Creation of a Nanobody-Based Fluorescent Immunosensor Mini Q-body for Rapid Signal-On Detection of Small Hapten Methotrexate

Abstract

Metrics & MoreArticle Recommendations * sı Supporting Information ABSTRACT: "Quenchbody (Q-body)" is a quench-based fluorescent biosensor labeled with a fluorescent dye near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules quickly by simply mixing with a sample. However, the development of Q-bodies using V HH -nanobodies derived from camelid heavy-chain antibodies has not been reported despite their favorable characteristics. Here, we report a "mini Q-body" that can detect the chemotherapy agent methotrexate (MTX) by using anti-MTX nanobody. Three kinds of constructs each encoding an N-terminal Cys-tag and anti-MTX VHH gene with a different length of linker (GGGS) n (n = 0, 2, and 4) between them were prepared followed by the expression in Escherichia coli and labeling with several dye maleimides. When the fluorescence intensities in the presence of varied MTX concentrations were measured, TAMRA-labeled nanobodies showed a higher response than ATTO520-or R6Glabeled ones. Especially, TAMRA C6-labeled mini Q-body with no linker showed the highest response of ∼6-fold and a low detection limit of 0.56 nM. When each Trp residue in the mini Q-body was mutated to address the quenching mechanism, the major role of Trp34 at CDR1 in quenching was revealed. Furthermore, the mini Q-body could detect MTX in 50% human serum with a low detection limit of 1.72 nM, showing its applicability to therapeutic drug monitoring. This study is expected to become the basis of the construction of highly responsive mini Q-bodies for sensitive detection of many molecules from small haptens to larger proteins, which will lead to broader applications such as point-of-care tests.

Evaluation and Selection of Potent Fluorescent Immunosensors by Combining Fluorescent Peptide and Nanobodies Displayed on Yeast Surface

“…In addition, the yeast-displayed MTX nanobody showed antigen-binding activity (K D : 22.4 nM) (Supplementary Fig. S2), which was in good agreement with that previously described 11 . This suggests that a su cient amount of E4 peptide and MTX nanobody to evaluate the Q-body was displayed on the yeast cell surface without preventing antigen-binding activity.…”

“…Assembly of Q-body on the yeast cell surface First, 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 bene cial in the cloud of the yeast cell surface. In addition, the MTX-recognizing nanobody was previously proven to function as a Q-body with 6-fold 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. Also, an E4/K4 peptide pair was selected as a labeling tag because its high speci city and a nity will be suitable for assembling Q-bodies on the yeast cell surface, where various endogenous proteins coexist.…”

Recent Advances in Quenchbody, a Fluorescent Immunosensor

“…However, their applicability to camelid heavy-chain antibody variable domain (V HH or nanobody) has been elusive. Recently, nanobodies have received considerable attention owing to their small size (~14 kd) compared to scFv or Fab, and their high stability and resultant high production yield when produced in E. coli [49]. Since their first report, many examples of nanobodies that bind antigens specifically and strongly have been reported.…”

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

“…“Quenchbody (Q-body)” is an antibody-based novel fluorescent immunosensor, in which a fluorophore is labeled near the antigen-binding site of an antibody fragment such as a single-chain variable region (scFv) or Fab fragment 10 . Recently, an anti-methotrexate (MTX) nanobody (V HH ) has been proven to become a smaller Q-body (mini Q-body) 11 . We also found that several V H monodomains can be converted to mini Q-bodies (Banwait et al, in preparation).…”