A novel dipstick developed for rapid Bet v 1-specific IgE detection: recombinant allergen immobilized via a monoclonal antibody to crystalline bacterial cell-surface layers.
The incidence of allergy to airborne proteins derived from tree and grass pollen, feces of mites, spores of molds, and pet dander has been increasing over the last decades. Since precise diagnosis is a prerequisite for successful immunotherapy, there is a rising demand for rapid, reliable, and inexpensive screening methods such as dipstick assays. With the purified recombinant major birch-pollen allergen rBet v 1a as model protein, crystalline bacterial cell-surface layers (S-layers) were tested for their applicability as an immobilization matrix for dipstick development. For this purpose, S-layers were deposited on a mechanically stable microporous support, cross-linked with glutaraldehyde, and free carboxylic acid groups of the S-layer protein were activated with carbodiimide. In the present test system, rBet v 1a was immobilized via the monoclonal mouse antibody BIP 1, which, unlike the allergen, is too large to enter the pores of the S-layer lattice, and which therefore formed a closed monolayer on the outermost surface of the crystal lattice. Moreover, BIP 1 is known to modulate IgE binding to the allergen. After incubation of the dipsticks in serum, washing of the reaction zone under tap water, and binding of an anti-IgE alkaline phosphatase conjugate, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium was used as substrate, forming an IgE concentration-dependent colored precipitate on the S-layer surface. The investigation of patient sera previously tested with the CAP system confirmed the specificity of the S-layer-based dipstick assay. Since the dipstick is easy to handle and the whole test procedure takes only 90 min, this test system should be applicable for rapid determination of specific IgE and for first screening in the doctor's practice.