Mechanisms of Allergen-Antibody Interaction of Cockroach Allergen Bla g 2 with Monoclonal Antibodies That Inhibit IgE Antibody Binding

BACKGROUND: Cockroach allergy is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2. METHODOLOGY/PRINCIPAL FINDINGS: Mutational analysis of selected residues in both epitopes was performed based on the X-ray crystal structures of the allergen with mAb Fab/Fab' fragments, to investigate the structural basis of allergen-antibody interactions. The epitopes of Bla g 2 for the mAb 7C11 or 4C3 were mutated, and the mutants were analyzed by SDS-PAGE, circular dichroism, and/or mass spectrometry. Mutants were tested for mAb and IgE antibody binding by ELISA and fluorescent multiplex array. Single or multiple mutations of five residues from both epitopes resulted in almost complete loss of mAb binding, without affecting the overall folding of the allergen. Preventing glycosylation by mutation N268Q reduced IgE binding, indicating a role of carbohydrates in the interaction. Cation-π interactions, as well as electrostatic and hydrophobic interactions, were important for mAb and IgE antibody binding. Quantitative differences in the effects of mutations on IgE antibody binding were observed, suggesting heterogeneity in epitope recognition among cockroach allergic patients. CONCLUSIONS/SIGNIFICANCE: Analysis by site-directed mutagenesis of epitopes identified by X-ray crystallography revealed an overlap between monoclonal and IgE antibody binding sites and provided insight into the B cell repertoire to Bla g 2 and the mechanisms of allergen-antibody recognition, including involvement of carbohydrates

Razine alergena Bla g 1 (Blatella germanica) u kućnoj prašini s područja grada Zagreba

Cockroach allergy is a health problem in many parts of the world. In urban environments, indoor exposure to cockroach allergens involves a risk of asthma. The aim of this study was to measure the mass fraction of Bla g 1, a major allergen of the German cockroach (Blatella germanica) in 30 house samples, collected at random from Zagreb area households, Croatia. Dust samples were collected on cellulose filters by vacuuming living rooms floors. After extraction, Bla g 1 was detected using the commercial enzyme-linked immunosorbent assay (ELISA). Only four of the thirty households had detectable Bla g 1 levels, and only in one was its concentration higher than 2.0 U g-1, the threshold associated with sensitisation. The Bla g 1 ELISA proved highly sensitive, with the detection limit of 0.12 U g-1. The within- and between-assay imprecision was 8.9 % and 14.4 %, respectively, and accuracy 85 % to 120 %. Low Bla g 1 levels in the household dust support previously reported low prevalence of skin sensitisation to B. germanica among Zagreb residents. Further monitoring should reveal if there are differences in cockroach allergen exposure and sensitisation between households from other geographic areas in Croatia.Alergija na žohare zdravstveni je problem u mnogim dijelovima svijeta. Prisutnost alergena žohara u ućanstvu važan je faktor rizika za nastanak astme u gradskoj sredini. Cilj ovoga rada bio je odrediti maseni djel Bla g 1, glavnog alergena smeđeg žohara (Blatella germanica) u 30 uzoraka kućne prašine uzorkovanih neselektivno izabranim kućanstvima sa šireg područja Zagreba. Uzorci prašine skupljeni su usisavanjem prašine s poda u dnevnom boravku na celulozne fi ltre. Nakon ekstrakcije, Bla g 1 je određen komercijalnim enzim-imunokemijskim testom. Maseni udjeli alergena Bla g 1 bili su mjerljivi u samo 4 od 30 kućanstava (13,3 %). Koncentracija Bla g 1 > 2 U g-1 koja se povezuje s pojavom senzibilizacije u ljudi ustanovljena je u jednom od 30 kućanstava (3,3 %). Bla g 1 ELISA vrlo je osjetljiva metoda s granicom detekcije od 0.12 U g-1, a dobiveni su zadovoljavajući rezultati za analitičku nepreciznost i netočnost. Rezultati ove pilot-studije pokazuju da je razina alergena Bla g 1 u kućnoj prašini s područja Zagreba vrlo niska, što je sukladno ranijim podatcima o niskoj učestalosti kožne reaktivnosti na smeđeg žohara u odrasloj populaciji iz istog područja. Zbog moguće razlike u izloženosti i senzibilizaciji stanovništva alergenima žohara ovisno o geografskom području, trebalo bi provesti daljnja ispitivanja