Cytokine detection

Cytokine Detection Service – Accredited Multiplex, ELISA and Flow Cytometry Analysis for Global Markets

Our internationally accredited laboratory delivers a comprehensive cytokine detection service that empowers pharmaceutical developers, clinical research organisations, biopharmaceutical manufacturers, academic immunology laboratories and diagnostic kit producers worldwide to accurately quantify secreted, intracellular and membrane‑bound cytokines in a wide variety of biological matrices. Every measurement is performed within the rigorous framework of ISO/IEC 17025, and each report bearing the ILAC mark is unconditionally accepted by regulatory authorities, notified bodies and supply‑chain partners in all major economies. The cytokine detection service employs a suite of validated analytical platforms – including high‑sensitivity multiplex bead‑based immunoassays, classical enzyme‑linked immunosorbent assays, electrochemiluminescence‑based single‑analyte and multi‑array systems, and multiparameter intracellular flow cytometry – to provide the concentration, the secretion kinetics and the cellular origin of the key immune mediators. For a sponsor monitoring the inflammatory response in a phase‑II oncology trial, a cell‑therapy developer characterising the cytokine release syndrome potential of a CAR‑T product, or a vaccine manufacturer quantifying the T‑helper‑1/T‑helper‑2 balance as a potency marker, this service delivers the legally robust, defensible cytokine data that underpin the safety and the efficacy evaluation, the biomarker discovery and the regulatory submission.

Cytokine detection

Product Samples We Regularly Subject to Cytokine Detection

The sample‑receipt, processing, aliquoting and long‑term biobanking facilities in our laboratory accommodate a vast variety of biological and experimental matrices. The following categories represent the most frequently tested items:

Human and animal serum and plasma – the peripheral blood samples collected into the EDTA, the heparin, the citrate or the serum‑separator tubes from the preclinical toxicology studies, the clinical‑trial cohorts and the epidemiological surveys
Whole blood and the stimulated‑blood supernatants – the lithium‑heparin whole blood that has been incubated with the mitogens, the antigens or the drug candidates, and the harvested plasma supernatant for the assessment of the functional immune competence and the cytokine‑release potential
Cell‑culture and the bioreactor supernatants – the conditioned media from the T‑cell, the natural‑killer‑cell, the macrophage, the dendritic‑cell and the stem‑cell cultures, the organoid co‑cultures, the hollow‑fibre bioreactors and the CAR‑T production runs
Cerebrospinal fluid, synovial fluid, bronchoalveolar lavage and other compartmental fluids – the samples from the special populations or the localised disease indications where the cytokine profile in the target compartment provides the diagnostic or the pharmacodynamic biomarker information
Tissue homogenates and the biopsy extracts – the snap‑frozen and the formalin‑fixed tumour biopsies, the lymph‑node aspirates, the skin‑punch biopsies and the intestinal‑mucosal extracts, processed with the protein‑extraction buffers that are compatible with the immunoassay detection
Intracellular and the permeabilised cell preparations – the peripheral‑blood mononuclear cells, the splenocytes, the bone‑marrow aspirates and the disaggregated tissue preparations that have been stimulated with the phorbol‑myristate‑acetate/ionomycin, the antigenic peptides or the target cells, and then fixed and permeabilised for the intracellular cytokine staining
Urine, saliva, tears and other dilute biofluids – the samples that require the highly sensitive detection platforms, such as the single‑molecule‑counting or the electrochemiluminescence assays, to quantify the low‑abundance cytokines that are present at the sub‑picogram per millilitre concentrations

Multiplex Bead‑Based Immunoassay – High‑Throughput Cytokine Detection According to the Luminex xMAP and the Comparable Platforms

Simultaneous quantification of up to fifty cytokines, chemokines and growth factors in a single small‑volume sample by the magnetic‑bead‑based multiplex immunoassay according to the internal validated protocols and the principles of the Luminex xMAP technology: the sample is incubated with a mixture of the capture‑antibody‑conjugated, fluorescently‑encoded magnetic beads, and the captured analytes are detected with a biotinylated detection‑antibody cocktail and a streptavidin‑phycoerythrin reporter. The beads are analysed on a dual‑laser flow‑based instrument, and the concentration of each analyte in the picograms per millilitre is interpolated from a five‑parameter logistic standard curve. The cytokine detection service using the multiplex platform provides the comprehensive immune‑response profiling from a minimal sample volume, and it is the method of choice for the clinical‑trial biomarker studies, the vaccine‑immunogenicity screening and the systems‑immunology research.
Custom‑configured and the pre‑defined multiplex panels for the targeted cytokine detection: the customer selects the specific cytokines that are relevant to the biological hypothesis – for example, a Th1/Th2/Th17 panel, a pro‑inflammatory/anti‑inflammatory panel, a chemokine panel, a tumour‑microenvironment panel or a neuroinflammation panel – and the assay is configured to measure only those analytes, optimising the sensitivity and the dynamic range for the analytes of interest and reducing the cost per data point.
Determination of the lower limit of quantification, the cross‑reactivity and the spike‑recovery in the sample matrix: the multiplex panel is validated for each sample matrix, and the analytical performance parameters are reported, ensuring that the measured concentrations are accurate and that the matrix components do not cause the false‑positive or the false‑negative results. The spike‑recovery of the known amounts of the recombinant cytokines into the sample matrix is within 70 % to 130 % of the expected value, complying with the FDA and the EMA bioanalytical‑method‑validation guidelines.
Longitudinal and the dose‑response cytokine profiling: the samples from the multiple time‑points and the escalating‑dose cohorts are analysed in the same analytical run, and the temporal and the dose‑dependent changes in the cytokine network are visualised by the heat‑maps, the principal‑component analysis and the hierarchical clustering, providing the integrated, systems‑level data that the translational‑medicine team uses to identify the pharmacodynamic biomarkers and to guide the dosing‑regimen selection.

Enzyme‑Linked Immunosorbent Assay and Electrochemiluminescence Detection – Single‑Analyte and Ultra‑Sensitive Cytokine Detection

Quantitative determination of a specific cytokine by the classical sandwich ELISA according to the internal validated protocol and the principles of the ICH Q2(R1) guideline: the capture antibody is immobilised on a high‑binding microtitre plate, and the sample is incubated, followed by the detection antibody and the enzyme‑conjugated reporter. The colourimetric or the fluorescent signal is developed, and the cytokine concentration is interpolated from a standard curve that is run on the same plate. This cytokine detection service is used for the routine, high‑throughput measurement of the single cytokine when the full multiplex panel is not required, or as the orthogonal method to confirm the multiplex results.
Ultra‑sensitive electrochemiluminescence‑based single‑analyte and multi‑array detection according to the MSD‑ECL or the comparable platform: the capture antibody is coated on a carbon‑electrode microplate, and the detection antibody is labelled with a ruthenium chelate. The electrochemiluminescence signal is generated upon the electrical stimulation, and the concentration is measured with a sensitivity that is typically ten‑ to one‑hundred‑fold greater than the conventional ELISA. The assay is particularly suited to the detection of the low‑abundance cytokines in the cerebrospinal fluid, the tears and the dilute biofluids, and to the quantification of the phosphorylated signalling proteins in the cell‑lysate samples.
Single‑molecule‑counting and the digital ELISA for the sub‑femtogram‑per‑millilitre cytokine detection: the sample is incubated with the capture beads and the detection antibody that is labelled with a fluorescent or an enzymatic reporter, and the individual beads are loaded into an array of the femtolitre‑sized wells that are designed to trap a single bead per well. The wells that contain a fluorescent product are counted, and the cytokine concentration is calculated from the fraction of the active wells relative to the Poisson‑distribution model. The method achieves the limit of detection that is sufficient to measure the basal levels of the circulating cytokines in the healthy individuals, enabling the true quantification of the normal reference ranges and the subtle, treatment‑induced changes that are below the sensitivity of the conventional assays.
Neutralizing‑antibody and the drug‑interference assessment in the cytokine detection assays: for the samples that contain the therapeutic monoclonal antibodies or the soluble receptors that can interfere with the cytokine measurement, a pre‑treatment with the acid‑dissociation or the heterophilic‑blocking reagents is performed, and the recovery of the spiked cytokine is verified, ensuring that the reported cytokine concentration reflects the true biological state and not the analytical artefact.

Flow Cytometry and Intracellular Cytokine Staining – Cytokine Detection at the Single‑Cell Level

Identification and the quantification of the cytokine‑producing cell subsets by the multiparameter intracellular cytokine staining according to the internal validated protocol and the best‑practice guidelines of the Human Immunology Project Consortium: the cells are stimulated with the phorbol‑myristate‑acetate and the ionomycin, or with the antigen‑specific peptides, in the presence of the protein‑transport inhibitors, fixed, permeabilised and stained with the fluorochrome‑conjugated antibodies against the surface markers and the intracellular cytokines. The sample is acquired on a multi‑laser flow cytometer, and the percentage of the cytokine‑positive cells and the median fluorescence intensity within each gated population are reported. This cytokine detection service provides the direct, single‑cell resolution data that distinguish the polyfunctional T‑cells that simultaneously produce the multiple cytokines from the monofunctional cells, and it is the essential assay for the characterisation of the vaccine‑induced cellular immunity and the adoptive cell‑therapy products.
Activation‑induced marker assay and the cytokine‑secretion assay for the live‑cell sorting: the cells are stimulated, and the surface‑expressed activation markers or the captured secreted cytokines are stained without the fixation and the permeabilisation, allowing the viable, antigen‑specific cytokine‑producing cells to be sorted by the fluorescence‑activated cell sorting for the downstream transcriptomic, the proteomic or the functional analyses.
Mass cytometry and the spectral flow cytometry for the high‑dimensional cytokine detection: the cells are stained with the metal‑conjugated or the polymer‑dye‑conjugated antibodies, and the single‑cell cytokine and the transcription‑factor profiles are acquired with the resolution of more than forty parameters, providing the comprehensive immune‑phenotyping data that are analysed by the t‑distributed stochastic neighbour embedding, the FlowSOM clustering and the other machine‑learning algorithms.
Whole‑blood intracellular cytokine staining and the functional‑immune‑competence monitoring: the unfractionated whole blood is stimulated, and the cytokine production by the monocytes, the natural‑killer cells and the T‑cells is measured, providing the simple, rapid assessment of the global immune function that is used for the monitoring of the immunosuppressed transplant recipients, the sepsis patients and the critically ill subjects.

Report Acceptance and Global Regulatory Compliance

All measurements performed within our cytokine detection service are executed under the fully accredited scope of our ISO/IEC 17025 quality management system. Each test report that carries the ILAC mark is therefore automatically recognised by regulatory authorities, notified bodies, customs offices and supply‑chain partners in all major economies. For pharmaceutical developers, clinical‑research organisations, cell‑therapy manufacturers and academic immunology laboratories anywhere in the world, the report constitutes legally robust, internationally accepted evidence that the cytokine concentrations, the secretion kinetics, the cellular source and the multi‑analyte profiles have been determined in accordance with the applicable ICH, FDA, EMA and customer‑specified methods. The documentation can be directly used to support the investigational‑new‑drug application, the biologics‑license application, the biomarker‑qualification dossier, the issue of inspection certificates according to EN 10204 or equivalent national standards, and the resolution of commercial and technical disputes concerning the immune‑monitoring data and the cytokine‑release profiles of any therapeutic or preventive intervention.