Activation of circularly permutated ß-lactamase tethered to antibody domains by specific small molecules.

Regulation of enzyme activity either by its substrates or by effectors is generally known as allostery. However, it has been considered hard to alter its effector specificity, despite its potential utility as a sensitive molecular sensor. To this end, we made fusion proteins consisting of an antibody variable region Fv and a circularly permutated TEM-1 ß-lactamase cpBLA. Two expression vectors encoding Fv-cpBLA with different antigen specificities were made, in which cpBLA was inserted into the linker region of the single chain Fv that specifically binds either bone-related disease marker osteocalcin (BGP) C-terminal peptide or neonicotinoid insecticide imidacloprid (ICP). The cpBLA having new termini near the active site was activated upon binding with its cognate antigen, owing to the stabilization of tethered Fv by bound antigen. As a result, both Fv-cpBLA showed specific antigen binding as well as antigen-induced enhancement in catalytic activity. Moreover, E. coli cells expressing Fv-cpBLA for ICP showed ICP concentration dependent growth in the medium containing ampicillin. The system was also applied to select for Fv-cpBLA linker mutants that confer faster growth. This will be the first of an antibody-based small molecule indicator enzyme.

Noncompetitive detection of low molecular weight peptides by open sandwich immunoassay.

Small peptides with less than 1000 in molecular weight are not considered amenable to sandwich immunoassays due to their difficulty of simultaneous recognition by two antibodies. As an alternative, we attempted noncompetitive detection of small peptides by open sandwich enzyme-linked immunosorbent assay (OS-ELISA) utilizing the antigen-induced enhancement of antibody VH/VL interaction. Taking fragments of human osteocalcin (BGP), a major non-collagen peptide produced in bone, as model peptides, OS immunoassay was performed using the cloned VH and VL cDNAs from two anti-BGP monoclonal antibodies either recognizing the N- or C-terminal fragment, respectively. When the clones were used for OS-ELISA with immobilized VL fragment and phage-displayed VH fragment, enhanced VH/VL interaction upon BGP addition was observed. Especially the clone for the C-terminal fragment showed a superior detection limit as well as a wider working range than those of competitive assay. The result was reproduced with purified VH-alkaline phosphatase and MBP-VL fusion proteins, where the latter was directly immobilized onto the microplate wells. The minimum detectable fragment was the hexamer including the C-terminus. This simple approach with a single monoclonal antibody with a short measurement time may prove a useful tool in immunodiagnostics as well as in proteomics research.