scholarly journals Expanding Assay Dynamics: A Combined Competitive and Direct Assay System for the Quantification of Proteins in Multiplexed Immunoassays

2008 ◽  
Vol 54 (6) ◽  
pp. 956-963 ◽  
Author(s):  
Michael Hartmann ◽  
Monika Schrenk ◽  
Anette Döttinger ◽  
Sarah Nagel ◽  
Johan Roeraade ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Protein Microarray ◽  
Measurement Range ◽  
Multiplex Assay ◽  
Competitive System ◽  
Direct Immunoassay ◽  
Sandwich Configuration ◽  
Readout Channel ◽  
Autoantibody Detection ◽  
Detection And Quantification

Abstract Background: The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay. Methods: The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system’s dynamic range is possible. Results: We implemented the method in a planar protein microarray–based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray–based system reduced spot-to-spot variation and intraassay variation. Conclusions: By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.

scholarly journals In-Process Diameter Measurement Technique for Micro-Optical Fiber with Standing Wave Illumination

2021 ◽  
Author(s):  
Masaki Michihata ◽  
Zhao Zheng ◽  
Daiki Funaiwa ◽  
Sojiro Murakami ◽  
Shotaro Kadoya ◽  
...  
Keyword(s):  
Optical Fiber ◽  
Standing Wave ◽  
Dynamic Range ◽  
Scattered Light ◽  
High Dynamic Range ◽  
Measurement Range ◽  
Diameter Distribution ◽  
Process Measurement ◽  
Air Turbulence ◽  
Tapered Optical Fiber

AbstractIn this paper, we propose an in-process measurement method of the diameter of micro-optical fiber such as a tapered optical fiber. The proposed technique is based on analyzing optically scattered light generated by standing wave illumination. The proposed method is significant in that it requires an only limited measurement range and does not require a high dynamic range sensor. These properties are suitable for in-process measurement. This experiment verified that the proposed method could measure a fiber diameter as stable as ± 0.01 μm under an air turbulence environment. As a result of comparing the measured diameter distribution with those by scanning electron microscopy, it was confirmed that the proposed method has a measurement accuracy better than several hundred nanometers.

scholarly journals Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

2007 ◽  
Vol 73 (20) ◽  
pp. 6557-6565 ◽  
Author(s):  
Pascal E. Saikaly ◽  
Morton A. Barlaz ◽  
Francis L. de los Reyes
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Fate And Transport ◽  
Biological Warfare ◽  
Quantitative Real Time Pcr ◽  
Pcr Assays ◽  
Detection And Quantification

ABSTRACT Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R 2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R 2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.

Optical Distance Meter Using a Pulsed Laser Diode and Fast Avalanche Photo Diodes for Measurements of Molten Steel Levels

1996 ◽  
Vol 118 (4) ◽  
pp. 800-803 ◽  
Author(s):  
Tsutomu Araki ◽  
Haruhiko Yoshida
Keyword(s):  
Light Source ◽  
Laser Diode ◽  
Molten Steel ◽  
Dynamic Range ◽  
Pulsed Laser ◽  
Casting Process ◽  
Measurement Range ◽  
Steel Mold ◽  
Optical Distance ◽  
Fast Rise

An optical distance meter with a wide dynamic range, is proposed for measuring the level of molten steel in a steel mold. The meter operates by measuring the flight time of a short optical pulse that is propagated between the light source and the target. A pulsed laser diode with a fast rise time is utilized as a light source, and two avalanche photo-diodes are used as detectors. The optical distance meter performs at a measurement range and a standard deviation of the measurement error of 1 m and 1 mm for the black paper target, respectively. To test its reliability, the distance meter is used to monitor the level of molten steel during a continuous casting process. The results obtained were compared with those obtained using an eddy current sensor and a γ-ray sensor. The measurement range of the optical method is ten times those attained using other two sensors. Continuous monitoring of the steel level can be done from the initiation of the casting process with the proposed distance meter.

scholarly journals In-Situ Wireless Pressure Measurement Using Zero-Power Packaged Microwave Sensors

Sensors ◽  
2019 ◽  
Vol 19 (6) ◽  
pp. 1263 ◽  
Author(s):  
Julien Philippe ◽  
Maria De Paolis ◽  
Dominique Henry ◽  
Alexandre Rumeau ◽  
Antony Coustou ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Pressure Sensors ◽  
Measurement Range ◽  
Full Scale ◽  
Remote Measurement ◽  
Reverberant Environments ◽  
Indoor Wireless ◽  
Measurement Results ◽  
Radar Imagery ◽  
First Time

This paper reports the indoor wireless measurement of pressure from zero-power (or passive) microwave (24 GHz) sensors. The sensors are packaged and allow the remote measurement of overpressure up to 2.1 bars. Their design, fabrication process and packaging are detailed. From the measurement of sensor scattering parameters, the outstanding sensitivity of 995 MHz/bar between 0.8 and 2.1 bars was achieved with the full-scale measurement range of 1.33 GHz. Moreover, the 3D radar imagery technique was applied for the remote interrogation of these sensors in electromagnetic reverberant environments. The full-scale dynamic range of 4.9 dB and the sensitivity of 4.9 dB/bar between 0.7 and 1.7 bars were achieved with radar detection in a highly reflective environment. These measurement results demonstrate for the first time the ability of the radar imagery technique to interrogate fully passive pressure sensors in electromagnetic reverberant environments.

Development of a quantitative PD-L1 assay using laser capture microdissection (LCM)-based reverse phase protein microarray (RPPA) workflow: Implications for precision medicine.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 35-35
Author(s):  
Mariaelena Pierobon ◽  
Elisa Baldelli ◽  
K. Alex Hodge ◽  
Maria Isabella Sereni ◽  
Vienna Ludovini ◽  
...  
Keyword(s):  
Cell Signaling ◽  
Tumor Cells ◽  
Dynamic Range ◽  
Protein Microarray ◽  
Population Data ◽  
Response Prediction ◽  
Antigen Retrieval ◽  
Subjective Analysis ◽  
Lung Cancers ◽  
Cut Point

35 Background: FDA approved IHC-based companion/complementary assays are routinely used to measure PD-L1 for treatment selection. However, IHC cut-point values vary across platforms and are based on subjective analysis that requires antigen retrieval methods. Even in highly selected populations of PD-L1 “positive” patients, clinical benefits are seen only in subgroups of patients. We tested the feasibility of utilizing LCM and RPPA as a new methodology for quantitative, operator independent measurements of PD-L1 on tumor cells. Methods: PD-L1 quantification by RPPA was compared to IHC on 23 lung cancers (LC). Tumor cells were isolated from the surrounding microenvironment using LCM. The E1L3N clone from Cell Signaling was used to quantify PD-L1 expression by RPPA and IHC. Reproducibility across antibody clones was then assessed on LCM procured tumor epithelia from 10 FFPE LC and 71 snap-frozen ovarian cancers (OC). PD-L1 measurements were compared between the Cell Signaling E1L3N and the Ventana SP-142 clone. A quantitative PD-L1 calibrated assay was developed and assessed with the pilot population data. Results: Of the 23 LC, 5 were PD-L1 positive by IHC. The 5 IHC-positive patients had the greatest level of PD-L1 expression by RPPA indicating that the RPPA platform correlates with IHC. However, RPPA quantification showed a dynamic range ~ 4-fold across IHC negative samples. Correlation between PD-L1 detection with the E1L3N and the SP-142 clone in the OC was significant (R2 = 0.85), indicating great concordance across clones. Results were confirmed in the 10 LC samples (R2 = 0.87). Conclusions: The LCM-RPPA workflow captures PD-L1 expression on a continuous quantitative scale. This quantitative output correlated with IHC although it captures a much broader dynamic range in both IHC negative and positive populations. PD-L1 quantification by LCM-RPPA may be less dependent upon the clone used for the detection than IHC. Because the detection is unconstrained by antigen retrieval issues as well as subjectivity of IHC interpretation, this approach may generate a more accurate cut-point of therapeutic response prediction.

scholarly journals Large Dynamic Current Monitoring for UAV Steering Motor Using Twin Nonlinear Shunt

Electronics ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1240
Author(s):  
Bo Li ◽  
Shengbing Zhang ◽  
Hanlu Zhang ◽  
Shiyu Wang ◽  
Feng He
Keyword(s):  
Monitoring System ◽  
Health Monitoring ◽  
Dynamic Range ◽  
Measurement Range ◽  
Industrial Control ◽  
Sampling Device ◽  
Health Monitoring System ◽  
Working Principle ◽  
Current Sampling ◽  
Current Monitoring

Steering motor is of vital importance in UAV’s health-monitoring system, to which its supply current is the most critical characteristic representing health statue of UAV. In order to conduct continuous measuring on the steering motor’s current of large dynamic range, in this paper, a current measurement method is therefore proposed on the basis of twin nonlinear shunt. The proposed method adopts the twin diode as the current sampling device, which not only realizes measurement range and relative constant resolution, but also ensures continuity of the measurement due to the eliminated operation of range switching. The associated diode is used to compensate the temperature of core temperature of the shunt diode, and to make the nonlinear-shunt more adaptive for the case of junction being heated under larger current. The working principle, real-time compensation method and circuit implementation of our method are discussed in detail. Experimental test results suggest that the measurement error of the proposed method is less than 4.5% when the measurement current varies between 10 mA to 10 A, maintaining the relative resolution at an almost constant level, while preventing the conventional method of frequent range switching from generating glitches. In addition to the ensured continuity, information-rich details of the current are sustained, contributing to the UAV’s health-monitoring system. The proposal can also be applied to other applications concerning large dynamic current detection, including, but not limited to, industrial control, motor control, etc.

HIGH SENSITIVITY AND HIGH DYNAMIC RANGE OPTICAL MICRO-RADIATOR VACUUM SENSOR FABRICTED WITH CMOS TECHNOLOGY

2008 ◽  
Vol 05 (03) ◽  
pp. 189-196
Author(s):  
Y. MA ◽  
R. P. W. LAWSON ◽  
A. M. ROBINSON
Keyword(s):  
Dynamic Range ◽  
High Sensitivity ◽  
High Dynamic Range ◽  
Cmos Technology ◽  
Measurement Range ◽  
Power Switching ◽  
Pressure Sensing ◽  
Vacuum Sensor ◽  
Sensitivity Improvement ◽  
Silicon Cmos

A Complementary Metal Oxide Silicon (CMOS) optical micro-radiator vacuum sensor has been designed, tested and calibrated. The package is comprised of a micromachined radiator and a photodetector. The sensitivity improvement of the system over the conventional Pirani gauge is up to nine magnitudes depending on the operating power of the micro-radiator. To increase sensor's dynamic range, an automated power-switching system has been demonstrated for pressure sensing operated with constant photodetector output. Calibration of the system has been performed by comparison with secondary standards. Experimental results showed that the sensor's measurement range from 10-3 Pa to 105 Pa has been achieved as its relative error is less than 8%.

scholarly journals Flow Cytometric Microsphere-Based Immunoassay: Analysis of Secreted Cytokines in Whole-Blood Samples from Asthmatics

2001 ◽  
Vol 8 (4) ◽  
pp. 776-784 ◽  
Author(s):  
Christophe Camilla ◽  
Laurent Mély ◽  
Antoine Magnan ◽  
Brice Casano ◽  
Sabine Prato ◽  
...  
Keyword(s):  
Simultaneous Determination ◽  
Whole Blood ◽  
Dynamic Range ◽  
Internal Standard ◽  
Multiplex Assay ◽  
Interleukin 4 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Flow Cytometric ◽  
Ifn Γ

ABSTRACT The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P = 0.003) and less IFN-γ (P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-γ than that of atopic nonasthmatic subjects (P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.

scholarly journals Detection and Quantification of Infectious Avian Influenza A (H5N1) Virus in Environmental Water by Using Real-Time Reverse Transcription-PCR

2010 ◽  
Vol 76 (7) ◽  
pp. 2165-2174 ◽  
Author(s):  
C. I. Dovas ◽  
M. Papanastassopoulou ◽  
M. P. Georgiadis ◽  
E. Chatzinasiou ◽  
V. I. Maliogka ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Real Time ◽  
Internal Control ◽  
Reverse Transcription ◽  
H5n1 Virus ◽  
Dynamic Range ◽  
Detection System ◽  
Environmental Water ◽  
Qrt Pcr ◽  
Detection And Quantification

ABSTRACT Routes of avian influenza virus (AIV) dispersal among aquatic birds involve direct (bird-to-bird) and indirect (waterborne) transmission. The environmental persistence of H5N1 virus in natural water reservoirs can be assessed by isolation of virus in embryonated chicken eggs. Here we describe development and evaluation of a real-time quantitative reverse transcription (RT)-PCR (qRT-PCR) method for detection of H5N1 AIV in environmental water. This method is based on adsorption of virus particles to formalin-fixed erythrocytes, followed by qRT-PCR detection. The numbers of hemagglutinin RNA copies from H5N1 highly pathogenic AIV particles adsorbed to erythrocytes detected correlated highly with the infectious doses of the virus that were determined for three different types of artificially inoculated environmental water over a 17-day incubation period. The advantages of this method include detection and quantification of infectious H5N1 AIVs with a high level of sensitivity, a wide dynamic range, and reproducibility, as well as increased biosecurity. The lowest concentration of H5N1 virus that could be reproducibly detected was 0.91 50% egg infective dose per ml. In addition, a virus with high virion stability (Tobacco mosaic virus) was used as an internal control to accurately monitor the efficiency of RNA purification, cDNA synthesis, and PCR amplification for each individual sample. This detection system could be useful for rapid high-throughput monitoring for the presence of H5N1 AIVs in environmental water and in studies designed to explore the viability and epidemiology of these viruses in different waterfowl ecosystems. The proposed method may also be adapted for detection of other AIVs and for assessment of their prevalence and distribution in environmental reservoirs.

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