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Scalable Conjugation and Characterization of Immunoglobulins with Stable Mass Isotope Reporters for Single-Cell Mass Cytometry Analysis

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Mass Cytometry

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1989))

Abstract

The advent of mass cytometry (CyTOF®) has permitted simultaneous detection of more than 40 antibody parameters at the single-cell level, although a limited number of metal-labeled antibodies are commercially available. Here we present optimized and scalable protocols for conjugation of lanthanide as well as bismuth ions to immunoglobulin (Ig) using a maleimide-functionalized chelating polymer and for characterization of the conjugate. The maleimide functional group is reactive with cysteine sulfhydryl groups generated through partial reduction of the Ig Fc region. Incubation of Ig with polymer pre-loaded with lanthanide ions produces metal-labeled Ig without disrupting antigen specificity. Antibody recovery rates can be determined by UV spectrophotometry and frequently exceeds 60%. Each custom-conjugated antibody is validated using positive and negative cellular control populations and is titrated for optimal staining at concentrations ranging from 0.1 to 10 μg/mL. The preparation of metal-labeled antibodies can be completed in 4.5 h, and titration requires an additional 3–5 h.

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Acknowledgments

We are grateful to Scott Tanner for providing useful feedback on this manuscript and to Fluidigm for supplying the chelating polymer that was used to optimize the protocol described here. F.J.H received support from the Swiss National Science Foundation (SNF Early Postdoc. Mobility), the Novartis Foundation for medical-biological research (Research Fellowship), and EMBO (Long-Term Fellowship). Also, DP5OD023056, R21AG057224 from the NIH and the Parker Institute for Cancer Immunotherapy to M.H.S. and S10OD018040 Shared Instrumentation Grant to UCSF. E.F.S. is supported by a Damon Runyon Cancer Research Foundation Fellowship (DRG-2190-14). G.P.N. is supported by the NIH grants R01CA184968, 1R01GM10983601, 1R01NS08953301, 1R01CA19665701, R01HL120724, 1R21CA183660, R33CA0183692, 1R33CA183654-01, U19AI057229, 1U19AI100627, U54-UCA149145A, N01-HV-00242, HHSN26820100034C, and 5UH2AR067676; the NIH Northrop-Grumman Corporation Subcontract 7500108142; FDA grant HHSF223201210194C; DOD grants OC110674 and W81XWH-14-1-0180; NWCRA Entertainment Industry Foundation; and Bill and Melinda Gates Foundation grant OPP1113682. S.C.B. is supported by the Damon Runyon Cancer Research Foundation Fellowship (DRG-2017-09), the NIH grants 1DP2OD022550-01, 1R01AG056287–01, 1R01AG057915-01, 1-R00-GM104148-01, 1U24CA224309-01, 5U19AI116484-02, 1U24CA224309-01, The Bill and Melinda Gates Foundation, and a Translational Research Award from the Stanford Cancer Institute.

Competing Financial Interests:

S.C.B. and E.F.S. have been paid consultants for the company Fluidigm Sciences, the manufacturers that produced some of the reagents and instrumentation described in this manuscript.

Author information

Authors and Affiliations

  1. Department of Pathology, Stanford University, Stanford, CA, USA

    Felix J. Hartmann, Trevor Bruce, Luciene Borges & Sean C. Bendall

  2. Department of Neurology, University of California San Francisco, San Francisco, CA, USA

    Erin F. Simonds

  3. Immunology Program, Stanford University, Stanford, CA, USA

    Nora Vivanco & Sean C. Bendall

  4. Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA

    Garry P. Nolan

  5. Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA, USA

    Matthew H. Spitzer

Authors
  1. Felix J. Hartmann
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  2. Erin F. Simonds
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  3. Nora Vivanco
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  4. Trevor Bruce
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  5. Luciene Borges
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  6. Garry P. Nolan
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  7. Matthew H. Spitzer
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  8. Sean C. Bendall
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Corresponding author

Correspondence to Sean C. Bendall .

Editor information

Editors and Affiliations

  1. Discipline of Pathology, The University of Sydney, Camperdown, NSW, Australia

    Helen M. McGuire

  2. Sydney Cytometry Facility, The University of Sydney and Centenary Institute, Camperdown, NSW, Australia

    Thomas M. Ashhurst

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Hartmann, F.J. et al. (2019). Scalable Conjugation and Characterization of Immunoglobulins with Stable Mass Isotope Reporters for Single-Cell Mass Cytometry Analysis. In: McGuire, H., Ashhurst, T. (eds) Mass Cytometry. Methods in Molecular Biology, vol 1989. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9454-0_5

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  • DOI: https://doi.org/10.1007/978-1-4939-9454-0_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9453-3

  • Online ISBN: 978-1-4939-9454-0

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