PM Q-probe: A fluorescent binding protein that converts many antibodies to a fluorescent biosensor
Introduction
Quenchbody (Q-body) technology is a fluorescent biosensor-based homogenous immunoassay that does not need washing steps to separate the unbound antibodies (Abe et al., 2011). When a fluorescent dye is used as a label at a site near the antigen-binding pocket of an antibody fragment, it moves inside the antibody fragment. This results in the quenching of dye-fluorescence by amino acid residues, namely tryptophan (Trp), present on the antibody, through a photo-induced electron transfer mechanism. However, when an antigen is added to the quenched Q-body, the fluorescence of the dye is recovered upon antigen-binding. So far, Q-bodies using antibody fragments, such as the single chain variable region (scFv) and antigen binding fragments (Fab) have been reported. The first Q-body was fortuitously discovered when fluorescent dye-labeled aminoacyl transfer RNA was used to label an antibody fragment using a cell-free translation system based on unnatural amino acid incorporation (Abe et al., 2010). Subsequently, Q-bodies with better sensitivity, named Ultra Q-body, were made from Fab fragments, which were either expressed in vitro or in bacteria, and labeled with fluorescent dye(s) at the N-terminal cysteine-containing tag(s) (Abe et al., 2014). Q-bodies have been successfully used to detect not only small molecules, such as the bone Gla protein (BGP) peptide, recreational drugs (Abe et al., 2011), protein phosphorylation (Jeong et al., 2013), amyloid beta peptide (Dong et al., 2018), neonicotinoid insecticide (Zhao et al., 2018), and antidepressant fluvoxamine (Sasao et al., 2019), but also larger proteins, such as claudin-4 (Jeong et al., 2017a) and hemagglutinin from influenza viruses (Abe et al., 2014; Jeong et al., 2018). For the preparation of Q-bodies, thiol-maleimide reaction (Abe et al., 2014), transamination reaction (Dong et al., 2016), and photochemical crosslinking to nucleotide-binding sites on the antibody (Jeong et al., 2017b), have been successfully used. However, although the Trp residues in the antibody variable region are well conserved (>95%), not all antibodies can be converted to a Q-body that shows a good response. This is probably because of structural factors including the accessibility of the dye to Trp residues (Ohashi et al., 2016), but not much is known yet. Hence, a faster and more convenient method to predict the suitability of an antibody to be converted into a Q-body has been long-awaited.
So far, several antibody-binding proteins from bacteria have been reported. Protein A (PA), with a molecular weight of 42 kDa, a component of the cell wall of Staphylococcus aureus, has four antibody-binding domains, each of which binds strongly to the Fc region of IgG (Forsgren and Sjoquist, 1966; Langone, 1982) and weakly to some Fab fragments at the VH domain (Roben et al., 1995; Starovasnik et al., 1999). Protein G (PG), a component of the cell wall of groups C and G Streptococcal bacteria, also binds strongly to Fc fragments of IgG at its binding domains (Bjorck and Kronvall, 1984; Sjobring et al., 1991) but shows weaker binding affinity to the Fab region of IgG. Previously, we reported a fusion protein made of one binding domain each of PA and PG, which showed greater IgG binding activity than either of the domains, presumably due to bivalent binding to the Fab and/or Fc region (Dong et al., 2015). It was also used to convert Fab fragments to Q-bodies (AG Q-probe), which showed a limited but reproducible antigen-dependent fluorescence response of up to ~6% (Jeong et al., 2016). However, it could not convert the whole IgG to a Q-body, because of its stronger binding affinity to the Fc region than the Fab region. Moreover, only 12% of mouse Fab was reported to bind to PA (Graille et al., 2002), indicating its limited utility.
Recently, a novel antibody light chain binding protein Protein M (PM) was discovered in Mycoplasma genitalium. It has a larger domain size than PA and PG, and binds tightly to almost all the light chains from many species, with a high affinity at low nanomolar dissociation constants (Grover et al., 2014). Although these better properties are probably due to its wider binding area, the binding of PM to the antibody hinders the binding of protein antigens. However, its effect on small antigen binding is not yet known. Considering the structural proximity of the C-terminus of the PM to the antigen-binding site of the bound antibody, we engineered a PM with an additional cysteine (Cys) residue appended at its C-terminus via a short flexible linker. As a result of the fluorescence labeling of the protein at its C-terminus, a fluorescent probe (Q-probe) capable of converting Fab or IgG antibodies into a fluorescent sensor that acts like a Q-body showing a more than 100% increase in fluorescence was successfully constructed. The Q-probe was successfully used to screen several anti-small biomarker antibodies for their suitability as a Q-body, and an anti-thyroxine IgG was successfully applied to the detection of total thyroxine concentration in serum. Finally, the response of the Q-probe/anti-cortisol IgG complex was compared with the recombinantly made scFv Q-body to evaluate its performance both in a buffer and in serum, to show the potential of this approach in clinical diagnosis.
Section snippets
Materials
The materials used for recombinant protein preparation are described in the Supplementary Material. StrepTactin agarose and StrepTactin-Horseradish peroxidase (HRP) conjugate were from IBA (Goettingen, Germany) and Bio-Rad (Hercules, CA, USA), respectively. Fluorescent dye 5-TAMRA-C6-maleimide was purchased from Setareh Biotech, Inc. (OR, USA). Anti-cAMP monoclonal antibody (mAb) SPM486, anti-progesterone mAb (Pg.53), anti-testosterone mAb (4E1G2), anti-thyroxine mAb (ME.125),
Construction of PM probe expression vectors
PM has recently been discovered as an antibody-binding protein that binds tightly to almost all types (κ and λ of many species) of antibody light chains, predominantly at their variable region VL. The solved crystal structure of the PM-Fab complex shows that the C-terminal region of PM is located just above the antigen-binding pocket and interferes with the binding of the protein antigens, which potentially limits its utility as a probe for antibody labeling (Fig. 1A). However, since its
Conclusions
As a fluorescence-labeled antibody-based biosensor, Q-body can detect many targets—from small molecules to large proteins—with high sensitivity and selectivity. Moreover, because of its very simple detection principle, the assay requires only seconds to minutes to obtain a signal, which is suitable for real-time monitoring of antigens in situ. Q-body enables homogeneous immunoassay and has many merits. It requires no washing steps, nor a large sample volume. Hence, these unique properties
CRediT authorship contribution statement
Jinhua Dong: Investigation, Writing - original draft. Chihiro Miyake: Investigation. Takanobu Yasuda: Methodology, Investigation. Hiroyuki Oyama: Resources. Izumi Morita: Resources. Tomoya Tsukahara: Investigation. Masaki Takahashi: Investigation. Hee-Jin Jeong: Methodology. Tetsuya Kitaguchi: Project administration. Norihiro Kobayashi: Resources. Hiroshi Ueda: Conceptualization, Methodology, Writing - review & editing, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank Yuki Ohmuro-Matsuyama for discussion, Chiaki Toyama for experimental help, Kyowa Medex Co. and Fujifilm Co. for providing KTM219 and 22G8A2 antibodies, respectively, and the Division of Biomaterial Analysis, Technical Department, Tokyo Institute of Technology, for nucleic acid sequence analysis. This work was performed under the Research Program of “Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials” in “Network Joint Research Center for Materials and
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