3.1 Design and production of recombinant allergen tetramers for CytoBas
Immunodominant allergen components of BV and RGP were selected for generation of recombinant allergen tetramers (Api m 1, Lol p 1 and Lol p 5).20-22,27,28 The full-length Api m 1 protein was produced with the native leader sequence, and C-terminal 6-His and BirA tags for purification and biotinylation (Figure 1A ). The construct contained a H67Q mutation to prevent catalytic activity without affecting IgE reactivity.34 Similarly, the Lol p 1 and Lol p 5 constructs were generated with the Api m 1 leader sequence to target these for secretion. Lol p 1 catalytic activity was abolished by a H104V mutation.35 Purified allergens were probed by Western blot using an anti-6-His antibody and detected at expected sizes with typical diversity in glycosylation density (Figure 1B ). Recombinant allergens were biotinylated and tetramerized with a streptavidin-APC conjugate to generate Api m 1, Lol p 1 and Lol p 5 tetramers.
The ability of allergen tetramers to detect specific IgE on basophils using flow cytometry was assessed using whole blood samples of allergic patients and controls. Following 15 min incubation with the relevant allergen tetramer and mAbs, binding of allergen to CD123+IgE+ basophils was examined (Figure 1C ). The Api m 1 tetramer ([Api m 1]4-APC) specifically stained basophils from a BV-allergic individual and not a control (Figure 1D ). Similarly, [Lol p 1]4-APC and [Lol p 5]4-APC each positively stained basophils from an RGP-allergic patient, and not a control. Thus, these recombinant allergen tetramers can be used to detect allergen sensitization by binding specific IgE on the surface of basophils using flow cytometry (CytoBas).