Skip to main content
SpringerLink
Log in

Immunoassay for Quantitative Detection of Antibody Transcytosis Across the Blood-Brain Barrier In Vitro

  • Protocol
  • First Online:
Induced Pluripotent Stem Cells and Human Disease

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2549))

Abstract

Automated high-throughput immunoassays are emerging as reliable analytic techniques for the quantitative detection of proteins from a variety of sample types. Herein, we describe a method using the Protein Simple Wes capillary-based automated immunoassays platform for the quantification of His- and HA-tagged antibody transcytosis across an in vitro transwell blood-brain barrier (BBB) model. Compared to conventional ELISA, fluorescence, and Mass Spec-based detection approaches, Wes provides comparable datasets with additional information regarding size, aggregation, and potential degradation of samples before and after BBB transcytosis. In this chapter, we have benchmarked our Wes technique against ELISA and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), using known BBB crossing (FC5) and non-crossing (A20.1) single domain antibodies.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Stanimirovic DB, Sandhu JK, Costain WJ (2018) Emerging technologies for delivery of biotherapeutics and gene therapy across the blood-brain barrier. BioDrugs 32(6):547–559. https://doi.org/10.1007/s40259-018-0309-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Bagchi S, Chhibber T, Lahooti B et al (2019) In-vitro blood-brain barrier models for drug screening and permeation studies: an overview. Drug Des Devel Ther 13:3591–3605. https://doi.org/10.2147/DDDT.S218708

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Williams-Medina A, Deblock M, Janigro D (2021) In vitro models of the blood–brain barrier: tools in translational medicine. Front Med Technol 2:623950. https://doi.org/10.3389/fmedt.2020.623950

  4. Helms HC, Abbott NJ, Burek M et al (2016) In vitro models of the blood-brain barrier: an overview of commonly used brain endothelial cell culture models and guidelines for their use. J Cereb Blood Flow Metab 36(5):862–890. https://doi.org/10.1177/0271678X16630991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ribecco-Lutkiewicz M, Sodja C, Haukenfrers J et al (2018) A novel human induced pluripotent stem cell blood-brain barrier model: applicability to study antibody-triggered receptor-mediated transcytosis. Sci Rep 8:1873. https://doi.org/10.1038/s41598-018-19522-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Charlebois C, Huang J, Sodja C et al (2021) Development of a blood-brain barrier permeability assay using human induced pluripotent stem cell derived brain endothelial cells. Methods Mol Biol. https://doi.org/10.1007/7651_2021_393. Epub ahead of print

  7. Ohshima M, Kamei S, Fushimi H et al (2019) Prediction of drug permeability using in vitro blood-brain barrier models with human induced pluripotent stem cell-derived brain microvascular endothelial cells. Biores Open Access 8(1):200–209. https://doi.org/10.1089/biores.2019.0026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Erickson MA, Wilson ML, Banks WA (2020) In vitro modeling of blood–brain barrier and interface functions in neuroimmune communication. Fluids Barriers CNS 17:26. https://doi.org/10.1186/s12987-020-00187-3

    Article  PubMed  PubMed Central  Google Scholar 

  9. Simple Western Size Assay Development Guide Copyright © (2014) ProteinSimple, 3040 Oakmead Village Drive Santa Clara, CA

    Google Scholar 

  10. Ruano-Salguero JS, Lee KH (2020) Antibody transcytosis across brain endothelial-like cells occurs nonspecifically and independent of FcRn. Sci Rep 10:3685. https://doi.org/10.1038/s41598-020-60438-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Muruganandam A, Tanha J, Narang S, Stanimirovic D (2002) Selection of phage-displayed llama single-domain antibodies that transmigrate across human blood-brain barrier endothelium. FASEB J 16(2):240–242. https://doi.org/10.1096/fj.01-0343fje

    Article  CAS  PubMed  Google Scholar 

  12. Sade H, Baumgartner C, Hugenmatter A, Moessner E et al (2014) A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding. PLoS One 9(4):e96340. https://doi.org/10.1371/journal.pone.0096340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Haqqani AS, Caram-Salas N, Ding W et al (2013) Multiplexed evaluation of serum and CSF pharmacokinetics of brain-targeting single-domain antibodies using a NanoLC-SRM-ILIS method. Mol Pharm 10:1542–1556. https://doi.org/10.1021/mp3004995

    Article  CAS  PubMed  Google Scholar 

  14. Kondrat RW, McClusky GA, Cooks RG (1978) Multiple reaction monitoring in mass spectrometry/mass spectrometry for direct analysis of complex mixtures. Anal Chem 50:2017–2021

    Article  CAS  Google Scholar 

  15. Mishra M, Tiwari S, Gomes AV (2017) Protein purification and analysis: next generation Western blotting techniques. Expert Rev Proteomics 14(11):1037–1053. https://doi.org/10.1080/14789450.2017.1388167

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nguyen U, Squaglia N, Boge A et al (2011) The Simple Westernâ„¢: a gel-free, blot-free, hands-free Western blotting reinvention. Nat Methods 8. https://doi.org/10.1038/NMETH.F.353

  17. Chen J, Heldman M, Herrmann M et al (2013) Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system. Anal Biochem 442:97–103. https://doi.org/10.1016/j.ab.2013.07.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhang W, Liu QY, Haqqani AS et al (2020) Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human. Fluids Barriers CNS 17:47. https://doi.org/10.1186/s12987-020-00209-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Farrington GK, Caram-Salas N, Haqqani AS et al (2014) A novel platform for engineering blood-brain barrier-crossing bispecific biologics. FASEB J 28(11):4764–4778. https://doi.org/10.1096/fj.14-253369

    Article  CAS  PubMed  Google Scholar 

  20. Stanimirovic DB, Bani-Yaghoub M, Perkins M et al (2014) Blood-brain barrier models: in vitro to in vivo translation in preclinical development of CNS-targeting biotherapeutics. Expert Opin Drug Discov 10(2):141–155. https://doi.org/10.1517/17460441.2015.974545

    Article  CAS  PubMed  Google Scholar 

  21. Abulrob A, Sprong H, Van Bergen en Henegouwen P, Stanimirovic D (2005) The blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells. J Neurochem 95(4):1201–1214. https://doi.org/10.1111/j.1471-4159.2005.03463.x

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. National Research Council of Canada, Ottawa, ON, Canada

    Caroline Sodja, Deborah Callaghan, Arsalan S. Haqqani, Danica B. Stanimirovic, Willard J. Costain & Anna Jezierski

Authors
  1. Caroline Sodja
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Deborah Callaghan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arsalan S. Haqqani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Danica B. Stanimirovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Willard J. Costain
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anna Jezierski
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anna Jezierski .

Editor information

Editors and Affiliations

  1. Ottawa, ON, Canada

    Kursad Turksen

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sodja, C., Callaghan, D., Haqqani, A.S., Stanimirovic, D.B., Costain, W.J., Jezierski, A. (2022). Immunoassay for Quantitative Detection of Antibody Transcytosis Across the Blood-Brain Barrier In Vitro. In: Turksen, K. (eds) Induced Pluripotent Stem Cells and Human Disease. Methods in Molecular Biology, vol 2549. Humana, New York, NY. https://doi.org/10.1007/7651_2021_456

Download citation

  • DOI: https://doi.org/10.1007/7651_2021_456

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2584-2

  • Online ISBN: 978-1-0716-2585-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation