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

Milad Dagher,Grant Ongo, Nathaniel Robichaud,Jinglin Kong, Woojong Rho, Ivan Teahulos, Arya Tavakoli, Samantha Bovaird, Shahem Merjaneh,Andrew Tan, Kiran Edwardson, Christelle Scheepers,Andy Ng, Andy Hajjar, Baly Sow, Michael Vrouvides, Andy Lee,Philippe DeCorwin-Martin,Shafqat Rasool, JiaMin Huang, Timothy Erps, Spencer Coffin,Yu Han,Srinivas Niranj Chandrasekaran,Lisa Miller,Maria Kost-Alimova,Adam Skepner,Shantanu Singh,Anne E. Carpenter,Jeffrey Munzar,David Juncker

bioRxiv the preprint server for biology(2023)

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Abstract
We present the nELISA, a high-throughput, high-fidelity, and high-plex protein profiling platform. DNA oligonucleotides are used to pre-assemble antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection. Spatial separation between non-cognate antibodies prevents the rise of reagent-driven cross-reactivity, while 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 on performance vs 1-plex signals, with sensitivities as low as 0.1pg/mL and measurements spanning 7 orders of magnitude. We then performed a large-scale secretome perturbation screen of peripheral blood mononuclear cells (PBMCs), 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 ones, that were conserved across donors and stimulation conditions. We also validated the nELISA’s use in phenotypic screening, and propose its application to drug discovery. ### Competing Interest Statement The Authors declare the following competing interests: N.R., G.O., W.R., I.T., A.T., J.K., J.M., S.B., S.M., A.T., K.E., C.S., A.N., A.H., A.L., P.D.M., S.R., J.H. and M.D. are employees and have ownership interest in Nomic Bio, which markets the nELISA platform, D.J. has ownership interest in Nomic Bio. S.S. and A.E.C. serve as scientific advisors for companies that use image-based profiling and Cell Painting (A.E.C: Recursion, S.S.:Waypoint Bio, Dewpoint Therapeutics) and receive honoraria for occasional talks at pharmaceutical and biotechnology companies. All other authors declare no competing interests.
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Key words
High-Throughput Analysis,DNA nanotechnology,Multiplexed Assays,Nanoparticles
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