Single molecule array simoa

Single Molecule Array (SIMOA; Quanterix) bead-based tau aggregates assay was developed using a mouse anti-HT7 antibody (Thermo Fisher Scientific, RRID: AB_2314654) as both capture and detection. The assay was prepared according to the manufacturer’s protocol. Recombinant full length P301L tau aggregates were made as described [56 (link)] and were used as a calibrator and included in each run to generate standard curve. HD-X instrument, buffers, helper beads and streptavidin B-galactosidase, and enzyme substrate resorufin β-D-galactopyranoside were obtained from Quanterix. Assays were performed according to the manufacturer’s instructions. All samples were diluted in the Tau Calibrator Diluent (Quanterix).

A total of 500 μl of plasma was utilized to measure biomarker levels using the Single Molecule Array (Simoa) technology (Simoa; Quanterix, Lexington, MA, USA). From the materials provided, a recombinant NfL calibration curve was constructed. The calibration range was 0–500 pg/ml with a dynamic range of 0–2,000 pg/ml. The sample and control concentrations were calculated from 4PL curve fit. CV for NfL was 0.038 and Lowest Level of Detection (LLOD) was 0.038 pg/ml.
Utilizing Simoa technology, multiplexed detection of Aβ₄₂, Aβ₄₀, and t tau was conducted. Calibration ranges for Aβ₄₂, Aβ₄₀, and t tau were 0–60, 0–140, 0–100 pg/ml and dynamic ranges of 0–240, 0–560, 0–400 pg/ml, respectively. Aβ₄₂, Aβ₄₀, and t tau control samples (analog 87.0, 393, 99.5 pg/ml and digital 3.20, 22.4, 2.24 pg/ml, respectively). The sample and control concentrations were calculated from a 4PL curve fit. CVs for Aβ₄₂, Aβ₄₀, and t tau were 0.043, 0.043, and 0.061, respectively. LLODs for Aβ₄₂, Aβ₄₀, and t tau were 0.045, 0.196, and 0.019 pg/ml, respectively. Interplate CVs were derived for high and low pooled controls from the Quanterix automated system: NfL (high control CV = 0.035 and low control CV = 0.092); Aβ₄₂ (high control CV = 0.051 and low control CV = 0.040); Aβ₄₀ (high control CV =0.050 and low control CV = 0.042); and t tau (high control CV = 0.040 and low control CV = 0.047).