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).