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 gr ass pollen, feces of mites, spores of molds, and pet dander has been i ncreasing over the last decades. Since precise diagnosis is a prerequi site 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) w ere tested for their applicability as an immobilization matrix for dip stick development. For this purpose, S-layers were deposited on a mech anically 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 la was immobili zed via the monoclonal mouse antibody BLP 1, which, unlike the allerge n, is too large to enter the pores of the S-layer lattice, and which t herefore formed a closed monolayer on the outermost surface of the cry stal lattice. Moreover, BIP 1 is known to modulate IgE binding to the allergen. After incubation of the dipsticks in serum, washing of the r eaction zone under tap water, and binding of an anti-IgE alkaline phos phatase conjugate, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tet razolium was used as substrate, forming an IgE concentration-dependent colored precipitate on the S-layer surface. The investigation of pati ent sera previously tested with the CAP(TM) system confirmed the speci ficity 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 s ystem should be applicable for rapid determination of specific IgE and for first screening in the doctor's practice.