Quantitative assessment of AD markers using naked eyes: point-of-care testing with paper-based lateral flow immunoassay

Abstract

Aβ₄₂ is one of the most extensively studied blood and Cerebrospinal fluid (CSF) biomarkers for the diagnosis of symptomatic and prodromal Alzheimer's disease (AD). Because of the heterogeneity and transient nature of Aβ₄₂ oligomers (Aβ₄₂Os), the development of technologies for dynamically detecting changes in the blood or CSF levels of Aβ₄₂ monomers (Aβ₄₂Ms) and Aβ₄₂Os is essential for the accurate diagnosis of AD. The currently commonly used Aβ₄₂ ELISA test kits usually mis-detected the elevated Aβ₄₂Os, leading to incomplete analysis and underestimation of soluble Aβ₄₂, resulting in a comprised performance in AD diagnosis. Herein, we developed a dual-target lateral flow immunoassay (dLFI) using anti-Aβ₄₂ monoclonal antibodies 1F12 and 2C6 for the rapid and point-of-care detection of Aβ₄₂Ms and Aβ₄₂Os in blood samples within 30 min for AD diagnosis. By naked eye observation, the visual detection limit of Aβ₄₂Ms or/and Aβ₄₂Os in dLFI was 154 pg/mL. The test results for dLFI were similar to those observed in the enzyme-linked immunosorbent assay (ELISA). Therefore, this paper-based dLFI provides a practical and rapid method for the on-site detection of two biomarkers in blood or CSF samples without the need for additional expertise or equipment.

Keywords: Alzheimer’s disease; Aβ42 monomer; Aβ42 oligomer; Blood; Gold nanoparticle; Magnetic nanoparticles; Paper-based lateral flow immunoassay.

Scheme 1

The principle and test procedure of dLFI. a The blood samples were enriched by the mAb 1F12-modified MNPs and then eluted for dLFI analysis. Schematic representation of the working principle of the dLFI for the detection of Aβ₄₂Os (b), Aβ₄₂Ms (c), Aβ₄₂Ms and Aβ₄₂Os (d), or without Aβ₄₂Ms or/and Aβ₄₂Os (e)

Fig. 1

Characterization of conformation-specific antibodies for Aβ₄₂Ms and Aβ₄₂Os. a Confocal fluorescence images of mouse 5xFAD brain sections using Cy3-labeled anti-Aβ₄₂ monoclonal antibody 1F12 or 2C6 and thioflavin S. (Scale bar: 500 μm). b Parallel analysis of the morphology of Aβ₄₂Ms and Aβ₄₂Os by cryo-transmission electron microscopy. (Scale bar: 500 nm). The SDS-PAGE (c) and Western blotting (d) analysis of the prepared Aβ₄₂Ms and Aβ₄₂Os using 1F12 or 2C6. The binding selectivity of 1F12 (e) and 2C6 (f) to Aβ₄₂ and Aβ₄₀ was determined by competitive ELISA. g The logIC50 value of 1F12 or 2C6 to Aβ₄₂ and Aβ₄₀. The data are presented as means ± SD, n = 3 in e and f. One-way analysis of variance (ANOVA) was used for multigroup comparisons. Statistical significance is indicated in the figures by ****p < 0.0001

Fig. 2

Characterization of AuNP–1F12 conjugates. a TEM images of synthetic AuNP. (Scale bar: 200 nm). b The size distributions of AuNP before and after modification. c A 12% reduced SDS-PAGE gel analysis of AuNP–1F12 and 1F12. d ELISA was used to determine the bioactivity of AuNP–1F12 conjugates. The optimal volume of K₂CO₃ for the conjugation of 1F12 with AuNP was evaluated by the color (e) and OD₅₂₅ value (g). The color (f) and OD₅₂₅ value (h) were used to evaluate the optimal concentration of 1F12 of synthetic AuNP-1F12. For Fig. 3b, d, g, and h, the data are presented as mean ± SD, n = 3. Statistical significance is indicated in the figures by n.s. (indicating no significance)

Fig. 3

The performance of the dLFI. The visual result (a) and gray value (c) on the T line of the dLFI in the specificity assay, n = 3. b The sensitivity of dLFI for the simultaneous detection of Aβ₄₂Ms and Aβ₄₂Os. d The plotted linear curve of different Aβ₄₂ conformations, n = 3. e, f Correlation between results from dLFI (Y-axis) and sandwich ELISA (X-axis) in spiked samples, n = 12. Data are presented as means ± SD. One-way analysis of variance (ANOVA) was used for multigroup comparisons. Statistical significance is represented in the figures by ****p < 0.0001 and n.s. (indicating no significance)

Fig. 4

Simultaneous detection of Aβ₄₂Ms and Aβ₄₂Os in the blood of 3- and 9-month-old 5xFAD and C57BL/6 J mice. The visual results (a) and grey value (b, c) on the T-line of the dLFI in blood samples from 5xFAD (n = 8) and C57BL/6 J (n = 6) mice at 3 and 9 months old. d, e Correlation between blood Aβ₄₂Os level and Aβ plaque area (d, p < 0.0001) or soluble Aβ₄₂ level (e, p < 0.0001) in the brain of 5xFAD mice, a total of n = 25, of which n = 5 in each age group, including 1, 3, 6, 9, and 12 months old. f Confocal fluorescence images of Aβ plaques and Iba 1-positive cell staining in 3 or 9-month-old 5xFAD mice. (Scale bar: 100 μm). Data are presented as means ± SD. Unpaired t-test was used for two-group comparisons. Statistical significance is indicated in the figures by **p < 0.01, ***p < 0.001 and n.s. (indicating no significance)

Fig. 5

Characterization of antibody-modified NHS-magnetic nanoparticles (MNPs). a Schematic illustration of the working steps for the use of antibody-modified MNPs for the enrichment of Aβ₄₂Os and/or Aβ₄₂Ms. b A 12% reduced SDS-PAGE gel analysis of 1F12-MNPs. 1F12 and NMNs were used as controls. ELISA (c) and IP-Western blotting (d) analysis for the bioactivity of 1F12-MNPs. Data are presented as means ± SD, n = 3 in c

Fig. 6

Detection of Aβ₄₂Ms and/or Aβ₄₂Os in human blood samples by dLFI. The visual results (a) and grey value (b) of the Aβ₄₂Ms and/or Aβ₄₂Os level in the blood of AD patients (n = 8) or healthy controls (n = 7) were analyzed by dLFI. c The Aβ₄₂Ms and/or Aβ₄₂Os levels in the blood of AD patients (n = 8) or healthy controls (n = 7) were detected by sandwich ELISA. d Total Aβ₄₂ levels in human blood samples were detected by sandwich ELISA (n = 8 for AD patients and n = 7 for healthy controls). Data are presented as means ± SD. Unpaired t-test was used for two-group comparisons. Statistical significance is indicated in the figures by *p < 0.05, **p < 0.01 and n.s. (indicating no significance)