Development of Recombinant Immunoglobulin G-Binding Luciferase-Based Signal Amplifiers in Immunoassays

Abstract

In general immunoassays, secondary antibodies are covalently linked with enzymes and bind to the Fc region of targetbound primary antibodies to amplify signals of low-abundant target molecules. The antibodies themselves are obtained from large mammals and are further modified with enzymes. In this study, we developed novel recombinant immunoglobulin G (IgG)-binding luciferase-based signal amplifiers (rILSAs) by genetically fusing luciferase (Nluc) with antimouse IgG1 nanobody (MG1Nb) and antibody-binding domain (ABD), individually or together, in a mixand-match manner. We obtained three different highly pure rILSAs in large quantities using a bacterial overexpression system and one-step purification. Mouse-specific rILSA, MG1Nb-Nluc, and rabbit-specific rILSA, Nluc-ABD, selectively bound to target-molecule-bound mouse IgG1 and rabbit IgG primary antibodies, whereas the bispecific rILSA, MG1Nb-Nluc-ABD, mutually bound to both mouse IgG1 and rabbit IgG primary antibodies. All rILSAs exhibited an outstanding signal-amplifying capability comparable to those of conventional horseradish-peroxidase-conjugated secondary antibodies, regardless of the target molecules, in various immunoassay formats, such as enzyme-linked immunosorbent assay, Western blot, and lateral flow assays. Each rILSA was selected for its own individual purpose and applied to various types of target analytes, in combination with a variety of target-specific primary antibodies, effectively minimizing the use of animals as well as reducing the costs and time associated with the production and chemical conjugation of signal-amplifying enzymes.