Panel A shows flow cytometry plots with sequential gating through SSC-A versus FSC-A at 51.0 percent, single cells at 99.1 percent on SSC-H versus SSC-A, viability dye negative at 99.8 percent, CD19 positive at 8.36 percent, IgM negative CD19 positive at 21.9 percent, and IgG negative IgE positive at 38.9 percent with IgE-PE versus IgG-AF488 axes. Panel B shows a table listing three monoclonal antibodies eB13, eB20, and eC5 with their respective HV, HD, HJ, LV, and LJ genes, CDRH3 and CDRL3 sequences, and ELISA EC50 values of 0.01726, 0.008891, and 0.01266 micrograms per milliliter. Panel C shows a line graph of absorbance at 450 nanometers up to 4 across mAb concentrations from 10 to the power of negative 7 to 10 to the power of 1 micrograms per milliliter for IgE B13, B20, and C5, where all three show sigmoidal curves reaching approximately 4 between 10 to the power of negative 2 and 10 to the power of 0. Panel D shows a heatmap of absorbance at 450 nanometers for IgE C5, B13, and B20 against PR-10 and non PR-10 allergens, where Bet v1 shows the highest absorbance reaching 4 across all three antibodies and other allergens show minimal signal. Panel E shows biolayer interferometry binding curves for mAb eB13, eB20, and eC5 at concentrations from 37.5 to 300 nanomolars, with KD values of 8.81 times 10 to the power of negative 10, 5.82 times 10 to the power of negative 10, and 1.54 times 10 to the power of negative 9 molars respectively, with shift reaching approximately 0.2 to 0.3 nanometers at 300 nanomolars. Panel F shows a table listing EC50 and KD values for seven monoclonal antibodies, with EC50 ranging from 0.004265 to 0.01726 and KD ranging from 3.3 times 10 to the power of negative 10 to 1.6 times 10 to the power of negative 9. Panel G shows a dendrogram with hierarchical clustering of seven antibodies by isotype, where eC5 and eB13 cluster together as IgE, mAb8, and mAb5 cluster as IgG, eB20 and mAb10 form a separate cluster and mAb11 does not cluster with other antibodies. Panel H shows a heatmap of competitive binding results for IgE C5, B13, and B20 capture antibodies against ten competitor antibodies, where IgE C5 shows competitive binding with antibodies 5, 8, 10 and 5313 and non-competitive with antibodies 11, 5714, and 5715. IgE B13 shows partial or total competitive binding with all tested antibodies except 5714, 5715 and IgE B20. Finally, IgE B20 shows competitive binding with only 5714 antibody.
Human B cell sorting for antibody discovery and characterization of three novel human anti-Bet v1 IgE antibodies. (A) Gating strategy for the isolation of human CD19+IgM−IgE−IgG+ and CD19+IgM−IgE+IgG− sorted cells. (B) Characteristics of three human anti-Bet v1 IgE antibodies with variable domain genes (V), (D), and (J) for heavy (H) and light (L) chains, CDR3 amino acid sequences, and EC50 against Bet v1. (C) Anti-Bet v1 ELISA of the novel anti-Bet v1 antibodies (n = 2). (D) Specificity of anti-Bet v1 antibodies to PR-10 and non-PR-10 allergens assessed by ELISA. (E) BLI kinetics of each novel antibody immobilized on a protein A–loaded biosensor and Bet v1 in solution. Red lines represent fittings to the sensogram traces. Bet v1 concentrations used for each antibody are reported in nM. (F) Comparative table with binding and affinity to Bet v1 of novel IgG and IgE antibodies. (G) Hierarchical clustering of antibody variable domain sequences visualized as a dendrogram. Antibody isotypes are indicated alongside the dendrogram (blue: IgE; gray: IgG). (H) Competitive binding study between antibodies binding Bet v1 protein–coupled beads. Competitors induced either strong blocking (red boxes), partial competition (gray boxes), or noncompetitive binding (white boxes) with corresponding mAb to Bet v1. Data in C are represented as the mean ± SEM. CDR3, third complementarity-determining region.