Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface

Akihito Inoue,Takanobu Yasuda,Hiroshi Ueda,Bo Zhu,Akikazu Murakami,Tetsuya Kitaguchi

doi:10.1038/s41598-021-02022-7

Akihito Inoue, Takanobu Yasuda + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-02022-7

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Abstract

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based “mini Q-bodies” by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize human serum albumin was used as a model. The best variant, showing a 2.4-fold signal increase, was obtained through selection by flow cytometry. Furthermore, the same nanobody prepared from Escherichia coli also worked as a mini Q-body after dye labeling. The described approach will be applied to quickly obtain well-behaved Q-bodies and other fluorescent biosensors for various targets through directed evolutionary approaches.

Highlights

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody
A nanobody-based Q-body was chosen as a candidate for displaying the Q-body because of its low molecular weight and high stability, which could be beneficial in the cloud of the yeast cell surface
The MTX-recognizing nanobody was previously proven to function as a Q-body with sixfold response[11], which suggested that MTX nanobody could be a suitable candidate for the proof-of-concept of the Q-body on the yeast cell surface

Summary

Introduction

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Enzyme-linked immunosorbent assays (ELISAs) are generally used in clinical diagnosis since they were first reported in 1 9724 Despite their high sensitivity, the requirement of multiple reaction steps makes the process time-consuming and technically complex, which could be a drawback in point-of-care testing (POCT) applications. The Q-body works on the principle of antigen-dependent removal of the quenching effect on a fluorophore that has been quenched by intrinsic tryptophan (Trp) residues of an antibody fragment[14] Because it does not require bound/free separation and detects antigens in a noncompetitive manner, the Q-body-based assay is simple, rapid, and exhibits a relatively high sensitivity. It does not need a light source, allowing visual observation of the color change and easier integration to a smartphone-based device

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