Comparison of ELISA- and SIMOA-based quantification of plasma Aβ ratios for early detection of cerebral amyloidosis

Abstract

Background: Blood-based amyloid biomarkers may provide a non-invasive, cost-effective and scalable manner for detecting cerebral amyloidosis in early disease stages.

Methods: In this prospective cross-sectional study, we quantified plasma Aβ_1-42/Aβ_1-40 ratios with both routinely available ELISAs and novel SIMOA Amyblood assays, and provided a head-to-head comparison of their performances to detect cerebral amyloidosis in a nondemented elderly cohort (n = 199). Participants were stratified according to amyloid-PET status, and the performance of plasma Aβ_1-42/Aβ_1-40 to detect cerebral amyloidosis was assessed using receiver operating characteristic analysis. We additionally investigated the correlations of plasma Aβ ratios with amyloid-PET and CSF Alzheimer's disease biomarkers, as well as platform agreement using Passing-Bablok regression and Bland-Altman analysis for both Aβ isoforms.

Results: ELISA and SIMOA plasma Aβ_1-42/Aβ_1-40 detected cerebral amyloidosis with identical accuracy (ELISA: area under curve (AUC) 0.78, 95% CI 0.72-0.84; SIMOA: AUC 0.79, 95% CI 0.73-0.85), and both increased the performance of a basic demographic model including only age and APOE-ε4 genotype (p ≤ 0.02). ELISA and SIMOA had positive predictive values of respectively 41% and 36% in cognitively normal elderly and negative predictive values all exceeding 88%. Plasma Aβ_1-42/Aβ_1-40 correlated similarly with amyloid-PET for both platforms (Spearman ρ = - 0.32, p < 0.0001), yet correlations with CSF Aβ_1-42/t-tau were stronger for ELISA (ρ = 0.41, p = 0.002) than for SIMOA (ρ = 0.29, p = 0.03). Plasma Aβ levels demonstrated poor agreement between ELISA and SIMOA with concentrations of both Aβ_1-42 and Aβ_1-40 measured by SIMOA consistently underestimating those measured by ELISA.

Conclusions: ELISA and SIMOA demonstrated equivalent performances in detecting cerebral amyloidosis through plasma Aβ_1-42/Aβ_1-40, both with high negative predictive values, making them equally suitable non-invasive prescreening tools for clinical trials by reducing the number of necessary PET scans for clinical trial recruitment.

Trial registration: EudraCT 2009-014475-45 (registered on 23 Sept 2009) and EudraCT 2013-004671-12 (registered on 20 May 2014, https://www.clinicaltrialsregister.eu/ctr-search/trial/2013-004671-12/BE ).

Keywords: Biomarkers; Cerebral amyloidosis; ELISA; Immunoassay; Plasma; Preclinical Alzheimer’s disease; Prescreening; SIMOA; β-Amyloid.

Fig. 1

ROC curves of plasma Aβ_1–42/Aβ_1–40 and Aβ_1–42/t-tau to detect cerebral amyloidosis: ELISA versus SIMOA. ROC curves of plasma Aβ_1–42/Aβ_1–40 (left) and Aβ_1–42/t-tau (right) are shown with amyloid-PET status as the standard-of-truth in the entire study population (n = 199) (a, b) as well as in the CN (n = 161) (c, d) and aMCI subgroup (n = 38) (e, f), when Aβ isoforms were measured with either ELISA (blue) or SIMOA assays (orange). Note that the AUCs for ELISA and SIMOA are based on plasma biomarker measurements on their own, without inclusion of age or APOE-ε4 genotype in the model. Additionally, the ROC curve of the basic demographic model, including only age and APOE-ε4 genotype, is shown (black) on each plot together with its corresponding AUC for the respective subgroups. Amyloid-PET positivity as binary input for ROC was defined as a SUVR_comp above a predefined cut-off of 1.38 for [¹⁸F]flutemetamol PET [21] and 1.29 for [¹⁸F]florbetaben PET. For calculation of these cut-offs, we used the same methodology as the one employed in a previous study [22]. aMCI, amnestic mild cognitive impairment; AUC, area under curve; Aβ, β-amyloid; CI, confidence interval; CN, cognitively normal; ROC, receiver operating characteristic; SIMOA, single molecule array; t-tau, total tau

Fig. 2

Correlations of ELISA and SIMOA plasma Aβ_1–42/Aβ_1–40 and Aβ_1–42/t-tau with established PET- and CSF-based AD biomarkers. Plasma Aβ_1–42/Aβ_1–40 (a–d) and Aβ_1–42/t-tau (e–h) with ELISA (left, blue) and SIMOA (right, orange) Aβ measurements were plotted against amyloid-PET binding (i.e. Centiloid values) (n = 199: 161 CN controls and 38 aMCI patients) and CSF Aβ_1–42/t-tau (n = 56: 37 CN controls and 19 aMCI patients). Filled circles represent measurements in CN controls, and open circles represent measurements in aMCI patients. Spearman rank correlations were calculated for the entire study population as well as for the CN and aMCI subgroups. p values are indicated in bold when significant after correction for multiple comparisons (significance level α = 0.05/2 = 0.03). aMCI, amnestic mild cognitive impairment; Aβ, β-amyloid; CN, cognitively normal; ELISA, enzyme-linked immunosorbent assay; SIMOA, single molecule array; t-tau, total tau

Fig. 3

Correlations and commutability between ELISA and SIMOA measurements of β-amyloid (Aβ) isoforms Aβ_1–40 and Aβ_1–42. a, b Box and whisker plots of ELISA (left, blue) and SIMOA (right, orange) measurements of plasma Aβ_1–40 (a) and Aβ_1–42 (b) are shown. The middle line of the box represents the median. The lower and upper line represent, respectively, the first and third quartiles, and the whiskers represent the range. Individual data points are superimposed on the boxplot. Agreement between the two platforms is shown for both Aβ isoforms (c–f). Scatter plots and Passing-Bablok regression analysis of plasma Aβ_1–40 (c) and Aβ_1–42 (d) concentrations measured by SIMOA Amyblood in function of their concentrations measured by ELISA. The regression line is shown in black. Spearman rank correlations were calculated to assess the non-linear relationship between the two methods for both isoforms. e, f Non-parametric percentile method of Bland-Altman graphically shows the agreement between the two immunoassay platforms for respectively Aβ_1–40 (e) and Aβ_1–42 (f). The solid red line represents the median of differences between measurements of the two methods from the same subject. The upper and the lower red dashed lines represent respectively the 97.5th and 2.5th percentile of the measurement differences between which 95% of measurements is situated. Aβ, β-amyloid; ELISA, enzyme-linked immunosorbent assay; SIMOA, single molecule array