HRP-conjugated plug-and-playable IgG-binding nanobodies as secondary antibody mimics in immunoassays

•IgG-binding nanobody-based secondary antibody mimics are established using SpyTag/SpyCatcher protein ligation system.•Both HRP:MG1Nb and HRP:RNb specifically recognized target-bound mouse IgG1 and rabbit IgG, respectively, in western blots and ELISA.•Their species-specificity toward primary IgGs allowed the simultaneous detection and visualization of two different target molecules within a single sample of cell and tissue. Conventional secondary antibodies for immunoassays are generally obtained from live animals or expensive mammalian cell culture systems. These antibodies are then conjugated with enzymes that repeatedly convert inactive substrates to signal-generating active products by secondary antibody mimics of low-abundance target molecules in a concentration-dependent manner. In this study, we constructed recombinant immunoglobulin G (IgG)-binding nanobody-based secondary antibody mimics using a well-established SpyTag/SpyCatcher protein ligation system. We prepared SpyCatcher as a scaffold protein, chemically conjugated it with an activated horseradish peroxidase (HRP), and fused SpyTag to two IgG-binding nanobodies specific for the Fc regions of mouse IgG1 and rabbit IgG, which is denoted as MG1Nb and RNb, respectively. We selectively ligated HRP-conjugated SpyCatcher with SpyTag-fused MG1Nb or RNb depending on the original species of primary antibodies and amplified signals of target molecules in a plug-and-playable manner. We successfully demonstrated that HRP:MG1Nb and HRP:RNb selectively bind to target-bound mouse IgG1 or rabbit IgG, respectively, and serve as excellent target-specific signal amplifiers in various immunoassays such as western blot, enzyme-linked immunosorbent assay (ELISA), and multiplex tyramide signal amplification (TSA) cell and tissue imaging. These recombinant signal-amplifying IgG-binders can be simple and reliable alternatives to conventional secondary antibodies in various immunoassays.