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SEC-ICP-MS and on-line isotope dilution analysis for characterisation and quantification of immunochemical assays

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Abstract

This study presents a novel size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method for the characterisation and quantification of immunoassays with lanthanide-labelled antibodies. SEC-ICP-MS in combination with a double isotope dilution approach enabled facile validation of the antibodies’ integrity, the determination of the batch to batch labelling efficiency, monitoring of each labelling step, and quantification of the immunocomplexes after incubation with the target protein. The addition of oxygen into the dynamic reaction cell improved the detection of sulphur as a marker for the antibodies and target protein via mass-shifting (LOD = 3.7 ng/mL), whilst maintaining sufficient sensitivity for the analysis of the lanthanides. Ultra-high performance liquid chromatography (UHPLC) SEC ensured a rapid chromatographic method with separation times under 7 min of the labelled and unlabelled antibodies, the immunocomplexes, and the unconjugated polymer used to lanthanide-label the antibodies. SEC calibration estimated the molecular weights of all peaks and provided valuable insights in immunochemical reactions and the stoichiometry of the reactants and products. A novel on-line isotope dilution analysis (IDA) enabled absolute quantification of sulphur and lanthanide signals and the protein of interest. The chromatographic separation of immunocomplexes and labelled antibodies allowed the simultaneous determination of the antibody/metal stoichiometry and target protein concentration from a single mass flow chromatogram. An immunoglobulin protein was quantified after incubation with an 153Eu-labelled primary polyclonal antibody. The procedure was validated with direct labelling of the target protein with 156Gd for parallel, simultaneous quantification. The concentration determined via direct labelling of the protein deviated 1.9% from the immunochemical approach employing 153Eu-labelled polyclonal antibodies.

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References

  1. Becker JS, Jakubowski N. The synergy of elemental and biomolecular mass spectrometry: new analytical strategies in life sciences. Chem Soc Rev. 2009;38:1969–83. https://doi.org/10.1039/B618635C.

    Article  CAS  PubMed  Google Scholar 

  2. Mounicou S, Szpunar J, Lobinski R. Metallomics: the concept and methodology. Chem Soc Rev. 2009;38:1119–38. https://doi.org/10.1039/b713633c.

    Article  CAS  PubMed  Google Scholar 

  3. Peng W, Ling-Ni M, He-Fang W, Xue-Guang S, Xiu-Ping Y. A multidimensional sensing device for the discrimination of proteins based on manganese-doped ZnS quantum dots. Angew Chemie. 2011;123:8268–71. https://doi.org/10.1002/ange.201101882.

    Article  Google Scholar 

  4. Biju V, Itoh T, Ishikawa M. Delivering quantum dots to cells: bioconjugated quantum dots for targeted and nonspecific extracellular and intracellular imaging. Chem Soc Rev. 2010;39:3031–56. https://doi.org/10.1039/B926512K.

    Article  CAS  PubMed  Google Scholar 

  5. Yalow RS, Berson SA. Assay of plasma insulin in human subjects by immunological methods. Nature. 1959;184:1648.

    Article  CAS  PubMed  Google Scholar 

  6. Zhang C, Wu F, Zhang Y, Wang X, Zhang X. A novel combination of immunoreaction and ICP-MS as a hyphenated technique for the determination of thyroid-stimulating hormone (TSH) in human serum. J Anal At Spectrom. 2001;16:1393–6. https://doi.org/10.1039/B106387C.

    Article  CAS  Google Scholar 

  7. Mairinger T, Wozniak-Knopp G, Rüker F, Koellensperger G, Hann S. Element labeling of antibody fragments for ICP-MS based immunoassays. J Anal At Spectrom. 2016;31:2330–7. https://doi.org/10.1039/c6ja00252h.

    Article  CAS  Google Scholar 

  8. Esteban-Fernández D, Scheler C, Linscheid MW. Absolute protein quantification by LC-ICP-MS using MeCAT peptide labeling. Anal Bioanal Chem. 2011;401:657–66. https://doi.org/10.1007/s00216-011-5104-2.

    Article  CAS  PubMed  Google Scholar 

  9. Giesen C, Wang HA, Schapiro D, Zivanovic N, Jacobs A, Hattendorf B, et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods. 2014;11:417.

    Article  CAS  PubMed  Google Scholar 

  10. Majonis D, Ornatsky O, Kinach R, Winnik MA. Curious results with palladium- and platinum-carrying polymers in mass cytometry bioassays and an unexpected application as a dead cell stain. Biomacromolecules. 2011;12:3997–4010. https://doi.org/10.1021/bm201011t.

    Article  CAS  PubMed  Google Scholar 

  11. Majonis D, Herrera I, Ornatsky O, Schulze M, Lou X, Soleimani M, et al. Synthesis of a functional metal-chelating polymer and steps toward quantitative mass cytometry bioassays. Anal Chem. 2010;82:8961–9. https://doi.org/10.1021/ac101901x.

    Article  CAS  PubMed  Google Scholar 

  12. Lou X, Zhang G, Herrera I, Kinach R, Ornatsky O, Baranov V, et al. Polymer-based elemental tags for sensitive bioassays. Angew Chemie Int Ed. 2007;46:6111–4. https://doi.org/10.1002/anie.200700796.

    Article  CAS  Google Scholar 

  13. Angelo RM. Method for multiplexed sample analysis by photoionizing secondary sputtered neutrals. 2015.

  14. Schwarz G, Mueller L, Beck S, Linscheid MW. DOTA based metal labels for protein quantification: a review. J Anal At Spectrom. 2014;29:221–33. https://doi.org/10.1039/C3JA50277E.

    Article  CAS  Google Scholar 

  15. He Y, Esteban-Fernandez D, Neumann B, Bergmann U, Bierkandt F, Linscheid MW. Application of MeCAT-click labeling for protein abundance characterization of E. coli after heat shock experiments. J Proteome. 2016;136:68–76. https://doi.org/10.1016/j.jprot.2015.12.010.

    Article  CAS  Google Scholar 

  16. Hann S, Boeck K, Koellensperger G. Immunoaffinity assisted LC-ICP-MS—a versatile tool in biomedical research. 2010;25:18–20. https://doi.org/10.1039/B911462A.

  17. Terenghi M, Elviri L, Careri M, Mangia A, Lobinski R. Multiplexed determination of protein biomarkers using metal-tagged antibodies and size exclusion chromatography-inductively coupled plasma mass spectrometry. Anal Chem. 2009;81:9440–8. https://doi.org/10.1021/ac901853g.

    Article  CAS  PubMed  Google Scholar 

  18. Campanella B, Bramanti E. Detection of proteins by hyphenated techniques with endogenous metal tags and metal chemical labelling. Analyst. 2014;139:4124–53. https://doi.org/10.1039/C4AN00722K.

    Article  CAS  PubMed  Google Scholar 

  19. Mueller L, Mairinger T, Hermann G, Koellensperger G, Hann S. Characterization of metal-tagged antibodies used in ICP-MS-based immunoassays. Anal Bioanal Chem. 2014;406:163–9. https://doi.org/10.1007/s00216-013-7416-x.

    Article  CAS  PubMed  Google Scholar 

  20. Pérez E, Bierla K, Grindlay G, Szpunar J, Mora J, Lobinski R. Lanthanide polymer labels for multiplexed determination of biomarkers in human serum samples by means of size exclusion chromatography-inductively coupled plasma mass spectrometry. Anal Chim Acta. 2018;1018:7–15. https://doi.org/10.1016/j.aca.2018.02.056.

    Article  CAS  PubMed  Google Scholar 

  21. Benda D, Schwarz G, Beck S, Linscheid MW. Quantification of intact covalently metal labeled proteins using ESI-MS/MS. J Mass Spectrom. 2014;49:13–8. https://doi.org/10.1002/jms.3302.

    Article  CAS  PubMed  Google Scholar 

  22. Quinn TJ. Primary methods of measurement and primary standards. Metrologia. 1997;34:61.

    Article  Google Scholar 

  23. Rodriguez-Gonzalez P, Marchante-Gayon JM, Garcia Alonso JI, Sanz-Medel A. Isotope dilution analysis for elemental speciation: a tutorial review. Spectrochim Acta B At Spectrosc. 2005;60:151–207. https://doi.org/10.1016/j.sab.2005.01.005.

    Article  CAS  Google Scholar 

  24. Yan X, Xu M, Yang L, Qiuquan W. Absolute quantification of intact proteins via 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-trisacetic acid-10-maleimidoethylacetamide- europium labeling and HPLC coupled with species-unspecific isotope dilution ICPMS. Anal Chem. 2010;82:1261–9. https://doi.org/10.1021/ac902163x.

    Article  CAS  PubMed  Google Scholar 

  25. Clases D, Gonzalez de Vega R, Adlard PA, Doble PA. On-line reverse isotope dilution analysis for spatial quantification of elemental labels used in immunohistochemical assisted imaging mass spectrometry via LA-ICP-MS. J Anal At Spectrom. 2019. https://doi.org/10.1039/C8JA00324F.

  26. Kretschy D, Koellensperger G, Hann S. Elemental labelling combined with liquid chromatography inductively coupled plasma mass spectrometry for quantification of biomolecules: a review. Anal Chim Acta. 2012;750:98–110. https://doi.org/10.1016/j.aca.2012.06.040.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Balcaen L, Woods G, Resano M, Vanhaecke F. Accurate determination of S in organic matrices using isotope dilution ICP-MS/MS. J Anal At Spectrom. 2013;28:33–9. https://doi.org/10.1039/C2JA30265A.

    Article  CAS  Google Scholar 

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Acknowledgments

DC is funded by the Deutsche Forschungsgemeinschaft (DFG. German Research Foundation) – 417283954.

DPB is supported by an Australian Research Council Discovery Early Career Researcher Award (DE180100194). PAD is the recipient of an Australian Research Council Discovery Project (DP170100036).

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  1. David Clases and Raquel Gonzalez de Vega contributed equally to this work.

Authors and Affiliations

  1. The Atomic Medicine Initiative, University of Technology Sydney, 15 Broadway, Sydney, NSW, 2007, Australia

    David Clases, Raquel Gonzalez de Vega, David Bishop & Philip Doble

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  1. David Clases
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  2. Raquel Gonzalez de Vega
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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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Correspondence to Philip Doble.

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Clases, D., Gonzalez de Vega, R., Bishop, D. et al. SEC-ICP-MS and on-line isotope dilution analysis for characterisation and quantification of immunochemical assays. Anal Bioanal Chem 411, 3553–3560 (2019). https://doi.org/10.1007/s00216-019-01836-9

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