nELISA: A high-throughput, high-plex platform enables quantitative profiling of the secretome

Abstract

We present the nELISA, a miniaturised, high-throughput, and high-fidelity protein profiling platform. DNA oligonucleotides are used to pre-colocalize antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection while ensuring spatial separation between non-cognate antibody pairs. Read-out is performed cost-efficiently and at high-throughput using flow cytometry. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity or impact to performance vs 1-plex signals, with sensitivities as low as 0.1pg/mL and measurements across the platform spanning 8 orders of magnitude. We then performed a large-scale PBMC secretome screen, with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating ~1.5M protein datapoints in under a week, a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel cytokine responses, that were conserved across donors and stimulation conditions. We also validated its use in phenotypic screening, and proposed applications for the nELISA in drug discovery.

A versatile information retrieval framework for evaluating profile strength and similarity

“…The measurements can report on bulk properties or offer single-cell resolution, depending on the experimental design and research question asked. Thus, they convey information about the molecular (e.g., genomic, epigenomic, transcriptomic, proteomic, or metabolomic) or cellular (e.g., morphological, spatial, viability across cell lines) status of the system [4][5][6] . Some profiling experiments can be executed at high-throughput scale, where biological samples are subjected to various perturbations, usually chemical compounds or genetic reagents [6][7][8][9][10][11] .…”

“…Thus, they convey information about the molecular (e.g., genomic, epigenomic, transcriptomic, proteomic, or metabolomic) or cellular (e.g., morphological, spatial, viability across cell lines) status of the system [4][5][6] . Some profiling experiments can be executed at high-throughput scale, where biological samples are subjected to various perturbations, usually chemical compounds or genetic reagents [6][7][8][9][10][11] . Ultimately, by combining high-dimensional readouts, diverse biological samples, and multiple perturbations, profiling can reveal the mechanisms of biological processes and potential therapeutic avenues.…”

A morphology and secretome map of pyroptosis

“…36 Similar advances in ELISA technologies have enabled the simultaneous acquisition of hundreds of secreted markers to form systems biology representations of the secretome, and recent technology allows the simultaneous measurement of secretome and cell morphology from the same wells. 37,38 In this study, we applied Cell Painting and nELISA (Nomic Bio -187 secretome markers) to profile cell morphology and secretome representations of pyroptosis and apoptosis. We modified the Cell Painting assay to swap the cytoplasmic RNA stain with a marker for cleaved N-terminal Gasdermin D protein, which is the canonical marker of pyroptosis pore formation.…”

“…36 Similar advances in ELISA technologies have enabled the simultaneous acquisition of hundreds of secreted markers to form systems biology representations of the secretome, and recent technology allows the simultaneous measurement of secretome and cell morphology from the same wells. 37,38…”

Multititration: The New Method for Implementing Ultrasensitive and Quantitative Multiplexed In-Field Immunoassays Despite Cross-Reactivity?

“…Some approaches aim at separating in situ the different reagents and/or analytes, either by prior fractionation of the sample, which may provide a large-scale multiplexing, or on reagent colocalization, followed by an ELISA-like detection. These latter methods eliminate nonspecific binding between antibodies and drastically limit analyte-induced cross-reactivity by avoiding the mixing of capture and detection antibodies, e.g., through a specific spotting on microarrays. , Another alternative to detect multiple targets in a complex environment is the use of mass spectrometry, which may achieve high sensitivity and selectivity, e.g., through the commercial SISCAPA solution. , However, these methods are complex to implement and require expensive and nonportable equipment or are not compatible with the LFA technology . An approach to tackle this issue is the development of multicolor LFAs, where each target is revealed by different reporters exhibiting a unique color. ,,− …”