The ability to simultaneously quantify multiple signaling molecule protein levels from microscopic neural tissue samples would be of great benefit to deciphering how they affect brain function. This follows from evidence that indicates signaling molecules can be pleiotropic and can have complex interactive behavior that is regionally and cellularly heterogeneous. Multiplexed examination of tissue proteins has been exceedingly difficult because of the absence of available techniques. This void now has been removed by the commercial availability of bead-based immunoassays for targeted proteins that allow analyses of up to 100 (6-150 kDa) proteins from as little as 12 μl. Thus far used only for sera (human and mouse) and culture media, we demonstrate here that sensitive (as low as 2 pg/ml), wide-ranging (up to 2-32 000 pg/ml), accurate (8% intra-assay covariance) and reliable (4-7% inter-assay covariance) measurements can be made of nine exemplary cytokines (e.g., IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF-α) simultaneously not only from rat serum but, for the first time, also brain tissue. Furthermore, we describe animal handling procedures that minimize stress as determined by serum glucocorticoid levels since they can influence cytokine expression.
This prospective study used antibody suspension bead arrays to identify biomarkers capable of predicting post-operative recurrence with distal metastasis in patients with colorectal cancer. One hundred colorectal cancer patients who underwent surgery were enrolled in this study. The median follow-up period was 3.9 years. The pre-operative plasma concentrations of 24 angiogenesisrelated molecules were analyzed with regard to the TNM stage and the development of post-operative recurrence. The concentrations of half of the examined molecules (13 ⁄ 24) increased significantly according to the TNM stage (P < 0.05). Meanwhile, a multivariate logistic regression analysis revealed that the concentrations of vascular cell adhesion molecule 1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) were significantly higher in the post-operative recurrence group. The VCAM-1 and PAI-1 model discriminated post-operative recurrence with an area under the curve of 0.82, a sensitivity of 0.75, and a specificity of 0.73. A leave-one-out cross-validation was applied to the model to assess the prediction performance, and the result indicated that the cross-validated error rate was 12.5% (12 ⁄ 96). In conclusion, our results demonstrate that antibody suspension bead arrays are a powerful tool to screen biomarkers in the clinical setting, and the plasma levels of VCAM-1 and PAI-1 together may be a promising biomarker for predicting post-operative recurrence in patients with colorectal cancer. (Cancer Sci 2010; 101: 1886-1890 C olorectal cancer (CRC) is one of the leading causes of death in Japan (http://ganjoho.ncc.go.jp/public/statistics/ index.html) and Western countries. (1) Despite recent advances in adjuvant chemotherapy and surgical techniques, 20-40% of patients die because of metastasis after curative surgery. (2) Tumor-node-metastasis (TNM) staging is well established and the most reliable system for predicting the outcome of CRC. In particular, the TNM staging system works very well for predicting the outcome of early stage I cancers and advanced stage IV cancers. However, the 5-year survival rate varies from 44% to 83% within TNM stage III, indicating that a wide variation in outcomes exists within each stage as a result of biological heterogeneity. (3) Thus, highly accurate predictors of post-operative recurrence are needed for patients with CRC who undergo curative surgery, as such predictors would likely contribute to the further improvement of the 5-year survival rate by justifying the addition of intensive adjuvant chemotherapy to the therapeutic regimens of subgroups with a high risk of post-operative recurrence. Therefore, the prediction of post-operative recurrence is regarded as one of the most important research themes in clinical settings and has been extensively studied, with particular attention given to the investigation of various molecular prognostic factors.In addition to the TNM stage, the carcinoembryonic antigen (CEA) level is routinely used to monitor recurrence in patients with CRC. (4) A large clinic...
Bio-Plex Pro(tm) cytokine, chemokine, and growth factors assays are magnetic bead-based multiplex assays designed to measure these cell signaling proteins in diverse matrices like serum, plasma and tissue culture supernatants. The abstract describes the development and validation of a Bio-Plex Pro(tm) Human Cytokine Assay Group II Panel. The assays are based on the Luminex® xMAP® technology capable of simultaneously quantifying 23 targets. Performance characteristics, including assay sensitivity, working ranges, precision, accuracy, linearity of dilution and parallelism are reported. Three assays were compared with commercially available ELISA kits using human serum samples (N=25). A good correlation was observed between the Bio-Plex Pro(tm) assays and the commercially available ELISA kits. Cross-panel-plexing between Bio-Plex Pro(tm) Human Cytokine Assay Group II and Group I panel was also investigated. The study showed that 16 of the 23 targets in Group II panel could be multiplexed with the 27-plex Group I panel. Analysis of normal and disease human serum samples using the Bio-Plex Pro(tm) human cytokine assays reported data consistent with published literatures.
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