title: Nanowell-based immunoassays for measuring single-cell secretion: characterization of transport and surface binding.
abstract: Arrays of subnanoliter wells (nanowells) provide a useful system to isolate single cells and analyze their secreted proteins. Two general approaches have emerged: one that uses open arrays and local capture of secreted proteins, and a second (called microengraving) that relies on closed arrays to capture secreted proteins on a solid substrate, which is subsequently removed from the array. However, the design and operating parameters for efficient capture from these two approaches to analyze single-cell secretion have not been extensively considered. Using numerical simulations, we analyzed the operational envelope for both open and closed formats, as a function of the spatial distribution of capture ligands, their affinities for the protein, and the rates of single-cell secretion. Based on these analyses, we present a modified approach to capture secreted proteins in-well for highly active secreting cells. This simple method for in-well detection should facilitate rapid identification of cell lines with high specific productivities.
authors.affiliation: Department of Chemical Engineering, ‡Department of Biological Engineering, and §Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Alexis J
authors.initials: AJ
authors.lastname: Torres
authors.forename: Abby S
authors.initials: AS
authors.lastname: Hill
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Love
mesh_terms: D000818:Animals; D001665:Binding Sites; D002645:Chickens; D006825:Hybridomas / Q000166:cytology / Q000378:metabolism*; D007118:Immunoassay; D007074:Immunoglobulin G / Q000032:analysis* / Q000276:immunology; D049329:Nanostructures / Q000737:chemistry*; D010047:Ovalbumin / Q000276:immunology; D059010:Single-Cell Analysis / Q000379:methods*; D013499:Surface Properties
chemical_list: D007074:Immunoglobulin G; D010047:Ovalbumin