to Profilins Bet v 2, Art v 4 and Amb a 8. Krzysztof Kowal1, Agnieszka Pampuch1, Ewa ... RESULTS: A high affinity Der p 2-specific IgE mAb 2G1 was isolated,.
AB260 Abstracts
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The Effect of Protein Methylation on Binding of IgE to Profilins Bet v 2, Art v 4 and Amb a 8.
Krzysztof Kowal1, Agnieszka Pampuch1, Ewa Sacharzewska1, Pawel Bielecki1, Caleb Schlachter2, Lesa R. Offermann2,3, and Maksymilian Chruszcz2; 1Medical University of Bialystok, Bialystok, Poland, 2University of South Carolina, Columbia, SC, 3Davidson College, Davidson, NC. RATIONALE: To evaluate the effect of protein methylation on IgE binding of profilins Bet v 2, Art v 4 and Amb a 8. METHODS: ELISA and inhibition ELISA with recombinant birch, mugwort and ragweed profilins and their methylated counterparts were performed using sera from allergic rhinitis patients. The reductive methylation protocol led to modification of all profilin lysine residues. The methylation was confirmed using mass spectrometry. The crystal structure of methylated rAmb a 8 was determined. RESULTS: In 32 patients with positive ELISA the intensity of IgE binding to individual profilins was different. In 15 patients, IgE binding to rBet v 2 was significantly less (30-60%) than to mugwort or ragweed profilins. In those patients, soluble rBet v 2 abolished IgE binding to birch but only attenuated (30-50%) binding to mugwort or ragweed profilins. IgE binding to methylated profilins was significantly less (25-60%) than to the corresponding counterparts. Inhibition with rBet v 2 completely blocked IgE binding to all three methylated profilins. Amino acid sequence analysis revealed the unique substitution of Q37 in Bet v 2 by K37 in Art v 4 and Amb a 8. The analysis of 3D structures of profilins allowed for identification of highly conserved and variable fragments on the surface of these allergens. CONCLUSIONS: In profilins, lysines participate in formation of IgE epitopes. K37 may participate in formation of IgE epitopes specific for weeds. Mapping of the sequence conservation on profilin structure also suggests that a region of the molecule containing K54 may be responsible for binding species specific IgE’s.
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First Naturally Occurring Human IgE Antibody Against Mite Allergen Der p 2
MONDAY
Anna Pomes, PhD, FAAAAI1, Jill Glesner, BS1, Dennis J. Horvath, PhD2, Martin D. Chapman, PhD, FAAAAI1, and Scott A. Smith, MD, PhD2; 1Indoor Biotechnologies, Inc., Charlottesville, VA, 2Vanderbilt University Medical Center, Nashville, TN. RATIONALE: The identification of molecular determinants for IgE binding on environmental allergens is limited because IgE antibodies are polyclonal, are present at low concentration in serum, and the frequency of circulating IgE B cells is exceedingly low. The goal was to isolate and analyze naturally occurring human monoclonal IgE antibodies to mite group 2 allergens for molecular analysis of allergenic epitopes. METHODS: Human B cells from peripheral blood of allergic patients were cultured and fused with a human myeloma cell line (HMMA2.5) using electrical cytofusion. Allergen-specific IgE antibodies were identified by ELISA, expressed and purified. The relative position of epitopes for IgE and three mouse monoclonal antibodies (mAb), and the IgE recognition of isoforms were analyzed by ELISA. RESULTS: A high affinity Der p 2-specific IgE mAb 2G1 was isolated, which bound allergen with an EC5057.1 ng/ml. The IgE mAb bound Der p 2.0101, Der p 2.0103 and Der f 2. Two mAbs that bind to opposite sides of Der p 2 (1D8 and 7A1) did not interfere with the IgE binding by two-site ELISA. The IgE mAb 2G1 and murine mAb aDpX (which recognizes both isoforms and Der f 2) appeared to bind to overlapping epitopes. CONCLUSIONS: The first human anti-Der p 2 IgE antibody that has the natural pairing of heavy and light chains was isolated by human hybridoma technology. This unique tool facilitates mapping of allergenic epitopes recognized by naturally occurring human IgE antibodies, geared to produce new hypoallergens for immunotherapy.
J ALLERGY CLIN IMMUNOL FEBRUARY 2017
