title: An optimized homogeneous noncompetitive immunoassay based on the antigen-driven enzymatic complementation.
abstract: We describe an optimized noncompetitive and homogeneous immunoassay based on the antigen-dependent reassociation of antibody variable domains and beta-galactosidase (beta-gal) complementation (open sandwich enzymatic complementation immunoassay, OS-ECIA). The reassociation of two fusion proteins, an antibody heavy chain variable region fragment tethered to an N-terminal deletion mutant of beta-gal, V(H)Deltaalpha, and the light chain variable region fragment tethered to a C-terminal deletion mutant of beta-gal, V(L)Deltaomega, was monitored by the enzymatic complementation between the two. With the use of anti-hen egg lysozyme (HEL) antibody HyHEL10, an antigen-dependent enhancement in the enzymatic activity was clearly observed. To optimize the assay, the lengths of the linkers connecting the two domains of each fusion protein were varied, and the optimal pair V(H)(G(4)S)(2)Deltaalpha/V(L)(G(4)S)Deltaomega showed much improved antigen-responsive beta-gal activity. After various optimizations, almost 1000-fold improvement in sensitivity compared with that of our corresponding homogeneous open sandwich (OS) assays based on the energy transfer was observed, possibly due to lower V(H)/V(L) concentration and background heterodimer association.
authors.affiliation: School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656 Bunkyo, Japan. hueda@bio.t.u-tokyo.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
authors.forename: Tomoichi
authors.initials: T
authors.lastname: Yokozeki
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Arai
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
authors.forename: Izumi
authors.initials: I
authors.lastname: Kumagai
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
mesh_terms: D006241:Haptens / Q000032:analysis*; D007124:Immunoenzyme Techniques / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D001616:beta-Galactosidase
chemical_list: D006241:Haptens; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase