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A useful guide for analysis of immune markers by fluorochrome (Luminex) technique
1School of Health Sciences, Department of Natural Science and Biomedicine, The Biomedical Platform, Jönköping University, SE-551 11, Jönköping, Sweden, maria.faresjo@hhj.hj.se.
Insights
The Luminex technique enables simultaneous measurement of multiple cytokines using a bead-based immunoassay. This sensitive method aids in characterizing cytokine profiles from various biological samples, including peripheral blood mononuclear cells (PBMC).
Area of Science:
- Immunology
- Biotechnology
- Analytical Chemistry
Background:
- Characterizing cytokine profiles is crucial for understanding immune responses.
- Traditional methods for cytokine detection can be time-consuming and require large sample volumes.
- The Luminex technique offers a multiplexing solution for simultaneous immune marker analysis.
Purpose of the Study:
- To describe the Luminex technique for multiplex cytokine detection.
- To highlight the importance of pre-analytical factors in immune marker assays.
- To demonstrate the adaptability of the Luminex method for various biomarkers.
Main Methods:
- Utilizes a bead-based sandwich immunoassay combining ELISA principles with flow cytometry.
- Employs microspheres (beads) coated with monoclonal antibodies for specific cytokine capture.
- Analyzes samples using a dual-laser flow analyzer for simultaneous detection of up to 100 analytes.
Main Results:
- The Luminex technique allows for sensitive and simultaneous detection of multiple cytokines.
- It can analyze immune markers in serum, plasma, and cell culture supernatants (e.g., from PBMC).
- Pre-analytical factors like cell separation and cryopreservation significantly impact detection outcomes.
Conclusions:
- The Luminex technique is a versatile and efficient tool for cytokine profiling.
- Careful attention to pre-analytical sample handling is essential for reliable results.
- The method is adaptable for detecting a wide range of biomarkers in biological samples.
Abstract:
The fluorochrome Luminex technique is a bead-based sandwich immunoassay that combines the enzyme-linked immuno sorbent assay (ELISA) with flow cytometry. The Luminex technique allows multiple cytokines to be measured simultaneously in small volumes and provides a convenient and sensitive tool for the detection of a large number of, e.g., extracellular secreted cytokines to characterize cytokine profiles.The technique is based on the so-called microspheres (beads) that serve as a solid phase for molecular detection. These individually dyed micro-beads have monoclonal antibodies directed against the cytokines and chemokines of interest and allow simultaneous detection of up to nearly 100 cytokines and chemokines in a dual-laser flow analyzer. Immune markers 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 multiple cytokines by magnetic bead sandwich immunoassay, with a special focus on some important pre-analytical factors, such as cell separation, cryopreservation, and PBMC thawing that may affect the detection outcome of immune markers. This method can also be easily adapted to measuring other biomarkers in biological samples.
