Abstract
Immune cells and their mediators are key players in human cancer progression involving alternation in the number and function of immune cells, both peripheral and at the site of tumor. Through reliable predictive biomarkers, cancer can be predicted, and progression and response to therapy followed. Thereby immune biomarkers, e.g., cytokines and chemokines can serve as intermediate mediators of cancer diagnostics. Multiplex analysis of immune mediators in small blood volumes allows for rapid quantification of large number of circulating analytes. The fluorochrome (Luminex) technique is a bead-based sandwich immunoassay that combines the enzyme-linked immunosorbent assay (ELISA) with flow cytometry. The Luminex technique allows multiple immune mediators to be measured simultaneously in small volumes, and provides a convenient and sensitive tool for the detection of a large number of extracellular secreted cytokines and chemokines to be used in prediction and therapy prognosis of cancer.
The technique is based on so-called microspheres (beads) that serve as a solid phase for molecular detection. These individually dyed microbeads have monoclonal antibodies directed against the cyto- and chemokines of interest and allow a simultaneous detection of up to nearly 100 cyto- and chemokines in a dual-laser flow analyzer. Immune mediators can be detected in serum and plasma samples as well as in cell culture supernatants from in vitro stimulated peripheral blood mononuclear cells (PBMC). This chapter describes the Luminex technique for detection of immune mediators in cancer by using magnetic bead sandwich immunoassay, with focus on some important pre-analytic factors, e.g., cell separation and cryopreservation and thawing of PBMC that may affect the outcome of detection of immune mediators. The Luminex technique thus represents a very suitable method to identify immune mediators in cancer tissues in order to diagnose and improve clinical outcome of cancer.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
De Jager W, te Velthuis H, Prakken BJ et al (2003) Simultaneous detection of 15 human cytokines in a single sample of stimulated peripheral blood mononuclear cells. Clin Diagn Lab Immunol 10:133–139
Lagrelius M, Jones P, Franck K et al (2006) Cytokine detection by multiplex technology useful for assessing antigen specific cytokine profiles and kinetics in whole blood cultured up to seven days. Cytokine 33:156–165
Tompa A, Nilsson-Bowers A, Faresjö M (2018) Subsets of CD4+, CD8+, and CD25hi lymphocytes are in general not influenced by isolation and long-term cryopreservation. J Immunol 201:1799–1809
Bull M, Lee D, Stucky J et al (2007) Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J Immunol Methods 322:57–69
Axelsson S, Faresjö M, Hedman M et al (2008) Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children. Cryobiology 57:201–208
Kivling A, Nilsson L, Faresjö M (2009) How and when to pick up the best signal from markers associated with T-regulatory cells? J Immunol Methods 345:29–39
Rydén A, Faresjö M (2011) Efficient expansion of cryopreserved CD4+CD25+CD127lo/− cells in type 1 diabetes. Res Immunol 1:36–44
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Faresjö, M. (2020). A Useful Guide for Analysis of Immune Mediators in Cancer by Fluorochrome (Luminex) Technique. In: Vancurova, I., Zhu, Y. (eds) Immune Mediators in Cancer. Methods in Molecular Biology, vol 2108. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0247-8_1
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0247-8_1
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0246-1
Online ISBN: 978-1-0716-0247-8
eBook Packages: Springer Protocols