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Screening individual hybridomas by microengraving to discover monoclonal antibodies

Nature Protocols volume 4, pages 767–782 (2009)Cite this article

Abstract

The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (∼105 cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 1–3 d (9–12 d including the steps for preparing arrays of microwells).

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Figure 1: Schematic diagram of the processes described in this protocol.
Figure 2: Design of an array of microwells for microengraving.
Figure 3: Retrieval of cells from microwells with a micromanipulator.
Figure 4: Microarrays of antibodies produced by microengraving.
Figure 5: 'Curtain' western blot of 12.5% polyacrylamide gel loaded with 200 μg of complete COS-1 cell lysate with 10 μg HBSAg (ViroStat).
Figure 6: Examples of potential variations in the quality of the microarrays produced by microengraving.

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Acknowledgements

This research was supported by the Eli and Edythe L. Broad Institute, and used facilities at the Center for Nanoscale Systems at Harvard University supported by the NSF under the National Nanotechnology Infrastructure Network, and services at the Stanford Microfluidics Foundry. E.P. was an NSERC postgraduate fellow. C.M.S. thanks Gordon College for sabbatical leave. The authors thank J. Ronan and J.H. Choi for helpful discussions on this protocol.

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Authors and Affiliations

  1. Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, 02139, Massachusetts, USA

    Adebola O Ogunniyi & J Christopher Love

  2. Department of Biology, Gordon College, 255 Grapevine Rd., Wenham, 01984, Massachusetts, USA

    Craig M Story

  3. Harvard/MIT Health Science and Technology Institute, 77 Massachusetts Ave., Cambridge, 02139, Massachusetts, USA

    Eliseo Papa

  4. Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, 02139, Massachusetts, USA

    Eduardo Guillen

  5. The Eli and Edythe L. Broad Institute, Seven Cambridge Center, Cambridge, 02139, Massachusetts, USA

    J Christopher Love

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  1. Adebola O Ogunniyi
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Correspondence to J Christopher Love.

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Ogunniyi, A., Story, C., Papa, E. et al. Screening individual hybridomas by microengraving to discover monoclonal antibodies. Nat Protoc 4, 767–782 (2009). https://doi.org/10.1038/nprot.2009.40

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