Abstract
BackgroundCytokines and chemokines are relevant biomarkers of pathology and immunity to infectious diseases such as malaria. Several commercially available kits based on quantitative suspension array technologies allow the profiling of multiple cytokines and chemokines in small volumes of sample. However, kits are being continuously improved and information on their performance is lacking.Methodology/Principal FindingsDifferent cytokine/chemokine kits, two flow cytometry-based (eBioscience® FlowCytomix™ and BD™ Cytometric Bead Array Human Enhanced Sensitivity) and four Luminex®-based (Invitrogen™ Human Cytokine 25-Plex Panel, Invitrogen™ Human Cytokine Magnetic 30-Plex Panel, Bio-Rad® Bio-Plex Pro™ Human Cytokine Plex Assay and Millipore™ MILLIPLEX® MAP Plex Kit) were compared. Samples tested were supernatants of peripheral blood mononuclear cells of malaria-exposed children stimulated with Plasmodium falciparum parasite lysates. Number of responses in range that could be detected was determined and reproducibility of duplicates was evaluated by the Bland-Altman test. Luminex® kits performed better than flow cytometry kits in number of responses in range and reproducibility. Luminex® kits were more reproducible when magnetic beads were used. However, within each methodology overall performance depended on the analyte tested in each kit. Within the Luminex® kits, the Invitrogen™ with polystyrene beads had the poorer performance, whereas Invitrogen™ with magnetic beads had the higher percentage of cytokines/chemokines with both readings in range (40%), followed by Bio-Rad® with magnetic beads (35%). Regarding reproducibility, the Millipore™ kit had the highest percentage (60%) of cytokines/chemokines with acceptable limits of agreement (<30%), followed by the Invitrogen™ with magnetic beads (40%) that had tighter limits of agreement.Conclusions/SignificanceCurrently available kits for cytokine and chemokine quantification differ in reproducibility and concentration range of accurate detection. Luminex®-based kits with magnetic beads perform the best. Data highlights the importance of testing different kits before each study to choose the most appropriate, depending on the priority of the cytokines assessed.
Highlights
Complex infectious diseases such as malaria induce intricate immune responses that may result in pathogenesis or protection
Multiple cytokines and chemokines are involved in pathological and immunological processes [1], reflecting the different capacities and functions of immune cells such as immunoregulation, proliferation, activation or cytotoxicity. Immune assays such as ELIspot, intracellular cytokine staining and flow cytometry or ELISA are limited in the number of variables able to measure at the same time in a single assay, whereas quantitative suspension array technology allows the simultaneous measurement of different cytokines/chemokines
The aim of this study is to compare the performance of six human cytokine/chemokine multiplex kits currently available using culture supernatants from peripheral blood mononuclear cells (PBMC) stimulated with malaria parasite lysates
Summary
Complex infectious diseases such as malaria induce intricate immune responses that may result in pathogenesis or protection. Multiple cytokines and chemokines are involved in pathological and immunological processes [1], reflecting the different capacities and functions of immune cells such as immunoregulation, proliferation, activation or cytotoxicity Immune assays such as ELIspot, intracellular cytokine staining and flow cytometry or ELISA are limited in the number of variables able to measure at the same time in a single assay, whereas quantitative suspension array technology allows the simultaneous measurement of different cytokines/chemokines. This technology is based on a capturedetection sandwich type assay using fluorescent microspheres, and only requires small volumes of samples. Kits are being continuously improved and information on their performance is lacking
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