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[en] The batch assay has been conventionally used for radioimmunoassay (RIA) because of its technical robustness and practical convenience. However, it has limitations in terms of the relative lag of report time due to the necessity of multiple assays in a small number of samples compared with the random assay technique. In this study, we aimed to verify whether the random assay technique can be applied in RIA and is feasible in daily practice. The coefficients of variation (CVs) of eight standard curves within a single kit were calculated in a CA-125 immunoradiometric assay (IRMA) for the reference of the practically ideal CV of the CA-125 kit. Ten standard curves of 10 kits from 2 prospectively collected lots (pLot) and 85 standard curves of 85 kits from 3 retrospectively collected lots (Lot) were obtained. Additionally, the raw measurement data of both 170 control references and 1123 patients' sera were collected retrospectively between December 2015 and January 2016. A standard curve of the first kit of each lot was used as a master standard curve for a random assay. The CVs of inter-kits were analyzed in each lot, respectively. All raw measurements were normalized by decay and radioactivity. The CA-125 values from control samples and patients' sera were compared using the original batch assay and random assay. In standard curve analysis, the CVs of inter-kits in pLots and Lots were comparable to those within a single kit. The CVs from the random assay with normalization were similar to those from the batch assay in the control samples (CVs % of low/high concentration; Lot1 2.71/1.91, Lot2 2.35/1.83, Lot3 2.83/2.08 vs. Lot1 2.05/1.21, Lot2 1.66/1.48, Lot3 2.41/2.14). The ICCs between the batch assay and random assay using patients' sera were satisfactory (Lot1 1.00, Lot2 0.999, Lot3 1.00). The random assay technique could be successfully applied to the conventional CA-125 IRMA kits. The random assay showed strong agreement with the batch assay. The random assay procedure could increase the flexibility and decrease the turnaround time of the radioimmunoassay technique
[en] Primary biliary cirrhosis (PBC) is a chronic and slowly progressive cholestatic liver disease of autoimmune etiology. A number of questions regarding its etiology are unclear. CD4+CD25+ regulatory T cells (Tregs) play a critical role in self-tolerance and, for unknown reasons, their relative number is reduced in PBC patients. B-cell-activating factor (BAFF) is a key survival factor during B-cell maturation and its concentration is increased in peripheral blood of PBC patients. It has been reported that activated B cells inhibit Treg cell proliferation and there are no BAFF receptors on Tregs. Therefore, we speculated that excessive BAFF may result in Treg reduction via B cells. To prove our hypothesis, we isolated Tregs and B cells from PBC and healthy donors. BAFF and IgM concentrations were then analyzed by ELISA and CD40, CD80, CD86, IL-10, and TGF-β expression in B cells and Tregs were measured by flow cytometry. BAFF up-regulated CD40, CD80, CD86, and IgM expression in B cells. However, BAFF had no direct effect on Treg cell apoptosis and cytokine secretion. Nonetheless, we observed that BAFF-activated B cells could induce Treg cell apoptosis and reduce IL-10 and TGF-β expression. We also showed that BAFF-activated CD4+ T cells had no effect on Treg apoptosis. Furthermore, we verified that bezafibrate, a hypolipidemic drug, can inhibit BAFF-induced Treg cell apoptosis. In conclusion, BAFF promotes Treg cell apoptosis and inhibits cytokine production by activating B cells in PBC patients. The results of this study suggest that inhibition of BAFF activation is a strategy for PBC treatment
[en] Molecular analyses of the c-kit and PDGFRα genes have contributed greatly to our understanding of the development of gastrointestinal stromal tumors (GISTs), but little is known about their malignant potential. The aim of our study was to evaluate cell cycle regulators as potential prognostic markers in GISTs. We investigated 104 KIT positive GISTs from various tumor sites in immunoassays on CD34, Ki67 and particularly on P53, BCL-2 and Cyclin D1. The results were compared with tumor size, mitotic rate, proliferative activity, histological subtype, nuclear atypia and risk assessment according to Fletcher and Miettinen. Occurrence of metastases and survival were also taken into account. The expression of P53 was significantly correlated with high risk criteria towards malignancy and epithelioid differentiation in GISTs. Likewise P53 label correlated significantly with the established prognostic indicators: tumor size, mitotic rate, nuclear atypia and proliferative activity. Regarding the site of tumor presentation, P53 was not a decisive factor. BCL-2 and Cyclin D1 expression was not related to any of the prognostic indicators. The present data identified P53 being a recommendable marker for predicting the risk of malignancy in GISTs. In addition, we found P53 significantly correlated with epithelioid tumor differentiation, independent of tumor site. BCL-2 and Cyclin D1, however, did not prove to be deciding markers for diagnosis and prognosis
[en] The Physics and Advanced Technologies (PAT) Directorate was created in July 2000 by Bruce Tarter, Director of Lawrence Livermore National Laboratory (LLNL). The Director called for the new organization to execute and support programs that apply cutting-edge physics and advanced technology to develop integrated solutions to problems in national security, fusion energy, information science, health care, and other national grand challenges. When I was appointed a year later as the PAT Directorate's first Associate Director, I initiated a strategic planning project to develop a vision, mission, and long-term goals for the Directorate. We adopted the goal of becoming a leader in frontier physics and technology for twenty-first-century national security missions: Stockpile Stewardship, homeland security, energy independence, and the exploration of space. Our mission is to: (1) Help ensure the scientific excellence and vitality of the major LLNL programs through its leadership role in performing basic and applied multidisciplinary research and development with programmatic impact, and by recruiting and retaining science and technology leaders; (2) Create future opportunities and directions for LLNL and its major programs by growing new program areas and cutting-edge capabilities that are synergistic with, and supportive of, its national security mission; (3) Provide a direct conduit to the academic and high-tech industrial sectors for LLNL and its national security programs, through which the Laboratory gains access to frontier science and technology, and can impact the science and technology communities; (4) Leverage unique Laboratory capabilities, to advance the state universe. This inaugural PAT Annual Report begins a series that will chronicle our progress towards fulfilling this mission. I believe the report demonstrates that the PAT Directorate has a strong base of capabilities and accomplishments on which to build in meeting its goals. Some of the highlights include: (1) Leadership of the Laboratory's Physical Data Research Program that provides fundamental physics information for the Stockpile Stewardship Program. (2) Development of the handheld Microbead Immunoassay Dipstick System that will allow relatively untrained first-responders to run sophisticated onsite diagnostics for pathogens, including those associated with biowarfare agents, by using a simple, one-step measurement. (3) Major advances in target design for inertial fusion energy research using both laser and ion-beam drivers. (4) Development of the Advanced Technology Kill Vehicle concept for use as a high-performance interceptor in a broad range of missile defense programs. Over the course of the past decade, the Laboratory has seen its major program evolve from weapons research, development, and testing, to Stockpile Stewardship. Today, the country's national security priorities are changing rapidly: nuclear security is becoming a broader set of missions, and the Laboratory is being asked to contribute to a range of new mission areas from countering bioterrorism to ensuring information security. As we embark on the twenty-first century, the new PAT Directorate is poised to help lead the Laboratory's response to the country's changing national security needs
[en] A powerful route to utilizing magnetic nanoparticles as labels in magnetic immunoassays is to exploit their non-linear response when they are exposed to a multi-frequency alternating magnetic field. We have upgraded this non-linear method allowing for the detection, discrimination and quantification of particles of two kinds when mixed together, with no need for spatial resolution. Each kind of particle is characterized by a specific magnetic signature based on d2B(H)/dH2. Appropriate data processing of the signature measured on a mixture of both particles allows for obtaining the amount of each particle. This will enable utilizing magnetic labels for multiparametric magnetic immunoassays.
[en] A new type of biosensor has been developed based on detection of nanosized superparamagnetic particles that serve as labels in bioreactions. The method is based on non-linear magnetic material detection by a magnetic field having components at two frequencies f 1 and f 2. The response is measured at the combinatorial frequencies f i=mf 1+nf 2, where m and n are integers, e.g., f i=f 1±2f 2. Several highly sensitive readers of superparamagnetic particles have been designed and used for development of various formats of immunoassays, including those compatible with immunoconcentration and magnetic enrichment of antigens
[en] The use of micro- and nanofluidic systems in modern analytical instruments allow you to implement a number of unique opportunities and achieve ultra-high measurement sensitivity. The possibility of manipulation of the individual biological objects (cells, bacteria, viruses, proteins, nucleic acids) in a liquid medium caused the development of devices on microchip platform for methods: chromatographic and electrophoretic analyzes; polymerase chain reaction; sequencing of nucleic acids; immunoassay; cytometric studies. Development of micro and nano fabrication technologies, materials science, surface chemistry, analytical chemistry, cell engineering have led to the creation of a unique systems such as “lab-on-a-chip”, “human-on-a-chip” and other. This article discusses common in microfluidics materials and methods of making functional structures. Examples of integration of nanoscale structures in microfluidic devices for the implementation of new features and improve the technical characteristics of devices and systems are shown. (paper)
[en] Highlights: • Rapid, sensitive, simple and quantitative detection of several biomarkers by magnetic labels. • Highly sensitive multi-channel quantification of magnetic particles in few reactions zones. • Four different approaches to multiplex magnetic biosensing on 3D porous structures. • Quick on-demand design of multiplex biosensors based on modular architecture. • Affordable inductively interrogated sensor chips with flat surface or micropillar microfluidics. Unique properties of magnetic nanoparticles (MNP) have provided many breakthrough solutions for life science. The immense potential of MNP as labels in advanced immunoassays stems from the fact that they, unlike optical labels, can be easily detected inside 3D opaque porous biosensing structures or in colored mediums, manipulated by an external magnetic field, exhibit high stability and negligible background signal in biological samples, etc. In this research, the magnetic nanolabels and an original technique of their quantification by non-linear magnetization have permitted development of novel methods of multiplex biosensing. Several types of highly sensitive multi-channel readers that offer an extremely wide linear dynamic range are developed to count MNP in different recognition zones for quantitative concentration measurements of various analytes. Four approaches to multiplex biosensing based on MNP have been demonstrated in one-run tests based on several 3D porous structures; flat and micropillar microfluidic sensor chips; multi-line lateral flow strips and modular architecture of the strips, which is the first 3D multiplexing method that goes beyond the traditional planar techniques. Detection of cardio- and cancer markers, small molecules and oligonucleotides were used in the experiments. The analytical characteristics of the developed multiplex methods are on the level of the modern time-consuming laboratory techniques. The developed multiplex biosensing platforms are promising for medical and veterinary diagnostics, food inspection, environmental and security monitoring, etc.