Quantum-dot antibody conjugation visualized at the single-molecule scale with high-speed atomic force microscopy

Umakoshi, Takayuki; Udaka, Hikari; Uchihashi, Takayuki; Ando, Toshio; Suzuki, Miho; Fukuda, Tsuguo

doi:10.1016/j.colsurfb.2018.04.015

Cited by 13 publications

(8 citation statements)

References 38 publications

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“…S2 show that fulllength Ig exhibits two representative images, Y-shaped (flat-on) and dumbbell-like (side-on) structures. The Fab and Fc regions are relatively distant (the average distance is 21 6 3 nm, mean 6 S.D., n = 10) compared with IgG1 structures previously reported (19). Moreover, Movie S1 shows that the connec- tion between Fab and Fc regions (two or three spots) of Ig particles are highly mobile, while the longest distances are 26 nm.…”

Section: Afm Analysis Of N-truncated Ig and Full-length Igmentioning

confidence: 71%

Molecular mechanism of the recognition of bacterially cleaved immunoglobulin by the immune regulatory receptor LILRA2

Yamazaki

Furukawa

Hirayasu

et al. 2020

Journal of Biological Chemistry

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Human leukocyte immunoglobulin-like receptors (LILRs) typically regulate immune activation by binding to the human leukocyte antigen class I molecules. LILRA2, a member of the LILR family, was recently reported to bind to other unique ligands, the bacterially degraded Igs (N-truncated Igs), for the activation of immune cells. Therefore, LILRA2 is currently attracting significant attention as a novel innate immune receptor. However, the detailed recognition mechanisms required for this interaction remain unclear. In this study, using several biophysical techniques, we uncovered the molecular mechanism of N-truncated Ig recognition by LILRA2. Surface plasmon resonance analysis disclosed that LILRA2 specifically binds to N-truncated Ig with weak affinity (Kd = 4.8 μm) and fast kinetics. However, immobilized LILRA2 exhibited a significantly enhanced interaction with N-truncated Ig due to avidity effects. This suggests that cell surface-bound LILRA2 rapidly monitors and identifies bi- or multivalent abnormal N-truncated Igs through specific cross-linking to induce immune activation. Van't Hoff analysis revealed that this interaction is enthalpy-driven, with a small entropy loss, and results from differential scanning calorimetry indicated the instability of the putative LILRA2-binding site, the Fab region of the N-truncated Ig. Atomic force microscopy revealed that N truncation does not cause significant structural changes in Ig. Furthermore, mutagenesis analysis identified the hydrophobic region of LILRA2 domain 2 as the N-truncated Ig-binding site, representing a novel ligand-binding site for the LILR family. These results provide detailed insights into the molecular regulation of LILR-mediated immune responses targeting ligands that have been modified by bacteria.

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Section: Afm Analysis Of N-truncated Ig and Full-length Igmentioning

confidence: 71%

Molecular mechanism of the recognition of bacterially cleaved immunoglobulin by the immune regulatory receptor LILRA2

Yamazaki

Furukawa

Hirayasu

et al. 2020

Journal of Biological Chemistry

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“…The conjugate of QDs and antibodies has been recognized as a powerful bioprobe and applied widely in ELISA and immunofluorescence (Goldman et al 2002;Umakoshi et al 2018). Given the excellent property of sdAb in terms of structure, stability, and production costs, sdAbs have been used successfully as an attractive alternative to conjugate with QDs for the imaging and diagnostic platforms (Sukhanova et al 2012;Wang et al 2015;Yin et al 2013).…”

Section: Discussionmentioning

confidence: 99%

Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus

Yang

Yin

et al. 2018

Appl Microbiol Biotechnol

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Single-domain antibody (sdAb) or nanobody possesses specific features non-accessible for conventional antibodies that make them suitable for research and biotechnological applications. Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets, resulting in great economic losses all over the world. To detect and isolate PEDV rapidly and accurately is important for the control and further research of the clinical PEDV strains. In this study, four sdAb fragments (sdAb-Mc19/29/30/37) targeting the membrane (M) protein of PEDV were selected from sdAb library that was constructed through M protein-immunized Camelus bactrianus. The selected sdAb-Mcs were solubly expressed in Escherichia coli. The functional characteristics analysis revealed that the recombinant sdAb-Mcs have excellent binding activity and specificity to M protein but have no neutralizing activity to PEDV. For further application, sdAb-Mc37 was conjugated with quantum dots to synthesize a nanoprobe for imaging PEDV in vero cells. The observed fluorescence in vero cells clearly reflects that PEDV virions can be reliably recognized and labeled by the nanoprobe. Furthermore, the sdAb-Mc29 was conjugated with superparamagnetic nanobeads to construct immunomagnetic nanobeads (IMNBs) used to isolate PEDV. One PEDV strain was successfully isolated from clinical fecal sample, suggesting IMNBs as a novel and efficient tool suitable for PEDV isolation from clinical samples. This study provided a novel application and substantiated the suitability of sdAb as a specific binder for the isolation of viruses.

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“…We note that the extinction coefficients at the first exciton peak are much lower than those at shorter wavelengths, in contrast to organic dyes which have their largest extinction coefficient at the peak of their absorption spectrum. Coupled with their broad excitation spectra which increase towards the UV, relatively long lifetimes ( > 10 ns) and resistance to chemical degradation, CdTe and CdSe QDs are excellent candidates for tracking time-dependent dynamic processes (154) , biomedical imaging including in vivo tumour detection (167,168) , deep tissue imaging (169) , environmental sensing (170,171) and antibody detection (172) .…”

Section: Quantum Dotsmentioning

confidence: 99%

A guide to small fluorescent probes for single-molecule biophysics

Leake

Quinn

2023

Chemical Physics Reviews

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The explosive growth of single-molecule techniques is transforming our understanding of biology, helping to develop new physics inspired by emergent biological processes, and leading to emerging areas of nanotechnology. Key biological and chemical processes can now be probed with new levels of detail, one molecule at a time, from the nanoscopic dynamics of nature's molecular machines to an ever-expanding range of exciting applications across multiple length and time scales. Their common feature is an ability to render the underlying distribution of molecular properties that ensemble averaging masks and to reveal new insights into complex systems containing spatial and temporal heterogeneity. Small fluorescent probes are among the most adaptable and versatile for single-molecule sensing applications because they provide high signal-to-noise ratios combined with excellent specificity of labeling when chemically attached to target biomolecules or embedded within a host material. In this review, we examine recent advances in probe designs, their utility, and applications and provide a practical guide to their use, focusing on the single-molecule detection of nucleic acids, proteins, carbohydrates, and membrane dynamics. We also present key challenges that must be overcome to perform successful single-molecule experiments, including probe conjugation strategies, identify tradeoffs and limitations for each probe design, showcase emerging applications, and discuss exciting future directions for the community.

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Quantum-dot antibody conjugation visualized at the single-molecule scale with high-speed atomic force microscopy

Cited by 13 publications

References 38 publications

Molecular mechanism of the recognition of bacterially cleaved immunoglobulin by the immune regulatory receptor LILRA2

Molecular mechanism of the recognition of bacterially cleaved immunoglobulin by the immune regulatory receptor LILRA2

Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus

A guide to small fluorescent probes for single-molecule biophysics

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