Bead-based Multiplex Research Articles

S.96. Development of a Robust, Specific and Sensitive Bead-Based Multiplex Assay for Quantification of Mouse Th17 Panel of Cytokines

S.96. Development of a Robust, Specific and Sensitive Bead-Based Multiplex Assay for Quantification of Mouse Th17 Panel of Cytokines

Performance evaluation of cytometric bead assays for the measurement of lung cytokines in two rodent models

There is a growing demand for a cost-effective, efficient, and high-throughput method for measuring cytokines. Currently, many studies are using flow cytometric bead-based multiplex assays in the measurement of cytokines. However, limited data are available regarding the performance of these cytometric bead assays versus enzyme-linked immunosorbent assay (ELISA) or correlation with mRNA expression using real time reverse transcriptase-polymerase chain reaction (RT-PCR). In one of our studies, cytometric bead array (CBA) was used to measure inflammatory cytokine protein levels in bronchoalveolar lavage (BAL) samples from mice exposed to welding fume, an inflammatory particulate. The results were then compared to whole lung mRNA levels of the same cytokines measured by real time RT-PCR in the same mouse model. It was found that the trends in cytokine profiles measured via CBA agreed with the whole lung mRNA results. In a separate experiment, we used a rat zymosan infectivity model to induce a pulmonary immunomodulatory response and determined cytokine concentrations in recovered BAL fluid by ELISA and two different types of cytometric bead-based assays, CBA and FlowCytomix (FC). The sample-to-sample correlation was good between ELISA and CBA with correlation coefficient R values of 0.76, 0.66, and 0.92 for rat IFN-γ, TNF-α, and IL-6, respectively. ELISA only correlated significantly with the FC assay for TNF-α with R = 0.43. Patterns of cytokine response in our rat model also differed among the assays but overall, the ELISA and CBA yielded similar results. For a method-to-method comparison, we assayed supplied cytokine standards from ELISA kits using both ELISA and CBA to determine the R values and found it to be greater than 0.90 for all the cytokines tested. It was found that the ELISA was more sensitive in the low range of the standard curve while the bead assays were capable of detecting higher protein concentrations, which would allow for direct measurement of concentrated samples. There was a lack of agreement between the absolute protein values for the ELISA and flow cytometric bead-based assays; in most cases, the latter method tended to give higher protein concentrations than ELISA. In conclusion, direct comparisons between absolute protein values did not agree among the assays tested in this study, but patterns of cytokine response generally agreed between ELISA and CBA. In the case of the mouse CBA, a companion measurement is recommended if samples with low concentrations of an analyte are reported and extrapolated below sensitivity or zero.

Cytokine expression profiles of patients with acute myelogenous leukemia (AML) and non-Hodgkin lymphoma (NHL) detected by multiplex assay

18530 Background: To determine the cytokine expression profiles of patients with AML and NHL using a sensitive bead-based Luminex multiplex assay in a routine clinical diagnostic setting. Methods: Blood (plasma/serum) samples were collected from ten AML and five NHL patients. Six control samples from patients diagnosed as non-neoplastic/non-autoimmune/non-inflammatory were also analyzed for comparison. All samples were frozen prior to analysis. Using a bead-based Luminex assay (Human Cytokine 8-Plex Assay, Bio- Rad, Hercules, CA) we analyzed these samples for a panel of cytokines (IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, and TNF-alpha). This assay uses polystyrene microspheres, which provides simultaneous quantitation of these cytokines in a single sample. The expression levels were presented in picograms/mL. Average values for each of these markers were obtained for each group of patients (AML versus NHL versus Controls), and their expression levels were compared using χ2 analysis. Results: Overall, there was a significant difference in the expression profiles of all these cytokines among three patients groups (χ2, P < 0.001). All cytokines were consistently expressed at low levels in NHL patients as compared to control group. However, the levels of IL-6 and IL-8 were increased by 2.7 and 5.8 times, respectively in AML patients as compared to controls. Conclusions: The low levels of cytokines in NHL and AML patients suggest suppressed immune system in these two disease conditions; however, these findings warrant further studies to explore the underlying mechanisms for the increased levels of IL-6 and IL-8 in AML patients. Currently, studies are in progress to compare the levels of cytokines measured by Luminiex assay in different stages of leukemias and lymphomas (initial, post treatment and recovery phase etc.). These studies are partially funded by grants from the National Institute of Health/National Cancer Institute (RO1-CA98932–01 and U24-CA086359). No significant financial relationships to disclose.

Platelet‐Rich Plasma: Growth Factors and Pro‐ and Anti‐Inflammatory Properties

Platelet-rich plasma (PRP) promotes regeneration of bone, presumably through the action of concentrated growth factors. However, it is not clear how PRP affects the inflammatory response. The purpose of this study was to analyze the growth factors in PRP and to study the effects of PRP on monocyte cytokine release and lipoxin A(4) (LXA(4)) generation. PRP was prepared from healthy donors. Platelet-derived growth factor (PDGF)-AB, PDGF-BB, transforming growth factor-beta1, insulin-like growth factor-I, fibroblast growth factor-basic (FGF-b), epidermal growth factor (EGF), vascular endothelial growth factor, interleukin-12 (p40/70), and regulated on activation, normal T-cell expressed and secreted (RANTES) levels were evaluated by enzyme-linked immunosorbent assay and bead-based multiplexing. Peripheral blood monocytes were isolated and cultured with or without PRP. Cytokine, chemokine, and LXA(4) levels as well as monocyte chemotactic migration were analyzed. Growth factors were increased significantly in PRP compared to whole blood (WB) and platelet-poor plasma. Monocyte chemotactic protein-1 (MCP-1) was suppressed significantly by PRP, whereas RANTES was increased significantly in monocyte cultures. LXA(4) levels were significantly higher in PRP compared to WB. PRP stimulated monocyte chemotaxis in a dose-dependent fashion, whereas RANTES, in part, was responsible for PRP-mediated monocyte migration. PRP is a rich source of growth factors and promoted significant changes in monocyte-mediated proinflammatory cytokine/chemokine release. LXA(4) was increased in PRP, suggesting that PRP may suppress cytokine release, limit inflammation, and, thereby, promote tissue regeneration.

Mobile phone base station‐emitted radiation does not induce phosphorylation of Hsp27

An in vitro study focusing on the effects of low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields act to induce phosphorylation and overexpression of heat shock protein hsp27. First, we evaluated the responses of human cells to microwave exposure at a specific absorption rate (SAR) of 80 mW/kg, which corresponds to the limit of the average whole-body SAR for general public exposure defined as a basic restriction in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. Second, we investigated whether continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) modulated signal RF fields at 2.1425 GHz induced activation or gene expression of hsp27 and other heat shock proteins (hsps). Human glioblastoma A172 cells were exposed to W-CDMA radiation at SARs of 80 and 800 mW/kg for 2-48 h, and CW radiation at 80 mW/kg for 24 h. Human IMR-90 fibroblasts from fetal lungs were exposed to W-CDMA at 80 and 800 mW/kg for 2 or 28 h, and CW at 80 mW/kg for 28 h. Under the RF field exposure conditions described above, no significant differences in the expression levels of phosphorylated hsp27 at serine 82 (hsp27[pS82]) were observed between the test groups exposed to W-CDMA or CW signal and the sham-exposed negative controls, as evaluated immediately after the exposure periods by bead-based multiplex assays. Moreover, no noticeable differences in the gene expression of hsps were observed between the test groups and the negative controls by DNA Chip analysis. Our results confirm that exposure to low-level RF field up to 800 mW/kg does not induce phosphorylation of hsp27 or expression of hsp gene family.

Comparison of Different Test Systems for Simultaneous Autoantibody Detection in Connective Tissue Diseases

The serological diagnosis of connective tissue diseases (CTDs) is based on the analysis of circulating autoantibodies to cytoplasmic and nuclear proteins (extractable nuclear antigens [ENAs]). The determination of autoantibody specificities supports the clinical diagnosis of the type of CTD and also often the prognosis of the disease. The former indirect immunofluorescence (IIF) technique still provides a useful screening method that currently is supplemented by a range of different techniques allowing the exact determination of single autoantibody specificities. These ENA profiling techniques include ELISA, immunoblotting, line-blot assays, and flow cytometric bead-based multiplex assays. The novel line immunoassay (LIA) from Mikrogen has been introduced in a recent study as a suitable technique for the simultaneous detection of autoantibodies in a routine clinical laboratory, providing comparable results as ELISA and ELiA (both from Pharmacia Diagnostics) (see Damoiseaux et al., this volume). In this study, LIAs from three different manufacturers were performed in 30 serum samples from patients with dermatological manifestations and 27 samples from SLE patients with renal involvement. The line assays from Mikrogen (recomLine ANA/ENA), Innogenetics (Inno-Lia ANA Update), and Imtec (ANA-LIA) were compared for antigen composition, handling, and statistical analysis including sensitivity and concordance. Autoantibody frequencies detected by the Mikrogen, Innogenetics, and Imtec line assays were 14.0%, 19.3%, and 15.8% for RNP; 14.0%, 22.8%, and 14.0% for Sm; 26.3%, 31.6%, and 40.3% for SSA; 3.5%, 12.3%, and 14.0% for SSB; and 3.5%, 14.0%, and 10.5% for histones. Our studies show that the line assay format is an easy-to-use, sensitive, and specific method for ENA antibody detection in human sera.