Visual Detection of Human Antibodies Using Sugar Chain-Immobilized Fluorescent Nanoparticles: Application as a Point of Care Diagnostic Tool for Guillain-Barré Syndrome

Abstract

Sugar chain binding antibodies have gained substantial attention as biomarkers due to their crucial roles in various disorders. In this study, we developed simple and quick detection method of anti-sugar chain antibodies in sera using our previously developed sugar chain-immobilized fluorescent nanoparticles (SFNPs) for the point-of-care diagnostics. Sugar chain structure on SFNPs was modified with the sugar moieties of the GM1 ganglioside via our original linker molecule to detect anti-GM1 antibodies. The structures and densities of the sugar moieties immobilized on the nanoparticles were evaluated in detail using lectins and sera containing anti-GM1 antibodies from patients with Guillain-Barré syndrome, a neurological disorder, as an example of disease involving anti-sugar chain antibodies. When optimized SFNPs were added to sera from patients with Guillain-Barré syndrome, fluorescent aggregates were able to visually detect under UV light in three hours. The sensitivity of the detection method was equivalent to that of the current ELISA method used for the diagnosis of Guillain-Barré syndrome. These results suggest that our method using SFNPs is suitable for the point-of-care diagnostics of diseases involving anti-sugar chain antibodies.

Fig 1. Preparation of GM1-Glc-immobilized fluorescent nanoparticles (GM1-Glc-SFNPs).

(A) The structures of synthesized ligand conjugates containing GM1 sugar chain moieties. (B) Scheme showing the preparation of SFNPs containing GM1 sugar moieties.

Fig 2. Characterization of GM1-Glc-immobilized fluorescent nanoparticles (GM1-Glc-SFNPs).

(A) UV-Vis (dotted line) and fluorescence (solid line) spectra of GM1-Glc-SFNPs. (B) Dynamic light scattering measurement of GM1-Glc-SFNPs. The average hydrodynamic diameter was 8.9 nm. (C) MALDI-TOF MS analysis of GM1-Glc-SFNPs. The detected peak was m/z: 1442.68 [M-H]⁻, corresponding to GM1-Glc-f-mono.

Fig 3. Interaction analysis of GM1-Glc-immobilized fluorescent nanoparticles (GM1-Glc-SFNPs) with lectins.

(A) Analysis of interactions between GM1-Glc-SFNPs and lectins. Visual image of the mixture of GM1-Glc-SFNPs and proteins under UV irradiation. (B) Fluorescence spectra of the supernatant monitored by an excitation wavelength at 360 nm.

Fig 4. Agglutination assay of GM1-Glc-immobilized fluorescent nanoparticles (GM1-Glc-SFNPs) with sera from GBS patients.

(A) Visual image of fluorescent aggregates under UV irradiation (samples 1 and 3: negative for anti-GM1/GD1a antibodies; sample 2: positive for anti-GD1a antibodies; samples 4, 5, and 6: positive for anti-GM1 IgG antibodies). (B) Fluorescence spectra of supernatants monitored by excitation at 360 nm. (C) Silver-stained SDS-PAGE of aggregates obtained from sample 6; 10% gel with 2-mercaptoethanol (2-ME; left) and 8% gel without reducing reagents (right). Lanes 1 and 4: GM1-Glc-SFNP; lanes 2 and 5: aggregates of GM1-Glc-SFNPs; lanes 3 and 6: diluted serum. (D) Western blotting analysis of fluorescent aggregates of GM1-Glc-SFNPs; 10% gel with 2-mercaptoethanol. Lane 1: aggregates of GM1-Glc-SFNPs; lanes 2: diluted serum.

Fig 5. Agglutination inhibition assay of GM1-Glc-immobilized fluorescent nanoparticles (GM1-Glc-SFNPs) with sera from GBS patients.

(A) Visual image of the inhibition caused by the addition of free GM1 sugar chains to the agglutination assay (concentration of free GM1 sugar chain: 0 to 4 mM). (B) Fluorescence spectra of the supernatants in the agglutination inhibition assay.

Fig 6. Agglutination assay of PNA using sugar chain-immobilized fluorescent nanoparticles (SFNPs) with different sugar-chain densities.

(A) Image from an agglutination assay using GM1-Glc/tetraethylene glycol-conjugated monovalent linker (TEG; 5:5)-SFNPs with peanut agglutinin (PNA). (B) Binding experiment using GM1-Glc/TEG-SFNPs (5:5) to calculate K _D values using a Scatchard plot. (C) Image from an agglutination assay using GM1-SFNPs with PNA. (D) Binding experiment using GM1-SFNPs to calculate K _D values using a Scatchard plot.

Fig 7. Agglutination assay of GM1-Glc-immobilized fluorescent nanoparticles (GM1-Glc-SFNPs) with different sugar-chain densities and sera from GBS patients.

(A–C) Images from an agglutination assay using GM1-Glc-SFNPs and serum from a GBS patient after incubation for (A) 1 h, (B) 3 h, or (C) 12 h. (D–F) Images from an agglutination assay using GM1-Glc/TEG (5:5)-SFNPs and serum from a GBS patient after incubation for (D) 1 h, (E) 3 h, or (F) 12 h. (G) Image from an agglutination assay of GM1-Glc/TEG (5:5)-SFNPs, GM1-SFNPs, or GM1-Glc/TEG-SFNPs using serial dilutions of serum, after incubation for 3 h.