Preparation of human IgG1 monoclonal antibody specific for house dust mite allergens Der p 2 and Der f 2 by single B cell PCR of VH and VL segments (HYP7P.275)

In allergic patients sensitive to house dust mite allergens, the inhaled allergenic proteins are dissolved in mucosal fluid, cross mucosal epithelia of the airway, and induce crosslinking of FcεRI-bound allergen-specific IgE on mast cells and basophils, leading to the degranulation of those inflammatory cells. Our group has proposed that allergen-specific IgG, delivered to the airway mucosa, can bind to the incoming allergens and preclude their entry into the mucosal layer. In this work, we prepared human monoclonal antibodies by isolating dust mite allergen-specific single B cells from patients with atopic dermatitis and performing PCR on the VH and VL cDNA of the B cells. One mAb, 1D11, an IgG1 (γ1, λ), bound strongly to both Der p 2 and Der f 2 and recognized a linear epitope in the peptide segment, a.a. residues #41-70, of the two proteins. We proceeded evaluating the ability of 1D11 to trap Der p 2 and Der f 2 in a culture of rat basophilic leukemic (RBL) SX-38 cells, which were charged with allergen-specific IgE contained in patient sera. In the culture, 1D11 was capable of inhibiting the degranulation of RBL SX-38 cells triggered by Der p 2 and Der f 2. Furthermore, the activation was reduced to the background level when protein A-conjugated Sepharose was employed to remove the 1D11-allergen complexes. These results indicate that mAb 1D11 is effective in trapping Der p 2 and Der f 2 allergens and prevent their activation of allergen-specific IgE-sensitized basophils.