinting as myosinlight chain 1 (MYL1) and myosin light chain 3 (MYL3) in cooked beef extract and are designated Bos d 13 isoallergens. MYL1 and MYL3 are produced recombinantly in Escherichia coli. ELISAs proved their IgE reactivity and circular dichroism analysis showed that they represent folded molecules with remarkable thermal stability. In vitro gastrointestinal digestion experiments showed the higher stability of rMYL1 as compared to rMYL3. Exposure of a monolayer of Caco-2 cells to rMYL1 indicated that the molecule is able to cross intestinal epithelial cells without disturbing the integrity of the tight junctions, suggesting the sensitizing capacity of MYL1. MYLs are identified as novel heat-stable bovine meat allergens.

Clinical analysis of food serum specific IgE in children with allergic rhinitis in Tianjin

Objective: To define the common food allergens in children with allergic rhinitis in Tianjin with detection of serum specific immunoglobulin E(IgE) against foods and inhalation.Method:Mediwiss(Screen Allergy) was used to determine serum specific IgE. The relationships between food allergy and gender, age, season were also analyzed.Result:Most common food allergens in children with AR in Tianjin are milk(17.28%), followed by egg white(13.79%), crab(9.56%) and cashew nuts(8.64%). Children in food-related allergy group were significantly less than that in the inhalation-related allergy group, the difference was statistically significant（P <0.05）. Among food-related allergy group, one hundred and ninty four cases are both sensitive to food allergens and inhalation allergens, and the other 34 cases are only sensitive to food allergens. There were significant differences about the positive rate of allergy between different age groups(P <0.01).There was no significant difference in the total food allergens sensitivity rate between different gender and seasonal groups（P >0.05）. The cashew and beef allergen sensitivity rates are different between male and female. The sensitivity rates of amaranth, egg, white milk, and crab are different among different age groups. The difference was statistically significant（P <0.05）.Conclusion: Milk, egg, crab, cashew nuts are the most common food allergens in children with AR in Tianjin area. Only food-related allergy is rare, food allergies are usually accompanied by inhalation-related allergy. Cashew allergen sensitivity rate was higher in male than female. Milk allergen sensitivity rate was higher in female than male. Sensitivity rate of amaranth allergen increased significantly, while that of egg milk and crab allergen decreased significantly with age.

Immunoproteomics of processed beef proteins reveal novel galactose-α-1,3-galactose-containing allergens.

Red meat allergy presents a novel form of food allergy with severe delayed allergic reactions where IgE antibodies are directed against the carbohydrate α-Gal epitope. Food preparation and processing can influence the allergenicity of proteins. The aim of this study was to characterize the proteomic profile of different beef preparations and to investigate their α-Gal reactivity and potential allergenicity. Extracts from raw, boiled, fried, and medium rare prepared beef were assessed by 2D PAGE for the comparison of protein profiles. IgE-binding proteins were identified using immunoblot-coupled proteomic analysis using sera from red meat-allergic patients. Presence of the α-Gal epitope was verified using anti-α-Gal antibody and IgE inhibition immunoblot with α-Gal. Multiple IgE-binding proteins were detected in the different beef preparations, many of which were also recognized by the anti-α-Gal antibody. Protein spots reacting with IgE in patient sera were analyzed by MS/MS, resulting in identification of 18 proteins with high identification scores. Seven of the 18 beef allergens identified using meat-allergic patient sera were also recognized by the anti-α-Gal monoclonal antibody, and four of them were stabile to thermal treatment. Furthermore, a dose-dependent inhibition of red meat-allergic patients' IgE to beef by α-Gal was demonstrated. We show that the α-Gal epitope is commonly present in IgE-reactive beef proteins recognized by meat-allergic patients. Seven novel α-Gal-containing IgE-binding proteins were identified, of which four were stable to heat treatment. Thus, the allergenicity of red meat proteins is preserved even upon different thermal cooking.

Effects of a High-Pressure Treatment on Bovine Gamma Globulin and Its Reduction in Allergenicity

The effects of a high-pressure treatment on the IgE-specific binding activity and structural changes to bovine gamma globulin (BGG), a beef allergen, were investigated. The allergenicity of pressure-treated BGG was also evaluated. We found that the IgE-specific binding activity and allergenicity of BGG were decreased by the high-pressure treatment. Almost no significant change in secondary structure was apparent for pressurized BGG, but a change in the tertiary structure was detected. The decreased IgE-specific binding activity and allergenicity of pressurized BGG were probably due to changes in the tertiary structure caused by pressurization. The results of this study suggest that a high-pressure treatment would be an effective food-processing technique to reduce the allergenicity of BGG.

식육알레르기 발생 경향 및 잠재적 우육알레르겐에 관한 연구

The prevalence of food allergies was investigated using questionnaires with 300 subjects whose ages ranged from 19 to 24 years old and the causative food allergens was analyzed using immunological analysis with serum of the subjects who answered that they have/had food allergy. The questionnaire showed that 11.33% of subjects have/had experience of food hypersensitivity, where the main causative foods were fish, beef, chicken, milk, egg, and pork in order. The meat allergy shared 4.65% (2.33% for beef, 1.66% for chicken, 0.66% for pork) in the prevalence of food allergies. The causative beef allergens were investigated with the serum of 6 subjects who have had beef allergy. Western blots were carried out with the serum of P6 subject who showed a positive reaction to beef extract in ELISA. The two specific bands were detected in beef extract on the PVDF membrane, and no band was detected in extracts of pork and chicken. A calculation of the distance of migration by SDS-PAGE enabled the molecular masses of the two bands to be estimated as 67kDa and 31kDa, respectively. The 67kDa was revealed as bovine serum albumin (BSA) which is one of the important beef allergens as reported previously though an analysis of the N-terminal amino acid sequence. However we could not identify the sequence of 31kDa, probably because they comprised several subunits and were modified proteins such as glycoprotein that were unlikely to be easily degraded by the Edman method. The 31kDa band were dyed with the PAS (periodic acid-schiff reagent), suggesting that it might be a glycoprotein. These results suggested that the 31kDa might be considered as a novel potential beef allergen which is not reported previously, although further studies are needed.

Analysis of Food Allergen Structures and Development of Foods for Allergic Patients

Food allergy is one of the important health problems, and countermeasures are socially required. We have been undertaking studies on wheat allergens and their epitopes, and have developed a method for producing hypoallergenic wheat flour by enzymatic modification. The hypoallergenic products are now provided to patients. More noteworthy, by taking hypoallergenic cupcakes over a long period, more than half of patients are hyposensitized and become able to eat normal wheat products. This suggests that the hypoallergenic wheat flour can act as anti-allergenic via allergen-specific immunotolerance. This series of studies was followed by expansive research on food allergy: analysis of epitopes of bovine serum albumin (the major beef allergen), isolation and identification of inhibitory peptides for allergen absorption at the intestine, evaluation of hesperetin as an inhibitor of degranulation of mast cells, and the development of PCR detection methods for verifying allergen labeling and for identifying hidden allergic ingredients in processed foods.

물리적 처리에 따른 우육추출물중의 BSA와 BGG단백질의 SDS-PAGE패턴 및 항원성의 변화

이전의 연구에서, 우육중의 주된 알레르겐은 우육추출물 중의 BSA, BGG라고 보고하였다. 본 연구에서는 이들 단백질의 열처리와 초고압처리와 같은 물리적 처리에 따른 SDS-PAGE상의 변화와 우육알레르기 환자의 혈청을 이용한 알레르겐성의 변화를 Western blots로 관찰하였다. 우육 추출물에 대한 열처리에 의한 알레르겐 단백질의 변화에서, 100℃ 가열처리군에서 BSA band는 큰 감소를 보였으나, BGG band는 완전히 제거되지 못하고 남아 있었고, 항원성 역시 대조군에 비하여 감소는 하였으나 여전히 유지되고 있었다. 120℃이상의 열처리는 추출물중의 BSA와 BGG band의 소실과 이에 따른 항원성의 큰 감소를 가져왔다. 우육추출물중의 BSA단백질은 열처리에서와 달리 초고압처리에 의하여 전기영동상에 거의 변화를 보이지 않았다. 그러나 BGG 단백질은 400 M㎩ 가압처리군부터 전기영동상 band가 감소하였고, 항원성 역시 유의적으로 감소하였다. 이는 초고압처리 정도에 따른 우육추출물중의 BSA와 BGG를 포함한 여러 분자의 SDS-PAGE상의 특이적 변화가 최초로 시사된 결과이다. 이러한 BGG 단백질의 고압처리에 의한 변화는 추출물중의 어떤 효소에 의한 것으로 추측되었고, 고압처리 후 37℃숙성 실험으로 이러한 가설이 확인되었다. 우육추출물에 존재하고 초고압처리로 활성화되어 BGG분자에 특이적으로 관여하는 효소 및 이와 관련된 메커니즘에 대한 연구가 앞으로 필요할 것으로 보인다.

高圧極限環境を利用した食品加工における新展開 高圧処理による食品タンパク質のアレルゲン性低減化

This paper describes general methods to eliminate or decrease the allergenicity of food protein, and also describes high pressure-induced elimination of the allergenicity of food protein, especially meat protein.The effect of high pressure treatment on the elimination of bovine serum albumin (BSA, the major beef allergen) allergenicity was evaluated on the basis of histamine release from human basophilic KU812F cells sensitized with sera from allergic patients, and the structural changes of BSA responsible for reducing allergenicity was estimated. BSA pressurized at pressures ranging from 300 to 600 MPa reduced histamine release from the cells sensitized with A5 serum with significance. The reducing effect of high-pressure treatment gradually increased with the increase of pressure applied to BSA. The pressure-induced structural changes of BSA were estimated by fluorescence spectra, circular dichroism (CD) spectra (the content of secondary structure), the amount of surface sulfhydryl (SH) group, and the surface aromatic hydrophobicity. The blue shift and decrease of the fluorescence of BSA gradually progressed with the increase of pressure applied. But no significant effect of pressure on CD spectra was observed. Pressure-treated BSA showed the maximum increase in the amount of SH group by pressure treatment at 100 MPa, and the aromatic hydrophobicity gradually decreased with the increase of high pressure applied. These results indicated that high pressure treatment induced the tertiary structural changes of BSA, but no effect on the secondary structure. We concluded that the pressure-induced elimination of BSA allergenicity seemed to be related to the tertiary structural change of BSA.

Hypoallergenic and T cell reactive analogue peptides of bovine serum albumin, the major beef allergen

We recently identified IgE-binding epitopes of bovine serum albumin (BSA), the major beef allergen, and found that the major epitope structure contains an EXXV motif. Here we report hypoallergenic analogue peptides of BSA that preserve T cell reactivity to retain the capacity to induce immunotolerance. Substitution of the glutamic acid in the EXXV sequence to aspartic acid leads to a remarkable reduction of IgE-binding ability and induces potent T cell proliferation. Moreover, in response to the substitutions, the production of INF-γ is markedly increased compared with that elicited by the native peptides. Therefore, these analogue peptides represent potential candidates for specific immunotherapy.

Anaphylaxis to beef, gelatin and Haemaccel

Rationale Meat-derived gelatin is commonly added to processed foods. 3 pts were seen initially, 1 allergic to topical & oral gelatin & 2 with anaphylaxis (Ax) to Haemaccel. All subsequently developed allergy to beef, suggesting that gelatin was a trigger. Methods 14 patients with suspected beef/gelatin Ax, 62 controls & 31 with idiopathic Ax (IAx; 14 prospectively) were investigated for gelatin sensitivity by skin testing (ST) & RAST. Results 8/14 patients with beef allergy reacted clinically to gelatin on separate occasions; gelatin RAST/ST were positive in 7 & 8/8, respectively. Gelatin ST were positive in 3 additional patients (6–20mm weals). Overall, 3/14 beef allergics had positive beef ST. 3 with negative gelatin ST/RAST had positive RAST to other beef proteins - BSA, bovine gammaglobulin & betalipoprotein IV, betalactobulin. 1/18 recalled IAx patients 1995–2001 had positive gelatin ST. By contrast, ST were negative in RJM (45 occasions) & 62 controls. 10/10 control sera had negative RAST. 92 patients with Ax were evaluated prospectively 2002–3. Triggers were animals, exercise, medication, idiopathic, stings, food allergy, healthy controls (1, 5, 5, 13,18, 47, 2 cases, respectively). Ax occurred after beef in 2/2 with positive gelatin ST, but only 1 had positive beef ST. The beef ST negative subject nonetheless reacted to deliberate Haemaccel challenge (∼1.1gm IVI gelatin). Conclusions Gelatin is an important beef allergen and may account for some cases of IAx. A positive ST convey risk for gelatin, meat & Haemaccel Ax which may be missed if one relies only on commercial beef allergens.

Accuracy of skin prick tests in IgE-mediated adverse reactions to bovine proteins

To review the recent literature on the diagnostic accuracy of skin prick tests (SPTs) in pediatric food allergy, focusing on adverse reactions to milk and beef. To present data about the test performance characteristics of beef extracts used in SPTs among children with atopic dermatitis (AD) reporting immediate hypersensitivity to beef. MEDLINE search using the following algorithm ["skin prick test" AND "food allergy" OR allergen; 1997-2002; English; all children]. Prospective sensitivity study of SPTs in 34 patients. Thirty-four children with AD (median age 2.29 years) were consecutively recruited between 1992 and 2000 because of immediate reactions to beef. On double-blind, placebo-controlled food challenges (entry criterion), 20 of the patients reacted to beef and 14 did not. Cut-off points for skin prick test wheal positivity was selected by receiving-operator characteristic analysis for fresh and commercial beef allergens. Sensitivity and specificity of skin tests and indices of reproducibility were calculated. In the literature, the positive predictive accuracies of skin prick tests vary between 69 and 100% and the negative predictive accuracies between 20 to 86% for cow's milk. In our series, SPTs with commercial beef extracts were highly diagnostic (100% sensitivity; 10% false positive rate) and SPTs with fresh beef were highly specific (100%), albeit with a false-positive rate of 21.42%. From the literature, we conclude that the diagnostic accuracy of SPTs with milk should be reappraised in the workup of cow's milk allergy. Carrying out commercial and fresh food SPTs at the same time substantially reduces costs and diagnostic work. Oral provocation is necessary in 20.68% of children with AD who have immediate symptoms to beef. Greater allergen standardization and streamlining of the workup of cow's milk allergy are desirable future goals.

Some human B and T cell epitopes of bovine serum albumin, the major beef allergen

Bovine serum albumin (BSA) is the major beef allergen. Since IgE and T cell recognitions are central to the specific immune response to allergens, the identification and immunologic characterization of B and T cell epitopes of BSA represent important steps in the development of treatments for beef allergy. Prior to our experiments, we hypothesized that BSA-specific antibodies and T cells react primarily with sequential epitopes in which the amino acid sequences differ greatly between bovine and human albumin. To clarify this hypothesis, 16 peptides corresponding to such regions were synthesized as candidate epitopes. Among them, at least two regions, aa336–345 and aa451–459, were found to be B cell (IgE-binding) epitopes. In inhibition ELISA experiments, EYAV (aa338–341) and LILNR (aa453–457) bound to patient IgE antibodies and were found to be the cores of the IgE-binding epitopes. Three regions, DDSPDLPKLKPDPNTLC (aa107–123), PHACYTSVFDKLKHLVDEP (aa364–382), and LSLILNRLC (aa451–459), were found to induce T cell proliferation in more than half of the patients tested. Of interest was that these three regions were also recognized by B cells. Information concerning human B and T cells epitopes can contribute greatly to the elucidation of the etiology of beef allergy.

Beef allergy in children

Beef allergy was poorly known before the ’90s. Since then, a number of papers appeared elucidating the nature, epidemiology, and symptoms of beef allergy in children allergic to cow’s milk and children suffering from atopic dermatitis. It is now clear that beef allergy is not an infrequent occurrence, with an incidence between 3.28% and 6.52% among children with atopic dermatitis, its incidence may be as much as 0.3% in the general population. A diagnosis of beef allergy must be supported by skin prick tests, RASTs, and challenges. The specificity and sensitivity according to type of test and the type of extract, however, remains to be evaluated. Despite the fact that other allergens can be sensitizing, the major beef allergen is bovine serum albumin (BSA). Beef-sensitive children are also sensitized to ovine serum albumin, as well as to other serum albumins; therefore, the use of alternative meats in beef-allergic children must be carefully evaluated on an individual basis. Because industrial heat processing is more efficient than domestic cooking in reducing reactivity in beef-sensitive children, freeze-drying and homogenization may support the introduction of processed beef into the diet of beef-allergic children.