Comparison of commercial kits to measure cytokine responses to Plasmodium falciparum by multiplex microsphere suspension array technology (original) (raw)
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PLoS ONE, 2013
Background: Cytokines 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 Findings: Different cytokine/chemokine kits, two flow cytometry-based (eBioscienceH FlowCytomix TM and BD TM Cytometric Bead Array Human Enhanced Sensitivity) and four LuminexH-based (Invitrogen TM Human Cytokine 25-Plex Panel, Invitrogen TM Human Cytokine Magnetic 30-Plex Panel, Bio-RadH Bio-Plex Pro TM Human Cytokine Plex Assay and Millipore TM MILLIPLEXH 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. LuminexH kits performed better than flow cytometry kits in number of responses in range and reproducibility. LuminexH 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 LuminexH kits, the Invitrogen TM with polystyrene beads had the poorer performance, whereas Invitrogen TM with magnetic beads had the higher percentage of cytokines/ chemokines with both readings in range (40%), followed by Bio-RadH with magnetic beads (35%). Regarding reproducibility, the Millipore TM kit had the highest percentage (60%) of cytokines/chemokines with acceptable limits of agreement (,30%), followed by the Invitrogen TM with magnetic beads (40%) that had tighter limits of agreement. Conclusions/Significance: Currently available kits for cytokine and chemokine quantification differ in reproducibility and concentration range of accurate detection. LuminexH-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.
BMC clinical pathology, 2018
Measuring expression profiles of inflammatory biomarkers is important in monitoring the polarization of immune responses; therefore, results should be independent of quantitation methods if they are to be accepted as validated clinical pathology biomarkers. To evaluate effects of differing quantitation methods, the seven major circulating Th1/Th2/Th17 cytokines interleukin 2 (IL-2), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), IL-4, IL-6, IL-10 and IL-17A were quantified in plasma of lipopolysaccharide (LPS)-treated mice with two different multiplex platforms. Female C57BL6 mice were treated orally with vehicle or dexamethasone, followed by LPS intravenously. Plasma samples were analyzed 0.5, 1, 2, 4, and 6 h post-LPS challenge with assays at Myriad-RBM and compared to assays performed on a BD Accuri C6 flow cytometer. IL-17A response to LPS was limited but sustained, and the response for the remaining cytokines were early and transient; dexamethasone reduced expression of...
Clinical Chemistry and Laboratory Medicine, 2011
Background: Multiplexed cytokine measurement offers many advantages over the conventional enzyme-linked immunosorbent assay (ELISA) format when applied in largescale epidemiological studies or clinical trials. In the present study we set out to define the reliability and consistency of a suspension multiplexed protein array, the cytometric bead array (CBA), in large-scale, longitudinal studies. Methods: The cytokines interleukin (IL)-5, IL-10, tumor necrosis factor-a (TNF-a) and interferon-g (IFN-g) were measured in pediatric samples from childhood asthma and allergy studies. Analytical performance of CBA was determined in sample supernatants and CBA was compared to conventional ELISA. Results: Within-run and total imprecision were between 5.2%-10.8% and 5.6%-13.2%, respectively, at three different concentrations for all cytokines. Slopes of dilution linearity were between 1.01 and 1.31 for the four cytokines. The recovery rate at two different concentrations of the cytokines was between 97% and 113%. Lower limits of detection and quantification as well as functional sensitivity were determined. Comparison of the multiplex array and solid phase method showed good correlation with r between 0.82 and 0.93. The
Standardization of cytokine flow cytometry assays
BMC immunology, 2005
Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well ...
An Evaluation of Commercial Fluorescent Bead-Based Luminex Cytokine Assays
2008
The recent introduction of fluorescent bead-based technology, allowing the measurement of multiples analytes in a single 25-50 ml sample has revolutionized the study of cytokine responses. However, such multiplex approaches may compromise the ability of these assays to accurately measure actual cytokine levels. This study evaluates the performance of three commercially available multiplex cytokine fluorescent bead-based immunoassays (Bio-Rad's Cytokine 17-plex kit; LINCO Inc's 29-plex kit; and RnD System's Fluorokine-Multi Analyte Profiling (MAP) base kit A and B). The LINCO Inc kit was found to be the most sensitive assay for measuring concentrations of multiple recombinant cytokines in samples that had been spiked with serial dilutions of the standard provided by the manufacturer, followed respectively by the RnD Fluorokine-(MAP) and Bio-Rad 17-plex kits. A positive correlation was found in the levels of IFN-c measured in antigen stimulated whole blood culture supernatants by the LINCO Inc 29-plex, RnD Fluorokine-(MAP) and RnD system IFN-c Quantikine ELISA kits across a panel of controls and stimulated samples. Researchers should take the limitation of such multiplexed assays into account when planning experiments and the most appropriate use for these tests may currently be as screening tools for the selection of promising markers for analysis by more sensitive techniques.
Multisite Comparison of High-Sensitivity Multiplex Cytokine Assays
Clinical and Vaccine Immunology, 2011
immune status, but low concentrations often require high-sensitivity assays to permit detection. The recent development of multiplex assays, which can measure multiple cytokines in one small sample, holds great promise, especially for studies in which limited volumes of stored serum or plasma are available. Four high-sensitivity cytokine multiplex assays on a Luminex (Bio-Rad, BioSource, Linco) or electrochemiluminescence (Meso Scale Discovery) platform were evaluated for their ability to detect circulating concentrations of 13 cytokines, as well as for laboratory and lot variability. Assays were performed in six different laboratories utilizing archived serum from HIV-uninfected and -infected subjects from the Multicenter AIDS Cohort Study (MACS) and the Women's Interagency HIV Study (WIHS) and commercial plasma samples spanning initial HIV viremia. In a majority of serum samples, interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha were detectable with at least three kits, while IL-1 was clearly detected with only one kit. No single multiplex panel detected all cytokines, and there were highly significant differences (P < 0.001) between laboratories and/or lots with all kits. Nevertheless, the kits generally detected similar patterns of cytokine perturbation during primary HIV viremia. This multisite comparison suggests that current multiplex assays vary in their ability to measure serum and/or plasma concentrations of cytokines and may not be sufficiently reproducible for repeated determinations over a long-term study or in multiple laboratories but may be useful for longitudinal studies in which relative, rather than absolute, changes in cytokines are important.
Laboratory Investigation, 2004
The multiple cellular and soluble elements of the immune system respond in a coordinated way, orchestrated by cytokines, to preserve the integrity of the organism. In this study, we describe a new and unique whole blood method that, with minimal sample manipulation, allows an overall evaluation of immune responses by simultaneously measuring cell activation and cytokine secretion. The identification of cells actively secreting cytokines is based on the stabilization of tumor necrosis factor a (TNFa) at the cell surface through the use of a specific inhibitor of the TNFa-converting enzyme. This inhibitor does not affect the release of cytokines other than TNFa and makes it possible to assess, in the same measurement, the phenotype of TNFa þ-secreting cells and quantify multiple secreted cytokines by using a specific and highly sensitive flow cytometry-based bead immunoassay. Upon stimulation of normal peripheral blood samples with either phorbol 12-myristate 13 acetate (PMA) plus ionomycin or lipopolysaccharide (LPS), both the number of TNFa þ cells and the amount of secreted cytokines progressively increased, the former becoming detectable first. After stimulation for 3 h with PMA plus ionomycin, cellular responses were associated with surface TNFa expression on the majority of CD3 þ T cells and secretion of Th1-associated cytokines: interferon c, interleukin (IL)-2, and to a lesser extent IL4. In turn, stimulation with LPS induced a response mainly by inflammatory cells. After 4 h of LPS-stimulation, the majority of CD14 þ monocytes showed surface TNFa expression; in parallel, high amounts of soluble IL1b, IL6, and IL8 became detectable. Likewise, stimulation of blood samples with cytomegalovirus (CMV) lysates induced viralspecific immune responses detectable in seropositive but not seronegative volunteers; such responses were associated with the detection of increased numbers of TNFa þ monocytes, TNFa þ /CD8 þ T cells and TNFa þ /CD8 À T lymphocytes in association with an increased secretion of IFNc, IL6 and TNFa.