- Top of page
- MATERIALS AND METHODS
- Author Contributions
- LITERATURE CITED
- Supporting Information
Commercially available assays for the simultaneous detection of multiple inflammatory and cardiac markers in porcine blood samples are currently lacking. Therefore, this study was aimed at developing a bead-based, multiplexed flow cytometric assay to simultaneously detect porcine cytokines [interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha], chemokines (IL-8 and monocyte chemotactic protein 1), growth factors [basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and platelet-derived growth factor-bb], and injury markers (cardiac troponin-I) as well as complement activation markers (C5a and sC5b-9). The method was based on the Luminex xMAP technology, resulting in the assembly of a 6- and 11-plex from the respective individual singleplex situation. The assay was evaluated for dynamic range, sensitivity, cross-reactivity, intra-assay and interassay variance, spike recovery, and correlation between multiplex and commercially available enzyme-linked immunosorbent assay as well as the respective singleplex. The limit of detection ranged from 2.5 to 30,000 pg/ml for all analytes (6- and 11-plex assays), except for soluble C5b-9 with a detection range of 2–10,000 ng/ml (11-plex). Typically, very low cross-reactivity (<3% and <1.4% by 11- and 6-plex, respectively) between analytes was found. Intra-assay variances ranged from 4.9 to 7.4% (6-plex) and 5.3 to 12.9% (11-plex). Interassay variances for cytokines were between 8.1 and 28.8% (6-plex) and 10.1 and 26.4% (11-plex). Correlation coefficients with singleplex assays for 6-plex as well as for 11-plex were high, ranging from 0.988 to 0.997 and 0.913 to 0.999, respectively. In this study, a bead-based porcine 11-plex and 6-plex assay with a good assay sensitivity, broad dynamic range, and low intra-assay variance and cross-reactivity was established. These assays therefore represent a new, useful tool for the analysis of samples generated from experiments with pigs. © 2013 International Society for Advancement of Cytometry
Pigs have been widely used as biomedical research models over the past decades . Based on their comparative anatomic and physiologic characteristics , they represent a suitable model species for investigation of a large number of human diseases and for technical developments in surgery or anesthesia. Animal experiments using pigs have made valuable contributions not only in the field of human medicine, including research of the cardiovascular [3, 4] system, but also in the field of critical and intensive care medicine [5, 6].
For the evaluation of such experiments, analyses of blood samples for the detection of cytokine profiles or organ-specific markers are indispensable tools. Cytokine levels are traditionally measured by enzyme-linked immunosorbent assay (ELISA) allowing analysis of only a single marker at a time. These tests may be rather expensive and time consuming. In particular, the assessment of multiple markers may require a considerable volume of serum or plasma. This may be a limiting factor in which minimal sample volume is available as is the case from microdialysis studies or long experiments that require serial testing of multiple parameters. Besides, cytokines themselves may alter each other's function and regulate the production of other cytokines , rendering cytokine profiles or ratios more valuable than single cytokine measurements . For these reasons, multiplex flow cytometry by the xMAP Technology has rapidly established itself [9, 10].
Multiplexed bead-based immunoassays for quantitation of human cytokines have been described in the past years [11-13], and several multiplexed cytokine detection assays are now commercially available to detect human, mouse, and rat cytokines based on xMAP technology. Multiplex kits for porcine cytokines are commercially available. However, no such kits for porcine markers combining cytokines, complement activation markers, and growth factors/angiogenesis factors are currently available. A major limitation for the development and use of porcine multiplex assays has been the lack of specific antibody pairs. Only relatively few reagents are currently available commercially for the detection of selected markers. In addition, microsphere-based multiplex assays for the detection of porcine cytokines have been recently reported [14-16].
In this article, we describe the development of a novel assay to detect porcine proinflammatory cytokines [interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein (MCP)-1], the anti-inflammatory cytokine IL-10, growth factors [basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor-bb (PDGF-bb)], complement activation markers [C5a, soluble (s)C5b-9], and a cardiac marker [cardiac troponin (cTn)-I]. We have developed an 11-plex assay including all parameters besides cTn-I (IL-1β, IL-6, IL-8, IL-10, TNF-α, C5a, sC5b-9, MCP-1, bFGF, VEGF, and PDGF-bb) and a 6-plex assay with a subset of the markers of the 11-plex assay plus cTn-I (IL-1β, IL-6, IL-8, IL-10, TNF-α, and cTn-I). As a multiplex for cytokine detection, this assay may serve as a tool to determine the “inflammation fingerprint” in a given pig experiment, and as a 6- or 11-plex may be particularly useful in, for example, the context of studying cardiac, limb, or general ischemia/reperfusion injury or models of shock.
- Top of page
- MATERIALS AND METHODS
- Author Contributions
- LITERATURE CITED
- Supporting Information
In this study, an 11-plex assay for the simultaneous detection of porcine IL-1β, IL-6, IL-8, IL-10, TNF-α, C5a, sC5b-9, MCP-1, bFGF, VEGF, and PDGF-bb as well as a 6-plex assay including five cytokines and cTn-I was set up following initial optimization of all individual parameters in a singleplex setting.
Although various studies have described the setup of multiplex assays for the detection of human neutralizing monoclonal antibodies  or human cytokines [13, 24, 25], only few studies have been currently published describing multiplex detection of porcine cytokines [14-16]. The potential advantage of the current assay, apart from including a larger number of cytokines, is the addition of complement activation markers and growth factors as well as tissue-specific injury markers (cTn-I). The multiplex assay may be used in more general porcine models of tissue injury or, with the addition of cTn-I, in particular for the investigation of cardiac injury, for example, in myocardial infarction. However, the assay sensitivity for the complement markers (C5a and sC5b-9) as well as growth factors (VEGF and PDGF-bb) was compromised when combined with cTn-I. In addition, with the increase of background values, the LLOD of these markers was high in the presence of cTn-I antibodies. Thus, two multiplex panels were developed as an 11-plex and an overlapping 6-plex with the addition of cTn-I.
Sensitivity of the assay (for cytokines) in terms of the lowest level of detection was comparable with commercial ELISAs. In addition, the dynamic detection range for all analytes was significantly broader in our bead-based assays. For cytokines, the range was higher than in commercial ELISAs, eliminating the need for multiple dilutions of high-concentration samples. Moreover, the simultaneous detection of analytes insured an internal consistency.
Standard cross-reactivity tests showed some recognition of recombinant PDGF-bb protein by all beads in the multiplex setting. As a certain amount of cross-reactivity was observed with all beads, this phenomenon is most likely due to nonspecific binding of MCP-1, bFGF, VEGF, and PDGF-bb to the beads than due to recognition of the protein by other capture antibodies. This may also explain the minimal cross-reactivity found in reporter cross-reactivity tests when analyzing all beads with detection antibodies. As far as intra-assay and interassay variance is concerned, the presented multiplex assay showed values comparable with and within an acceptable range when compared with those observed by others [13, 26].
Although recovery values for spiked plasma samples varied to a certain extent, the percentage of recovery was stable within each tested parameter. By prediluting the EDTA plasma in standard/sample diluent, the percent recovery of each parameter following spiking was dose dependently increased to a maximum. This indicates that the low recovery values were due to absorption of the respective recombinant protein following spiking into the plasma samples. Furthermore, there was a good correlation between the results obtained in singleplex versus 6-plex or singleplex versus 11-plex. Additionally, cytokine concentrations measured by 6-plex and 11-plex assays showed correlation coefficients (r2), 0.9720 for IL-8, 0.9736 for TNF-α, 0.9873 for IL-6, 0.9904 for IL-1β, and 0.9995 for IL-10, indicating for a good sensitivity of both assays. In contrast, recovery percentages in diluent-only spiked samples were more consistent and generally higher, most likely due to significantly reduced matrix effects. Taken together, interassay variance and spike recovery values of some analytes showed deviations from expected values; however, relative concentrations of each parameter were consistent.
Cytokine concentrations in the multiplex assay correlated well with those measured using commercial ELISAs. Although in certain longitudinal studies, such as in the illustrated model, relative cytokine values may be adequate to evaluate the follow-up, some experimental setups require exact values. In general, however, absolute concentrations of antigens as measured by ELISA or similar technique have to be regarded with caution as values may differ significantly among commercially available kits . In multiplex analysis, at least the issue of repeated testing (on different plates and different days) may be reduced as up to 80 samples, and in the current assay, up to 11 parameters can be measured simultaneously on one single plate.
The established 6-plex and 11-plex assays were validated using samples from in vivo porcine experiments in CPB (data not shown) and ECC models, respectively . The increase in IL-1β, IL-6, IL-8, TNF-α, and cTn-I on reperfusion post-CPB fit in with data from other authors. The course of IL-10 post-CPB has, in part, been shown to be biphasic, with an initial drop in early reperfusion . However, an early increase has been described by others . In the limb perfusion model with ECC, plasma samples were measured using 11-plex. IL-1β, IL-6, IL-8, MCP-1, C5a, and sC5b-9 as well as VEGF levels were increased significantly from baseline during ECC, and all markers were again back to normal at the end of the replantation period. Endothelial activation due to I/R injury induces inflammatory reactions, for example, shedding of endothelial glycocalyx, activation of complement and coagulation cascades, and increased cytokine release, causing endothelial dysfunction [31-34]. In this study, we observed that prolonged preservation of amputated pig limbs using ECC has no effects on I/R-induced injury as the results fit in with the expected course of inflammatory markers within this experimental setting .
In conclusion, we were able to establish a bead-based multiplex assay to detect porcine cytokines (IL-1β, IL-6, IL-10, and TNF-α), chemokines (IL-8 and MCP-1), growth factors (bFGF, VEGF, and PDGF-bb), injury markers (cTn-I), and complement activation markers (C5a and sC5b-9) in porcine blood samples with a good assay sensitivity, broad dynamic range, and low intra-assay variance. The simultaneous detection of analytes allows for an internal consistency and greatly increases the amount of information obtained from a single volume-limited sample. This method should prove a valuable tool in the analysis of porcine samples obtained from experimental setups using pig as a model animal to study various states of disease, including cardiovascular disease and shock.