2018 EBF, Barcelona, Spain

Francesco Piraino, Joseph Johnson, Dandan Shan, Ariel Vonk, Muriel Menendez, Yao Chen, Linan Song, Lei Chang, Milena Milutinovic, Daniella Svancara, Purvish Patel, Shazia Baig, Shuai Nie, Katie Beauregard, David Rissin, David Duffy Quanterix Corporation, 113 Hartwell Avenue, Lexington, MA 02421, USA

Abstract: Digital ELISA (Enzyme Linked Immunosorbent Assay) based on single molecule arrays (Simoa) has improved sensitivity of traditional ELISA from picomolar (10-12 M) to femtomolar (10 -15 M), increasing the quality and quantity of biomarkers that can be measured for health and disease. Digital ELISA counts signal generated from single immunocomplexes formed on superparamagnetic beads confined in arrays of femtoliter-sized wells in which fluorescent molecules are highly concentrated. We have commercialized digital ELISA in a fully-automated instrument (Simoa HD- Analyzer), ideal for use in pharmaceutical companies, drug discovery, clinical research and other areas necessitating full automation and high throughput. We have recently launched the SR-X benchtop reader, with a smaller footprint and more flexible workflow. Operators prepare assays in microtiter plates at the bench in a semi-automated format similar to traditional ELISA, with the notable exception that plates are preserved by drying after assay completion, and can be read immediately or the next day.

Methods: Cross Validation of SR-X and HD-1. We have compared performance of the following Simoa assays on SR-X to HD-: Aβ40; Aβ42; Cytokine 6-Plex Panel ; HIV p24; IFNγ; IL-6; IL-10; IL-13; IL-17A; IL-22; IL-33; mouse Tau; Neurology 4-Plex A; Neurofilament-light; PD-L1; PSA; Tau; and TNFα. Measured sample levels correlated with R2 values from 0.96 to .00, with average LOD and LLOQ within .4 and .5 fold of HD-, respectively. Inter-assay precision ranged from 4.0 to .2% CV across assays. Operators tested fullplates from start to finish within – 2 hours (/2 hour hands on time) and a read time of 2 hours (5 minutes hands on time). The more flexible work-flow of SR-X allows exploration of novel uses of Simoa, including nucleic acid testing.

Results: Ten matched serum and plasma samples from normal donors were measured on SR-X and HD-1 across 26 markers. Sample levels over 10 orders of magnitude correlated with an average R 2 of 0.9412. 

Limit of detection (LOD), lower limit of quantitation (LLOQ), intra-run and inter-run precision were compared for SR-X and HD-1. LOD was estimated as 2.5 standard deviations above the blank. LLOQ was determined as the lowest dilution of calibrator that showed CV < 20% and accuracy within 20%. Intra-run and Inter-run precision were measured with two controls and three serum / plasma panels over 6 runs across 2 instruments. SR-X LOD was 98% and LLOQ was 134% compared to HD-1 on average. Average intra-run precision was 6.4% and 6.0% for SR-X and HD-1 respectively; average inter-run precision was 7.0% and 6.0% for SR-X and HD-1 respectively.

Summary: The Quanterix SR-X benchtop single molecule detection system provides performance equivalent to the fully-automated HD-1 Analyzer for ultra-sensitive detection of fluid biomarkers at fg/ml concentrations in single-plex and multi-plex assay formats. The more compact benchtop form factor reduces the physical space and the semi-automated workflow of the SR-X has been optimized using conventional laboratory sample prep devices.