Anti‐betalactoglobulin IgG antibodies bind to a specific profile of epitopes when patients are allergic to cow's milk proteins

Background: β–Lactoglobulin (BLG) represents one of the major allergens causing cow’s milk allergy (CMA) – a disease with a wide spectrum of clinical symptoms. The aim of this study was to evaluate sequential B cell epitopes of BLG by the Pin–ELISA method. Furthermore, we wanted to investigate a possible association of the IgE recognition patterns in sera of patients with BLG sensitization and the type of clinical reactions following contact with cow’s milk. Methods: Overlapping sequential decapeptides corresponding to the amino acid sequence of BLG were used in Pin–ELISAs specific for human IgE. Tested sera were from 14 individuals with CMA, 8 of them with a history of immediate systemic reactions and 6 with delayed skin reactions following contact with cow’s milk. All of them showed specific IgE antibodies to BLG in the CAP–RAST. Control sera were from 5 healthy nonallergic individuals. Results: All sera from BLG–sensitized individuals showed IgE binding with one region of BLG corresponding to amino acids 95–113. Furthermore, individual sera showed reactions with two further regions, 12–27 and 124–135. Inhibition of IgE binding to BLG with one soluble synthetic peptide confirmed the major epitope. No differences were found in the B cell epitope recognition pattern to BLG in the two groups of patients with CMA, characterized by acute systemic or delayed skin reactions. Conclusions: Using IgE Pin–ELISAs we were able to confirm previously described sequential B cell epitopes of BLG. However, the recognition pattern of one of the major cow’s milk allergens is not predictive of the clinical type of reaction.

“…The same applies for the signal intensity to the whole protein as well as single epitopes. This is in contrast to the findings in IgG antibody testing [31]. …”

Section: Discussioncontrasting

confidence: 51%

Section: Discussionmentioning

confidence: 99%

The Recognition Pattern of Sequential B Cell Epitopes of Beta–Lactoglobulin Does Not Vary with the Clinical Manifestations of Cow’s Milk Allergy

Heinzmann¹,

Blattmann²,

Spuergin³

et al. 1999

“…One may assume that the IgE and IgG4 epitopes are mainly conformational (which agrees with the lack of IgE binding to Lep d 2-derived peptides [34]) whereas the IgG1 epitopes appear mostly linear. With another methodological approach, recent studies have suggested differences in the fine specificity of IgG Ab to Der p 1 and bovine β-lactoglobulin between allergics and healthy controls [35, 36]. In line with our IgG4 findings allergic subjects had IgG Ab preferentially directed to conformational epitopes.…”

Section: Discussionsupporting

confidence: 72%

IgG1, IgG4 and IgE Antibody Reactivity to Mutant Forms of the Major Dust Mite Allergen Lep d 2 among Atopic and Nonatopic Subjects Naturally Exposed to <i>Lepidoglyphus destructor</i>

Olsson

Hage

Magnusson

2001

Background: Lepidoglyphus destructor is a common dust mite causing IgE-mediated sensitization. The major allergen, Lep d 2, has previously been cloned and expressed as several double Cys to Ser mutants with the purpose of producing hypoallergenic variants for immunotherapy. Our aim was to investigate the reactivity pattern of IgG1, IgG4 and IgE antibodies to wild-type (wt) rLep d 2 and four mutants among atopic and nonatopic subjects in relation to sensitization and exposure to L. destructor. Methods: Inhibition and sandwich ELISA were used to compare IgG1, IgG4 and IgE antibody reactivities to rLep d 2 variants in serum of 20 atopic and 18 nonatopic farmers naturally exposed to L. destructor. A group of 22 urban subjects served as controls. Results: Atopic farmers demonstrated correlating IgE and IgG4 levels to rLep d 2(wt) (r_s = 0.70; p < 0.0001) which were significantly (p < 0.0001) higher than those of nonatopic farmers and urban controls. No IgG4 antibodies were detected in nonatopic farmers despite chronic allergen exposure. A parallel reactivity pattern of IgE and IgG4 to all rLep d 2 mutants was observed. The mutant lacking all 3 disulfide bonds, rLep d 2.6Cys, demonstrated neither any IgE nor IgG4 reactivity. In contrast, IgG1 antibodies had a different reactivity pattern and were detected among most subjects irrespective of atopy, exposure to L. destructor or disulfide impairments in rLep d 2. Moreover, IgG1 levels to rLep d 2(wt) and rLep d 2.6Cys correlated (n = 60; r_s = 0.65; p < 0.0001). Conclusions: IgE/IgG4 Ab to rLep d 2 were restricted to atopic farmers and demonstrated parallel recognition patterns of conformational epitopes. In contrast, IgG1 antibodies are ubiquitously found and mainly recognize sequential structures. The observed isotypic difference and interindividual variation in antibody specificities among atopic and nonatopic subjects imply careful investigation of hypoallergenic variants destined for immunotherapy.

“…Studies investigating BLG-specific IgG binding epitopes by ELISA, using both native and pepsin-digested products, have suggested that IgG antibodies from milk-allergic patients show a marked preference for conformational epitopes whereas normal individuals recognize both conformational and sequential ones [18]. While the presence of IgE antibodies to foods have been associated with clinical hypersensitivity, the IgG antibody formation to food is physiologic.…”

Section: Discussionmentioning

confidence: 99%

IgE and IgG Binding Epitopes on α-Lactalbumin and β-Lactoglobulin in Cow’s Milk Allergy

Järvinen

Chatchatee²,

Bardina³

et al. 2001

274

195

Background: Cow’s milk is one of the most common causes of food allergy in the first years of life. We recently defined IgE and IgG binding epitopes for α_s1-casein, a major cow’s milk allergen, and found an association between recognition of certain epitopes and clinical symptoms of cow’s milk allergy (CMA). Since α-lactalbumin (ALA) and β-lactoglobulin (BLG) are suspected to be significant allergens in cow’s milk, we sought to determine the structure of sequential epitopes recognized by IgE antibodies to these proteins. We further sought to assess the pattern of epitope recognition in association with the clinical outcome of CMA. Methods: According to the known amino acid sequence of ALA and BLG, 57 and 77 overlapping decapeptides (offset by two amino acids), respectively, were synthesized on a cellulose derivatized membrane. Sera from 11 patients 4–18 years of age with persistent CMA (IgE to cow’s milk >100 kU_A/l) were used to identify IgE binding epitopes. In addition, 8 patients <3 years of age and likely to outgrow their milk allergy (IgE to cow’s milk <30 kU_A/l) were used to investigate the differences in epitope recognition between patients with ‘persistent’ and those with ‘transient’ CMA. Seven patients 4–18 years of age were used for assessing the IgG binding regions. Results: In patients with persistent allergy, four IgE binding and three IgG binding regions were identified on ALA, and seven IgE and six IgG binding epitopes were detected on BLG. The younger patients that are likely to outgrow their allergy recognized only three of these IgE binding epitopes on BLG and none on ALA. Conclusions: The presence of IgE antibodies to multiple linear allergenic epitopes may be a marker of persistent CMA. The usefulness of IgE binding to distinct epitopes on whey proteins in defining the patients that would have a lifelong CMA needs to be investigated in further studies.