Monoclonal antibodies that recognize specific antigens of interest are used as therapeutic agents and as tools for biomedical research1. Discovering a single monoclonal antibody requires retrieval of an individual hybridoma from polyclonal mixtures of cells producing antibodies with a variety of specificities. The time required to isolate hybridomas by a limiting serial-dilution, however, has restricted the diversity and breadth of available antibodies. Here we present a soft lithographic method based on intaglio printing to generate microarrays comprising the secreted products of single cells. These engraved arrays enable a rapid (<12 h) and high-throughput (>100,000 individual cells) system for identification, recovery and clonal expansion of cells producing antigen-specific antibodies. This method can be adapted, in principle, to detect any secreted product in a multiplexed manner.
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We thank Jennifer Love (Whitehead Institute Center for Microarray Technology) and Jessie Dausman for technical assistance. This research was supported by grants from the National Institutes of Health and National Academies Keck Futures Initiative, and used facilities at the Center for Nanoscale Systems at Harvard University supported by the NSF under the National Nanotechnology Infrastructure Network. J.C.L. is a Gilead Sciences Fellow of the Life Sciences Research Foundation. G.M.G. is supported by a postdoctoral fellowship from the Netherlands Organization for Scientific Research (NWO).
The authors declare no competing financial interests.
Analysis of efficiency for loading microwells with cells. (DOC 169 kb)
Measurement of the viability of individual cells in a closed microenvironment over time. (DOC 26 kb)
Fluorescence micrographs of two microarrays of anti-H-2Kb (red) and anti-ovalbumin (green) prepared sequentially using the same array of microwells loaded with Y3 and Hyb 099-01 cells. (DOC 408 kb)
Fluorescence micrographs of the regions from which the images shown in Figure 3 of the manuscript are taken. (DOC 388 kb)
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Love, J., Ronan, J., Grotenbreg, G. et al. A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat Biotechnol 24, 703–707 (2006). https://doi.org/10.1038/nbt1210
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