Site‐Specific Photoconjugation of Beta‐Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split‐Enzyme Complementation

Abstract

Protein fragment complementation assays (PCA) rely on a proximity-driven reconstitution of a split reporter protein activity, typically via interaction between bait and prey units separately fused to the reporter protein halves. The PCA principle can also be formatted for use in immunossays for analyte detection, e.g., via the use of small immunoglobulin binding proteins (IgBp) as fusion partners to split-reporter protein fragments for conversion of pairs of antibodies into split-protein half-probes. However, the non-covalent binding between IgBp and antibodies is not ideal for development of robust assays. Here, the authors describe how split-enzyme reporter halves can be both site-specifically and covalently photoconjugated at antibody Fc-parts for use in homogeneous dual-antibody in vitro immunoassays based on analyte-dependent split-enzyme fragment complementation. The half-probes consist of parts of a beta-lactamase split-protein reporter fused to an immunoglobulin Fc binding domain equipped with a unique cysteine residue at which a photoactivable maleimide benzophenone group (MBP) is attached. Using such antibody conjugates the authors obtain an analyte-driven complementation of the reporter enzyme fragments monitored via conversion of a chromogenic substrate. Results from detection of human interferon-gamma and the extracellular domain of HER2 is shown. The described principles for site-specific conjugation of proteins to antibodies should be broadly applicable.

Affinity-Based Methods for Site-Specific Conjugation of Antibodies

“…This enabled analyte detection via split-enzyme complementation in a dual-antibody assay. 46 Perols et al used SPPS to introduce BPA or benzoylbenzoic acid (BBA, a benzophenone attached to a flexible linker) at positions 5 and 32 in Z, where Z32BPA could cross-link 41% of human IgG1 heavy chains and Z5BBA cross-linked 66% of mouse IgG1 heavy chains. 47 Further improving the cross-linking abilities of the Z domain, Hui et al reported up to 80% cross-linking of mouse IgG3 heavy chains by introducing BPA at position 17 in the protein domain and 47% conjugation efficiency for human IgG1 with BPA at position 35.…”

Engineering Protein Switches for Rapid Diagnostic Tests

“…The ability to easily utilize this vast source of “off-the-shelf” recognition elements within protein switches would improve the scope and uptake of the technology. Whole antibodies have only rarely been used for in vitro protein switches, but in the new commercially available Lumit immunoassay, antibodies are linked to each half of a proximity protein switch . Attachment is via chemical modification of the antibodies with synthetic ligands that bind a self-labeling protein tag (HaloTag) on the reporter fragments .…”

“…They have thus been superseded by enzymatic reporters that enhance sensitivity by signal amplification, as in ELISAs. Despite enormous possibilities, the same set of reporter enzymes encompassing proteases, , β-lactamase, ,,, glucose dehydrogenase, ,,,, and luciferases ,− ,,,,,,− are repeatedly used in protein switches, perhaps due to their history as reporters in other assays and the ease of recombinant production. Dependent on the enzyme and substrate, there are four key formats: colorimetric, fluorometric, luminescent, and electrochemical.…”

“…In proximity switches, analyte binding induces colocalization of sensor components, which results in a change in response (Figure ). The most common approach is split-enzyme complementation, in which an enzyme is split into two inactive fragments, usually directly but sometimes via mutations at a dimerization interface. ,,− ,, Each fragment is attached to recognition elements that bind nonoverlapping epitopes on the analyte, such that binding colocalizes the fragments and induces reconstitution of the active enzyme. ,,− ,, This approach is insensitive to the exact geometry of the analyte and binding domains, so long as linkers between the binder and fragment are sufficient. Once colocalized, the high effective concentration and residual affinity between fragments should drive reconstitution, regardless of exact orientations.…”

“…The NanoBit system was linked to antibodies to form the basis of the recent Lumit cell-lysate immunoassay . To utilize NanoBit for diagnostic assays, both Dixon et al and Ohmuro-Matsuyama et al further split the system, to give a polypeptide and two peptide fragments. , The two small peptide fragments could be produced more easily in fusion with recognition elements and the polypeptide could be held at a separately high concentration in the assay. , The sensitivity (pM), response dynamic range (up to 100 000%), and analyte linear range (>3 orders of magnitude) of these systems are superior to other in vitro split-enzyme assays. ,,− ,, These qualities are a testament to the engineering effort that enabled low background complementation and high luminescent activity recovery. Nevertheless, tests were in a maximum 5% serum matrix, and use as a general format for wide ranging analytes is yet to be shown.…”