Related Experiment Video
Updated: May 11, 2026
Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels
Published on: August 7, 2017
Optimising the quantification of cytokines present at low concentrations in small human mucosal tissue samples using
Emily Staples1, Richard James Michael Ingram, John Christopher Atherton
1Nottingham Digestive Diseases Biomedical Research Unit, Queen's Medical Centre, Nottingham, NG7 2UH, UK.
Insights
Sensitive measurement of cytokines in gastric biopsies is challenging. Luminex assays, particularly the MILLIPLEX kit with optimized manual disruption, offer a viable solution for detecting interleukin-17A (IL-17) and interferon-gamma (IFNγ) in small tissue samples.
Area of Science:
- Biotechnology
- Immunology
- Gastroenterology
Background:
- Measuring multiple cytokine profiles from small mucosal biopsies, like gastric samples, presents significant technical challenges.
- Multiplex assays, such as Luminex, are promising but lack standardized protocols for tissue applications.
- Accurate quantification of cytokines like IL-17 and IFNγ is crucial for understanding mucosal immunity and disease.
Purpose of the Study:
- To evaluate the utility of three commercial Luminex kits (VersaMAP, Bio-Plex, MILLIPLEX) for measuring IL-17 and IFNγ in human gastric biopsies.
- To optimize sample preparation methods for enhanced cytokine recovery from mucosal tissues.
- To establish a reliable protocol for sensitive cytokine detection in small tissue biopsies.
Main Methods:
- Assessed technical performance, sensitivity, and dynamic ranges of three Luminex kits.
- Spiked gastric biopsies with known cytokine concentrations to evaluate kit accuracy and precision.
- Compared manual versus automated tissue disruption and various extraction buffers.
- Validated the optimized protocol using Luminex and real-time PCR on inflamed and uninflamed gastric mucosa.
Main Results:
- The MILLIPLEX kit demonstrated the most consistent sensitivity, especially for low cytokine concentrations.
- Manual tissue disruption significantly improved cytokine recovery compared to automated methods.
- The MILLIPLEX kit showed consistent performance across the widest range of spiked cytokine concentrations.
- Optimized protocol successfully measured relative cytokine levels in gastric mucosa samples.
Conclusions:
- Luminex kits, particularly MILLIPLEX, are suitable for sensitive cytokine detection in mucosal biopsies with proper optimization.
- Optimized manual tissue processing enhances cytokine recovery from small tissue samples.
- This study provides a validated method to aid researchers in quantifying low-concentration cytokines in limited tissue volumes.
Abstract:
Sensitive measurement of multiple cytokine profiles from small mucosal tissue biopsies, for example human gastric biopsies obtained through an endoscope, is technically challenging. Multiplex methods such as Luminex assays offer an attractive solution but standard protocols are not available for tissue samples. We assessed the utility of three commercial Luminex kits (VersaMAP, Bio-Plex and MILLIPLEX) to measure interleukin-17A (IL-17) and interferon-gamma (IFNγ) concentrations in human gastric biopsies and we optimised preparation of mucosal samples for this application. First, we assessed the technical performance, limits of sensitivity and linear dynamic ranges for each kit. Next we spiked human gastric biopsies with recombinant IL-17 and IFNγ at a range of concentrations (1.5 to 1000 pg/mL) and assessed kit accuracy for spiked cytokine recovery and intra-assay precision. We also evaluated the impact of different tissue processing methods and extraction buffers on our results. Finally we assessed recovery of endogenous cytokines in unspiked samples. In terms of sensitivity, all of the kits performed well within the manufacturers' recommended standard curve ranges but the MILLIPLEX kit provided most consistent sensitivity for low cytokine concentrations. In the spiking experiments, the MILLIPLEX kit performed most consistently over the widest range of concentrations. For tissue processing, manual disruption provided significantly improved cytokine recovery over automated methods. Our selected kit and optimised protocol were further validated by measurement of relative cytokine levels in inflamed and uninflamed gastric mucosa using Luminex and real-time polymerase chain reaction. In summary, with proper optimisation Luminex kits (and for IL-17 and IFNγ the MILLIPLEX kit in particular) can be used for the sensitive detection of cytokines in mucosal biopsies. Our results should help other researchers seeking to quantify multiple low concentration cytokines in small tissue samples.
