Multiplex Cytokine Analysis
For multiplex cytokine analysis, different technologies can be proposed by Charles River, such as electrochemiluminescence or the Luminex xMAP technology. The latter utilizes fluorescently labelled magnetic microspheres conjugated to specific-capture antibodies to detect up to 100 different analytes simultaneously.
There are numerous applications for multiplex cytokine measurements. It allows evaluating the complexity and dynamic nature of inflammatory responses in humans during clinical trials, can be used to support the mechanism of action of immune modulatory compounds, or to investigate the effect of a therapy on the immune response in humans, after immune cells restimulation ex vivo to monitor changes that may not be apparent using direct measurement. For instance, specific antigens can be selected to measure different arms of the immune response with LPS commonly used to look at innate responses and SEB or anti-CD3/anti-CD28 to monitor adaptive responses. Blood samples are typically obtained prior to and following in vivo dosing with a test therapeutic.
- Before analyzing clinical samples using multiplex immunoassays, pre-analytical sample handling and the multiplex immunoassay must be validated.
- A fit-for-purpose validation plan is designed based on the trial logistics and the intended purpose of the data. The aim is to understand and minimize sources of variability to ensure that a robust, precise, and reliable data set is produced.
- Samples from a single patient will be preferably batched to run on a single multiplex cytokine assay plate to minimize the impact of any inter-assay variation on cytokine concentrations. In order to achieve this, samples may be stored for a period of time before analysis, and sample stability can be assessed as part of the validation package at a timeframe appropriate to the trial.
- Once the validation of the multiplex cytokine assay is complete, clinical samples can be assessed for cytokine concentrations. Pre- and post-dosing concentrations of cytokines can be compared providing information around the effect of the therapy on the immune system and providing valuable information to support the decision-making process to progress to the next phase in the clinical development pipeline.
Validation of Multiplex Cytokine Assays
Cytokine Data from Patients in a Phase I Clinical Trial Pre-Dosing by Multiplex Cytokine Assay
Measurement of cytokine biomarkers by multiplex analysis can provide valuable information for a multitude of diseases, including autoimmune, autoinflammatory, immuno-oncology, and cardiovascular, where systemic changes have been documented.
The multiplex cytokine assay data can be complemented by flow cytometry-based analysis of T cells and B cells to further investigate immune responses. Intracellular cytokine staining can provide information on which cell types the therapeutic is modulating. Molecular profiling by NanoString technology or qPCR can provide further measurement of target engagement and the impact on downstream pathways.
Frequently Asked Questions (FAQs) about Multiplex Cytokine Assays
What is multiplex cytokine analysis?
Multiplex cytokine assay technology allows the measurement of numerous analytes in a single sample, increasing speed and throughput in comparison to traditional ELISA techniques. Luminex multiplex immunoassays utilize fluorescently labelled magnetic microspheres conjugated to specific-capture antibodies to detect up to 100 different analytes simultaneously. This technology is routinely applied to the quantitation of inflammatory cytokines in biological samples.
While the simultaneous measurement of a large number of analytes is possible via a multiplex cytokine assay, we would typically recommend the selection of a more focused cytokine panel of 6-10 markers relevant to the disease or antigen of interest to increase the chances that the majority of these are within the measurable range.
What sample types can be analyzed via Luminex multiplex immunoassay?
The starting material for Luminex analysis is typically liquid biological samples, including serum/plasma, supernatants of cultured cells, or activated whole blood.
How sensitive are multiplex cytokine assays?
For multiplex cytokine assays, the lower limit of quantitation of the standard curve for many cytokines is ~1pg/ml. To investigate the effect of a therapy on the immune response in humans, immune cells present in whole blood can be stimulated ex vivo to monitor changes that may not be apparent using direct measurement of serum/plasma. Specific antigens can be selected to measure different arms of the immune response with LPS commonly used to look at innate responses and SEB or anti-CD3/anti-CD28 to monitor adaptive responses.