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Sample records for lateral flow assay

  1. Lateral flow strip assay

    DOEpatents

    Miles, Robin R [Danville, CA; Benett, William J [Livermore, CA; Coleman, Matthew A [Oakland, CA; Pearson, Francesca S [Livermore, CA; Nasarabadi, Shanavaz L [Livermore, CA

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  2. Lateral flow assays

    PubMed Central

    Koczula, Katarzyna M.

    2016-01-01

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  3. A lateral electrophoretic flow diagnostic assay.

    PubMed

    Lin, Robert; Skandarajah, Arunan; Gerver, Rachel E; Neira, Hector D; Fletcher, Daniel A; Herr, Amy E

    2015-03-21

    Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings.

  4. Quantifiable Lateral Flow Assay Test Strips

    NASA Technical Reports Server (NTRS)

    2003-01-01

    As easy to read as a home pregnancy test, three Quantifiable Lateral Flow Assay (QLFA) strips used to test water for E. coli show different results. The brightly glowing control line on the far right of each strip indicates that all three tests ran successfully. But the glowing test line on the middle left and bottom strips reveal their samples were contaminated with E. coli bacteria at two different concentrations. The color intensity correlates with concentration of contamination.

  5. Quantifiable Lateral Flow Assay Test Strips

    NASA Technical Reports Server (NTRS)

    2003-01-01

    As easy to read as a home pregnancy test, three Quantifiable Lateral Flow Assay (QLFA) strips used to test water for E. coli show different results. The brightly glowing control line on the far right of each strip indicates that all three tests ran successfully. But the glowing test line on the middle left and bottom strips reveal their samples were contaminated with E. coli bacteria at two different concentrations. The color intensity correlates with concentration of contamination.

  6. Battery operated preconcentration-assisted lateral flow assay.

    PubMed

    Kim, Cheonjung; Yoo, Yong Kyoung; Han, Sung Il; Lee, Junwoo; Lee, Dohwan; Lee, Kyungjae; Hwang, Kyo Seon; Lee, Kyu Hyoung; Chung, Seok; Lee, Jeong Hoon

    2017-07-11

    Paper-based analytical devices (e.g. lateral flow assays) are highly advantageous as portable diagnostic systems owing to their low costs and ease of use. Because of their low sensitivity and detection limits for biomolecules, these devices have several limitations in applications for real-field diagnosis. Here, we demonstrate a paper-based preconcentration enhanced lateral flow assay using a commercial β-hCG-based test. Utilizing a simple 9 V battery operation with a low power consumption of approximately 81 μW, we acquire a 25-fold preconcentration factor, demonstrating a clear sensitivity enhancement in the colorimetric lateral flow assay; consequently, clear colors are observed in a rapid kit test line, which cannot be monitored without preconcentration. This device can also facilitate a semi-quantitative platform using the saturation value and/or color intensity in both paper-based colorimetric assays and smartphone-based diagnostics.

  7. High frequency lateral flow affinity assay using superparamagnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Lago-Cachón, D.; Rivas, M.; Martínez-García, J. C.; Oliveira-Rodríguez, M.; Blanco-López, M. C.; García, J. A.

    2017-02-01

    Lateral flow assay is one of the simplest and most extended techniques in medical diagnosis for point-of-care testing. Although it has been traditionally a positive/negative test, some work has been lately done to add quantitative abilities to lateral flow assay. One of the most successful strategies involves magnetic beads and magnetic sensors. Recently, a new technique of superparamagnetic nanoparticle detection has been reported, based on the increase of the impedance induced by the nanoparticles on a RF-current carrying copper conductor. This method requires no external magnetic field, which reduces the system complexity. In this work, nitrocellulose membranes have been installed on the sensor, and impedance measurements have been carried out during the sample diffusion by capillarity along the membrane. The impedance of the sensor changes because of the presence of magnetic nanoparticles. The results prove the potentiality of the method for point-of-care testing of biochemical substances and nanoparticle capillarity flow studies.

  8. Analytical Tools to Improve Optimization Procedures for Lateral Flow Assays

    PubMed Central

    Hsieh, Helen V.; Dantzler, Jeffrey L.; Weigl, Bernhard H.

    2017-01-01

    Immunochromatographic or lateral flow assays (LFAs) are inexpensive, easy to use, point-of-care medical diagnostic tests that are found in arenas ranging from a doctor’s office in Manhattan to a rural medical clinic in low resource settings. The simplicity in the LFA itself belies the complex task of optimization required to make the test sensitive, rapid and easy to use. Currently, the manufacturers develop LFAs by empirical optimization of material components (e.g., analytical membranes, conjugate pads and sample pads), biological reagents (e.g., antibodies, blocking reagents and buffers) and the design of delivery geometry. In this paper, we will review conventional optimization and then focus on the latter and outline analytical tools, such as dynamic light scattering and optical biosensors, as well as methods, such as microfluidic flow design and mechanistic models. We are applying these tools to find non-obvious optima of lateral flow assays for improved sensitivity, specificity and manufacturing robustness. PMID:28555034

  9. Analytical Tools to Improve Optimization Procedures for Lateral Flow Assays.

    PubMed

    Hsieh, Helen V; Dantzler, Jeffrey L; Weigl, Bernhard H

    2017-05-28

    Immunochromatographic or lateral flow assays (LFAs) are inexpensive, easy to use, point-of-care medical diagnostic tests that are found in arenas ranging from a doctor's office in Manhattan to a rural medical clinic in low resource settings. The simplicity in the LFA itself belies the complex task of optimization required to make the test sensitive, rapid and easy to use. Currently, the manufacturers develop LFAs by empirical optimization of material components (e.g., analytical membranes, conjugate pads and sample pads), biological reagents (e.g., antibodies, blocking reagents and buffers) and the design of delivery geometry. In this paper, we will review conventional optimization and then focus on the latter and outline analytical tools, such as dynamic light scattering and optical biosensors, as well as methods, such as microfluidic flow design and mechanistic models. We are applying these tools to find non-obvious optima of lateral flow assays for improved sensitivity, specificity and manufacturing robustness.

  10. Aptamer-phage reporters for ultrasensitive lateral flow assays

    PubMed Central

    Adhikari, Meena; Strych, Ulrich; Kim, Jinsu; Goux, Heather; Dhamane, Sagar; Poongavanam, Mohan-Vivekanandan; Hagström, Anna E. V.; Kourentzi, Katerina; Conrad, Jacinta C.; Willson, Richard C.

    2015-01-01

    We introduce the modification of bacteriophage particles with aptamers for the use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin-biotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ~100 times lower than those of previously reported IgE assays. PMID:26456715

  11. Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays.

    PubMed

    Adhikari, Meena; Strych, Ulrich; Kim, Jinsu; Goux, Heather; Dhamane, Sagar; Poongavanam, Mohan-Vivekanandan; Hagström, Anna E V; Kourentzi, Katerina; Conrad, Jacinta C; Willson, Richard C

    2015-12-01

    We introduce the modification of bacteriophage particles with aptamers for use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin-biotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ∼100 times lower than those of previously reported IgE assays.

  12. Time-resolved luminescent lateral flow assay technology.

    PubMed

    Song, Xuedong; Knotts, Michael

    2008-09-26

    We here report a detection technology that integrates highly sensitive time-resolved luminescence technique into lateral flow assay platform to achieve excellent detection performance with low cost. We have developed very bright, surface-functionalized and mono-dispersed phosphorescent nanoparticles of long lifetime under ambient conditions. The phosphorescent nanoparticles have been used to conjugate with monoclonal antibody for C-reactive protein (CRP), an inflammatory biomarker. Lateral flow immunoassay devices have been developed using the conjugate for highly sensitive detection of CRP. The CRP assay can achieve a detection sensitivity of <0.2 ngmL(-1) in serum with a linear response from 0.2 to 200 ngmL(-1) CRP. We have also developed a low cost time-resolved luminescence reader for the lateral flow immunoassay (LFIA) devices. The reader does not use expensive band pass filter and still provide very low detection background and high detection sensitivity on solid substrates such as nitrocellulose membranes. The reader can detect less than 2.5 ng phosphorescent particles captured on a nitrocellulose membrane strip with more than three orders of magnitude linear detection dynamic range. The technology should find a number of applications, ranging from clinical diagnostics, detection of chemical and biological warfare agents, to food and environmental monitoring.

  13. A paper-based lateral flow assay for morphine.

    PubMed

    Teerinen, Tuija; Lappalainen, Timo; Erho, Tomi

    2014-09-01

    Morphine was used as a model analyte to examine the possibility of using cellulose, physically modified by papermaking and converting techniques, as a capillary matrix in a lateral flow type of diagnostic assay. This research was directed toward low-cost, disposable, and portable paper-based diagnostics, with the aim of addressing the analytical performance of paper as a substrate in the analysis for drugs of abuse. Antibody Fab fragments were used as sensing molecules, and gold nanoparticle detection was employed. Inkjet printing was used to pattern sensing biomolecules as detection zones on paper. To validate the usefulness of paper as a diagnostic platform, the principle of a direct sandwich assay, based on immunocomplex formation between morphine and the anti-morphine Fab fragment and detection of the formed immunocomplex by another Fab fragment, was implemented. Results were compared with that achieved by using nitrocellulose as a reference material. Possible interfering from the sample matrix on assay quality was investigated with spiked oral fluid samples. Under optimized conditions, a visually assessed limit of detection for the sandwich assay was 1 ng/mL, indicating that the paper-based test devices developed in this work can perform screening for drugs of abuse and can fulfill the requirement for a sensitive assay in diagnostically relevant ranges.

  14. Detection of Shiga Toxins by Lateral Flow Assay

    PubMed Central

    Ching, Kathryn H.; He, Xiaohua; Stanker, Larry H.; Lin, Alice V.; McGarvey, Jeffery A.; Hnasko, Robert

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD) for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing. PMID:25855129

  15. Quantitative Lateral Flow Assays for Salivary Biomarker Assessment: A Review

    PubMed Central

    Miočević, Olga; Cole, Craig R.; Laughlin, Mary J.; Buck, Robert L.; Slowey, Paul D.; Shirtcliff, Elizabeth A.

    2017-01-01

    Saliva is an emerging biofluid with a significant number of applications in use across research and clinical settings. The present paper explores the reasons why saliva has grown in popularity in recent years, balancing both the potential strengths and weaknesses of this biofluid. Focusing on reasons why saliva is different from other common biological fluids such as blood, urine, or tears, we review how saliva is easily obtained, with minimal risk to the donor, and reduced costs for collection, transportation, and analysis. We then move on to a brief review of the history and progress in rapid salivary testing, again reviewing the strengths and weaknesses of rapid immunoassays (e.g., lateral flow immunoassay) compared to more traditional immunoassays. We consider the potential for saliva as an alternative biofluid in a setting where rapid results are important. We focus the review on salivary tests for small molecule biomarkers using cortisol as an example. Such salivary tests can be applied readily in a variety of settings and for specific measurement purposes, providing researchers and clinicians with opportunities to assess biomarkers in real time with lower transportation, collection, and analysis costs, faster turnaround time, and minimal training requirements. We conclude with a note of cautious optimism that the field will soon gain the ability to collect and analyze salivary specimens at any location and return viable results within minutes. PMID:28660183

  16. Performance of the Cryptococcal Antigen Lateral Flow Assay in Non-HIV-Related Cryptococcosis.

    PubMed

    Jitmuang, Anupop; Panackal, Anil A; Williamson, Peter R; Bennett, John E; Dekker, John P; Zelazny, Adrian M

    2016-02-01

    The cryptococcal antigen lateral flow assay (CrAg LFA) was evaluated for the diagnosis of cryptococcosis in HIV-negative patients. The sensitivity was excellent, suggesting that this assay can replace conventional testing based on latex agglutination (LA). CrAg LFA and LA titers were correlated but were not directly comparable, with implications for conversion between assays.

  17. Performance of the Cryptococcal Antigen Lateral Flow Assay in Non-HIV-Related Cryptococcosis

    PubMed Central

    Jitmuang, Anupop; Panackal, Anil A.; Williamson, Peter R.; Bennett, John E.; Dekker, John P.

    2015-01-01

    The cryptococcal antigen lateral flow assay (CrAg LFA) was evaluated for the diagnosis of cryptococcosis in HIV-negative patients. The sensitivity was excellent, suggesting that this assay can replace conventional testing based on latex agglutination (LA). CrAg LFA and LA titers were correlated but were not directly comparable, with implications for conversion between assays. PMID:26607986

  18. Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey.

    PubMed

    Posthuma-Trumpie, Geertruida A; Korf, Jakob; van Amerongen, Aart

    2009-01-01

    Lateral flow (immuno)assays are currently used for qualitative, semiquantitative and to some extent quantitative monitoring in resource-poor or non-laboratory environments. Applications include tests on pathogens, drugs, hormones and metabolites in biomedical, phytosanitary, veterinary, feed/food and environmental settings. We describe principles of current formats, applications, limitations and perspectives for quantitative monitoring. We illustrate the potentials and limitations of analysis with lateral flow (immuno)assays using a literature survey and a SWOT analysis (acronym for "strengths, weaknesses, opportunities, threats"). Articles referred to in this survey were searched for on MEDLINE, Scopus and in references of reviewed papers. Search terms included "immunochromatography", "sol particle immunoassay", "lateral flow immunoassay" and "dipstick assay".

  19. Sensitive Detection of Norovirus Using Phage Nanoparticle Reporters in Lateral-Flow Assay

    PubMed Central

    Hagström, Anna E. V.; Garvey, Gavin; Paterson, Andrew S.; Dhamane, Sagar; Adhikari, Meena; Estes, Mary K.; Strych, Ulrich; Kourentzi, Katerina; Atmar, Robert L.; Willson, Richard C.

    2015-01-01

    Noroviruses are recognized worldwide as the principal cause of acute, non-bacterial gastroenteritis, resulting in 19-21 million cases of disease every year in the United States. Noroviruses have a very low infectious dose, a short incubation period, high resistance to traditional disinfection techniques and multiple modes of transmission, making early, point-of-care detection essential for controlling the spread of the disease. The traditional diagnostic tools, electron microscopy, RT-PCR and ELISA require sophisticated and expensive instrumentation, and are considered too laborious and slow to be useful during severe outbreaks. In this paper we describe the development of a new, rapid and sensitive lateral-flow assay using labeled phage particles for the detection of the prototypical norovirus GI.1 (Norwalk), with a limit of detection of 107 virus-like particles per mL, one hundred-fold lower than a conventional gold nanoparticle lateral-flow assay using the same antibody pair. PMID:25978622

  20. Sensitive detection of norovirus using phage nanoparticle reporters in lateral-flow assay.

    PubMed

    Hagström, Anna E V; Garvey, Gavin; Paterson, Andrew S; Dhamane, Sagar; Adhikari, Meena; Estes, Mary K; Strych, Ulrich; Kourentzi, Katerina; Atmar, Robert L; Willson, Richard C

    2015-01-01

    Noroviruses are recognized worldwide as the principal cause of acute, non-bacterial gastroenteritis, resulting in 19-21 million cases of disease every year in the United States. Noroviruses have a very low infectious dose, a short incubation period, high resistance to traditional disinfection techniques and multiple modes of transmission, making early, point-of-care detection essential for controlling the spread of the disease. The traditional diagnostic tools, electron microscopy, RT-PCR and ELISA require sophisticated and expensive instrumentation, and are considered too laborious and slow to be useful during severe outbreaks. In this paper we describe the development of a new, rapid and sensitive lateral-flow assay using labeled phage particles for the detection of the prototypical norovirus GI.1 (Norwalk), with a limit of detection of 107 virus-like particles per mL, one hundred-fold lower than a conventional gold nanoparticle lateral-flow assay using the same antibody pair.

  1. A lateral flow assay (LFA) for the rapid detection of extraparenchymal neurocysticercosis using cerebrospinal fluid.

    PubMed

    Fleury, Agnes; Sastre, Patricia; Sciutto, Edda; Correia, Silvia; Monedero, Alejandro; Toledo, Andrea; Hernandez, Maricela; Harrison, Leslie J S; Parkhouse, R Michael E

    2016-10-27

    A lateral flow assay (LFA) for the diagnosis and monitoring of extraparenchymal neurocysticercosis, has been developed. The assay is based on the use of the monoclonal antibody HP10, and when applied to cerebrospinal fluid, correctly identified 34 cases of active extraparenchymal neurocysticercosis, but was negative with 26 samples from treated and cured neurocysticercosis patients and with 20 samples from unrelated neurological diseases. There was complete agreement between the HP10 Ag-ELISA results and the HP10-LFA. The HP10-LFA thus has utility for diagnosis and treatment of extraparenchymal neurocysticercosis, frequently a more dangerous form of the infection.

  2. Comparison of conventional lateral-flow assays and a new fluorescent immunoassay to detect influenza viruses.

    PubMed

    Leonardi, Gary P; Wilson, Adele M; Zuretti, Alejandro R

    2013-05-01

    Sofia, a novel, fluorescent lateral-flow immunoassay was compared with two conventional colorimetric assays, Quickvue Influenza A+B and Directigen FLU A+B, to identify influenza viral antigen from patient nasopharyngeal specimens. A total of 118 frozen original influenza-positive specimens and 57 prospective specimens were examined. Using rt-PCR as a referee assay, sensitivity values (%) for influenza A/B of 80.0/74.8, 73.3/59.3 and 73.3/40.7 were obtained using the Sofia, Quickvue and Directigen assays, respectively. All assays demonstrated reduced sensitivity for influenza B as compared with influenza A virus. With respect to the Sofia assay, the sensitivity of influenza B for the Directigen assay was significantly diminished. False positive results were not observed in the Sofia and Directigen assays. The Quickvue assay produced 3 false-positive results (2 influenza A and 1 influenza B) resulting in a specificity (%) of 96 and 98 for influenza A and B, respectively. Cross-reactivity to other respiratory viruses was not observed among immunoassays. A sensitivity rank (highest to low) of rt-PCR>culture>Sofia>Quickvue>Directigen was established using dilutions of influenza A and B. Sofia provides enhanced sensitivity and objective result interpretation over conventional colorimetric immunoassays.

  3. From Lateral Flow Devices to a Novel Nano-Color Microfluidic Assay

    PubMed Central

    Assadollahi, Saied; Reininger, Christiane; Palkovits, Roland; Pointl, Peter; Schalkhammer, Thomas

    2009-01-01

    Improving the performance of traditional diagnostic lateral flow assays combined with new manufacturing technologies is a primary goal in the research and development plans of diagnostic companies. Taking into consideration the components of lateral flow diagnostic test kits; innovation can include modification of labels, materials and device design. In recent years, Resonance-Enhanced Absorption (REA) of metal nano-particles has shown excellent applicability in bio-sensing for the detection of a variety of bio-molecular binding interactions. In a novel approach, we have now integrated REA-assays in a diagnostic microfluidic setup thus resolving the bottleneck of long incubation times inherent in previously existing REA-assays and simultaneously integrated automated fabrication techniques for diagnostics manufacture. Due to the roller-coating based technology and chemical resistance, we used PET-co-polyester as a substrate and a CO2 laser ablation system as a fast, highly precise and contactless alternative to classical micro-milling. It was possible to detect biological binding within three minutes – visible to the eye as colored text readout within the REA-fluidic device. A two-minute in-situ silver enhancement was able to enhance the resonant color additionally, if required. PMID:22454573

  4. A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.

    PubMed

    Liu, Wei; Liu, Hui-Xin; Zhang, Lin; Hou, Xue-Xia; Wan, Kang-Lin; Hao, Qin

    2016-08-03

    A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully applied in B. burgdorferi detection, and its specific amplification was only achieved from the genomic DNA of B. burgdorferi. The detection limit of the new assay was about 25 copies of the B. burgdorferi genomic DNA. Twenty Lyme borreliosis patients' serum samples were detected by LF-RPA assay, real-time qPCR and nested-PCR. Results showed the LF-RPA assay is more effective than nested-PCR for its shorter reaction time and considerably higher detection rate. This method is of great value in clinical rapid detection for Lyme borreliosis. Using the RPA assay might be a megatrend for DNA detection in clinics and endemic regions.

  5. Detection of Viruses By Counting Single Fluorescent Genetically Biotinylated Reporter Immunophage Using a Lateral Flow Assay

    PubMed Central

    Kim, Jinsu; Adhikari, Meena; Dhamane, Sagar; Hagström, Anna E. V.; Kourentzi, Katerina; Strych, Ulrich; Willson, Richard C.; Conrad, Jacinta C.

    2015-01-01

    We demonstrated a lateral flow immunoassay (LFA) for detection of viruses using fluorescently-labeled M13 bacteriophage as reporters and single-reporter counting as the readout. AviTag-biotinylated M13 phage were functionalized with antibodies using avidin-biotin conjugation and fluorescently labeled with AlexaFluor 555. Individual phage bound to target viruses (here MS2 as a model) captured on an LFA membrane strip were imaged using epi-fluorescence microscopy. Using automated image processing, we counted the number of bound phage in micrographs as a function of target concentration. The resultant assay was more sensitive than enzyme-linked immunosorbent assays and traditional colloidal-gold nanoparticle LFAs for direct detection of viruses. PMID:25581289

  6. Fluorescence-based lateral flow assays for rapid oral fluid roadside detection of cannabis use.

    PubMed

    Plouffe, Brian D; Murthy, Shashi K

    2017-02-01

    With the recent worldwide changes in the legalization of marijuana, there is a significant need for rapid, roadside screening test for driving under the influence of drugs. A robust, sensitive, lateral flow assay has been developed to detect recent use via oral-fluid testing for Δ(9) -tetrahydrocannabinol (THC). This proof-of-concept assay uses a fluorescent-based immunoassay detection of polymeric beads, conjugated to antibodies against native THC. The fluorescent technique allows for significantly lower limits of detection and higher precision determination of recent marijuana use without the use of urine or blood sampling-thus allowing for roadside identification. Detection levels of 0.01 ng/mL were distinguished from background and the lower limit of quantification was determined to approach 1 ng/mL. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. The cryptococcal antigen lateral flow assay: A point-of-care diagnostic at an opportune time.

    PubMed

    Tang, Michele W; Clemons, Karl V; Katzenstein, David A; Stevens, David A

    2016-08-01

    Cryptococcal meningitis is a devastating HIV-related opportunistic infection, affecting nearly 1 million individuals and causing over 500 000 deaths each year. The burden of disease is greatest in sub-Saharan Africa and Southeast Asia, where cryptococcal disease is the most common cause of meningitis. Rapid, accurate and affordable diagnosis of cryptococcal disease has been lacking in many of the most heavily affected areas. Here, we review a point-of-care assay for cryptococcal disease, the dipstick-formatted cryptococcal antigen lateral flow assay (LFA) (IMMY, Norman, OK). In comparison to culture, the assay is 99.5% sensitive and 98% specific. In comparison to other commercially available tests for cryptococcal antigen, the LFA has equal or superior sensitivity and specificity in CSF, plasma and serum samples. We discuss potential applications for the use of the assay in resource-limited settings, including what is likely to be an important role of the LFA in screening for early cryptococcal infection before clinical disease and in evaluating pre-emptive treatment.

  8. Rapid screening test for detection of oxytetracycline residues in milk using lateral flow assay.

    PubMed

    Naik, Laxmana; Sharma, Rajan; Mann, Bimlesh; Lata, Kiran; Rajput, Y S; Surendra Nath, B

    2017-03-15

    A rapid, semi-quantitative lateral flow assay (LFA) was developed to screen the oxytetracycline (OTC) antibiotics residues in milk samples. In this study a competitive immuno-assay format was established. Colloidal gold nano-particles (GNP) were prepared and used as labelling material in LFA. Polyclonal antibodies were generated against OTC molecule (anti-OTC), purified and the quality was assessed by enzyme linked immuno sorbet assay. For the first time membrane components required for LFA in milk system was optimized. GNP and anti-OTC stable conjugate preparation method was standardized, and then these components were placed over the conjugate pad. OTC coupled with carrier protein was placed on test line; species specific secondary antibodies were placed on the control line of the membrane matrix. Assay was validated by spiking OTC to antibiotic free milk samples and results could be accomplished within 5min. without need of any equipment. The visual detection limit was 30ppb. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India

    PubMed Central

    Vanithamani, Shanmugam; Shanmughapriya, Santhanam; Narayanan, Ramasamy; Raja, Veerapandian; Kanagavel, Murugesan; Sivasankari, Karikalacholan; Natarajaseenivasan, Kalimuthusamy

    2015-01-01

    Background Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area. Methods/Principal Findings In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients’ sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05). Conclusion The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative. PMID:26340095

  10. Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India.

    PubMed

    Vanithamani, Shanmugam; Shanmughapriya, Santhanam; Narayanan, Ramasamy; Raja, Veerapandian; Kanagavel, Murugesan; Sivasankari, Karikalacholan; Natarajaseenivasan, Kalimuthusamy

    2015-01-01

    Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area. In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients' sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05). The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.

  11. Mobile Phone Sensing of Cocaine in a Lateral Flow Assay Combined with a Biomimetic Material.

    PubMed

    Guler, Emine; Yilmaz Sengel, Tulay; Gumus, Z Pinar; Arslan, Mustafa; Coskunol, Hakan; Timur, Suna; Yagci, Yusuf

    2017-08-28

    Lateral flow assays (LFAs) are an ideal choice for drug abuse testing favored by their practicability, portability, and rapidity. LFA based on-site rapid screening devices provide positive/negative judgment in a short response time. The conventionally applied competitive assay format used for small molecule analysis such as abused drugs restricts the quantitation ability of LFA strips. We report herein, for the first time, a new strategy using the noncompetitive assay format via a biomimetic material, namely, poly(p-phenylene) β-cyclodextrin poly(ethylene glycol) (PPP-CD-g-PEG) combined with gold nanoparticle (AuNP) conjugates as the labeling agent to recognize the target cocaine molecule in the test zone. The intensities of the visualized red color in the test line indicate that the cocaine concentrations were analyzed via a smartphone application. Significantly, a combination of this platform with a smartphone application provides quantitative data on the cocaine amount, making it a very inventive and attractive approach especially for on-site applications at critical points such as traffic stops and the workplace.

  12. Increasing Binding Efficiency via Reporter Shape and Flux in a Viral Nanoparticle Lateral-Flow Assay.

    PubMed

    Kim, Jinsu; Vu, Binh; Kourentzi, Katerina; Willson, Richard C; Conrad, Jacinta C

    2017-03-01

    To identify factors controlling the performance of reporter particles in a sensitive lateral-flow assay (LFA), we investigated the effect of the flux and shape of filamentous bacteriophage (phage) on the performance of phage LFAs. Phage of three different lengths and diameters were modified with biotin and AlexaFluor 555 as binding and read-out elements, respectively. The binding efficiencies of the functionalized phage were tested in a fibrous glass LFA membrane modified with avidin. The total binding rate, quantified using real-time particle counting and particle image velocimetry, decreased monotonically with the average bulk flux of phage through the membrane. At the pore scale, more phage bound in regions with faster local flow, confirming that both average and local flux increased binding. The number of bound phage increased with the aspect ratio of the phage and scaled with the phage surface area, consistent with a binding interaction controlled by the number of recognition elements on the surface. Together, these results indicate that increasing the likelihood that recognition elements on the surface of phage encounter the fibers enhances the assay binding efficiency and suggests one origin for the improved performance of nonspherical phage reporters.

  13. Point-of-care diagnosis and prognostication of cryptococcal meningitis with the cryptococcal antigen lateral flow assay on cerebrospinal fluid.

    PubMed

    Kabanda, Taseera; Siedner, Mark J; Klausner, Jeffrey D; Muzoora, Conrad; Boulware, David R

    2014-01-01

    The cryptococcal antigen (CRAG) lateral flow assay (LFA) had 100% sensitivity and specificity on cerebrospinal fluid samples. Pretreatment LFA titers correlated with quantitative cultures (R(2) = 0.7) and predicted 2- and 10-week mortality. The CRAG LFA is an accurate diagnostic assay for CSF and should be considered for point-of-care diagnosis of cryptococcal meningitis.

  14. Detection of mycobacterial DNA by a specific and simple lateral flow assay incorporating cadmium selenide quantum dots.

    PubMed

    Cimaglia, Fabio; Liandris, Emmanouil; Gazouli, Maria; Sechi, Leonardo; Chiesa, Maurizio; De Lorenzis, Enrico; Andreadou, Margarita; Taka, Styliani; Mataragka, Antonia; Ikonomopoulos, John

    2015-12-01

    Cadmium selenide quantum dots have been incorporated to a lateral flow assay for the specific and very simple detection of different mycobacterial DNA targets within only a few minutes, bypassing the complexity of conventional DNA hybridization assays. The method extends our previous work on protein detection using an identical procedure.

  15. Development and validation of a lateral flow assay for the detection of crustacean protein in processed foods.

    PubMed

    Koizumi, Daisuke; Shirota, Kazuya; Akita, Ryoko; Oda, Hiroshi; Akiyama, Hiroshi

    2014-05-01

    We developed and validated a novel lateral flow assay for the detection of crustacean protein in processed foods. This assay had high sensitivity; the visual detection limit for shrimp protein extract was 25μg/L, equivalent to 1μg/g protein in a food sample, and results could be obtained within 20min without sophisticated procedures or expensive equipment. Concordance between our assay and another validated quantitative enzyme-linked immunosorbent assay was 97% for commercially processed foods. This assay is rapid, simple, reliable, and highly correlated with validated enzyme-linked immunosorbent assays and is thus suitable for monitoring of food products, especially in food-processing facilities.

  16. Persistent Luminescence Strontium Aluminate Nanoparticles as Reporters in Lateral Flow Assays

    PubMed Central

    2015-01-01

    Demand for highly sensitive, robust diagnostics and environmental monitoring methods has led to extensive research in improving reporter technologies. Inorganic phosphorescent materials exhibiting persistent luminescence are commonly found in electroluminescent displays and glowing paints but are not widely used as reporters in diagnostic assays. Persistent luminescence nanoparticles (PLNPs) offer advantages over conventional photoluminescent probes, including the potential for enhanced sensitivity by collecting time-resolved measurements or images with decreased background autofluorescence while eliminating the need for expensive optical hardware, superior resistance to photobleaching, amenability to quantitation, and facile bioconjugation schemes. We isolated rare-earth doped strontium aluminate PLNPs from larger-particle commercial materials by wet milling and differential sedimentation and water-stabilized the particles by silica encapsulation using a modified Stöber process. Surface treatment with aldehyde silane followed by reductive amination with heterobifunctional amine-poly(ethylene glycol)-carboxyl allowed covalent attachment of proteins to the particles using standard carbodiimide chemistry. NeutrAvidin PLNPs were used in lateral flow assays (LFAs) with biotinylated lysozyme as a model analyte in buffer and monoclonal anti-lysozyme HyHEL-5 antibodies at the test line. Preliminary experiments revealed a limit of detection below 100 pg/mL using the NeutrAvidin PLNPs, which was approximately an order of magnitude more sensitive than colloidal gold. PMID:25247754

  17. Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay

    PubMed Central

    Jauset-Rubio, Miriam; Svobodová, Markéta; Mairal, Teresa; McNeil, Calum; Keegan, Neil; Saeed, Ayman; Abbas, Mohammad Nooredeen; El-Shahawi, Mohammad S.; Bashammakh, Abdulaziz S.; Alyoubi, Abdulrahman O.; O´Sullivan, Ciara K.

    2016-01-01

    Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10−11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need. PMID:27886248

  18. Orientational binding modes of reporters in a viral-nanoparticle lateral flow assay.

    PubMed

    Kim, Jinsu; Poling-Skutvik, Ryan; Trabuco, João R C; Kourentzi, Katerina; Willson, Richard C; Conrad, Jacinta C

    2016-12-19

    Using microscopy and image analysis, we characterize binding of filamentous viral nanoparticles to a fibrous affinity matrix as models for reporter capture in a lateral flow assay (LFA). M13 bacteriophage (M13) displaying an in vivo-biotinylated peptide (AviTag) genetically fused to the M13 tail protein p3 are functionalized with fluorescent labels. We functionalize glass fiber LFA membranes with antibodies to M13, which primarily capture M13 on the major p8 coat proteins, or with avidin, which captures M13 at the biotin-functionalized tail, and compare orientational modes of reporter capture for the side- versus tip-binding recognition interactions. The number of captured M13 is greater for side-binding than for tip-binding, as expected from the number of recognition groups. Whereas two-thirds of side-bound M13 captured by an anti-M13 antibody bind immediately after colliding with the membrane, tip-bound M13 prominently exhibit three additional orientational modes that require M13 to reorient to enable binding. These results are consistent with the idea that the elongated M13 shape couples with the complex flow field in an open and disordered fibrous LFA membrane to enhance capture.

  19. Apolipoprotein E genotyping using PCR-GoldMag lateral flow assay and its clinical applications

    PubMed Central

    Lian, Ting; Hui, Wenli; Li, Xianying; Zhang, Chao; Zhu, Juanli; Li, Rui; Wan, Yinsheng; Cui, Yali

    2016-01-01

    A polymerase chain reaction-gold magnetic nanoparticles lateral flow assay (PCR-GoldMag LFA) has been developed via integrating multiplex amplification refractory mutation system PCR (multi-ARMS-PCR) with GoldMag-based LFA for the visual detection of single-nucleotide polymorphisms (SNPs). This assay was applied to genotype Apolipoprotein E (ApoE). ApoE genotyping is important due to the predictive value for the development of coronary artery disease and Alzheimer's disease. The method requires two steps: i) Simultaneous amplifications of the two polymorphic codons (ApoE 158 and 112), performed in separated reactions using multi-ARMS-PCR; and ii) detection of the wild-type and mutant PCR products via dual immunoreactions, which can be performed in ~5 min. Within two LFAs, anti-digoxin antibody-conjugated GoldMag probes bind digoxin-labeled wild-type PCR products, and anti-fluorescein isothiocyanate (FITC) antibody-conjugated GoldMag probes bind FITC-labeled mutant PCR products. All PCR products are biotin labeled and are detected by streptavidin-coated regions on the LFA strip, resulting in a red color. The current approach is capable of detecting the SNPs of ApoE in ~1.5 h, with a broad detection range from 10–1,000 ng of genomic DNA. Thus, the present protocol may facilitate simple, fast and cost-effective screening for important SNPs, as demonstrated by the evaluation of the prevalence of ApoE variants in a Han Chinese cohort. PMID:27665864

  20. Quantitative lateral flow strip assays as User-Friendly Tools To Detect Biomarker Profiles For Leprosy

    PubMed Central

    van Hooij, Anouk; Tjon Kon Fat, Elisa M.; Richardus, Renate; van den Eeden, Susan J. F.; Wilson, Louis; de Dood, Claudia J.; Faber, Roel; Alam, Korshed; Richardus, Jan Hendrik; Corstjens, Paul L. A. M.; Geluk, Annemieke

    2016-01-01

    Leprosy is a debilitating, infectious disease caused by Mycobacterium leprae. Despite the availability of multidrug therapy, transmission is unremitting. Thus, early identification of M. leprae infection is essential to reduce transmission. The immune response to M. leprae is determined by host genetics, resulting in paucibacillary (PB) and multibacillary (MB) leprosy associated with dominant cellular or humoral immunity, respectively. This spectral pathology of leprosy compels detection of immunity to M. leprae to be based on multiple, diverse biomarkers. In this study we have applied quantitative user friendly lateral flow assays (LFAs) for four immune markers (anti-PGL-I antibodies, IL-10, CCL4 and IP-10) for whole blood samples from a longitudinal BCG vaccination field-trial in Bangladesh. Different biomarker profiles, in contrast to single markers, distinguished M. leprae infected from non-infected test groups, patients from household contacts (HHC) and endemic controls (EC), or MB from PB patients. The test protocol presented in this study merging detection of innate, adaptive cellular as well as humoral immunity, thus provides a convenient tool to measure specific biomarker profiles for M. leprae infection and leprosy utilizing a field-friendly technology. PMID:27682181

  1. Multisite validation of cryptococcal antigen lateral flow assay and quantification by laser thermal contrast.

    PubMed

    Boulware, David R; Rolfes, Melissa A; Rajasingham, Radha; von Hohenberg, Maximilian; Qin, Zhenpeng; Taseera, Kabanda; Schutz, Charlotte; Kwizera, Richard; Butler, Elissa K; Meintjes, Graeme; Muzoora, Conrad; Bischof, John C; Meya, David B

    2014-01-01

    Cryptococcal meningitis is common in sub-Saharan Africa. Given the need for data for a rapid, point-of-care cryptococcal antigen (CRAG) lateral flow immunochromatographic assay (LFA), we assessed diagnostic performance of cerebrospinal fluid (CSF) culture, CRAG latex agglutination, India ink microscopy, and CRAG LFA for 832 HIV-infected persons with suspected meningitis during 2006-2009 (n = 299) in Uganda and during 2010-2012 (n = 533) in Uganda and South Africa. CRAG LFA had the best performance (sensitivity 99.3%, specificity 99.1%). Culture sensitivity was dependent on CSF volume (82.4% for 10 μL, 94.2% for 100 μL). CRAG latex agglutination test sensitivity (97.0%-97.8%) and specificity (85.9%-100%) varied between manufacturers. India ink microscopy was 86% sensitive. Laser thermal contrast had 92% accuracy (R = 0.91, p<0.001) in quantifying CRAG titers from 1 LFA strip to within <1.5 dilutions of actual CRAG titers. CRAG LFA is a major advance for meningitis diagnostics in resource-limited settings.

  2. Multisite Validation of Cryptococcal Antigen Lateral Flow Assay and Quantification by Laser Thermal Contrast

    PubMed Central

    Rolfes, Melissa A.; Rajasingham, Radha; von Hohenberg, Maximilian; Qin, Zhenpeng; Taseera, Kabanda; Schutz, Charlotte; Kwizera, Richard; Butler, Elissa K.; Meintjes, Graeme; Muzoora, Conrad; Bischof, John C.; Meya, David B.

    2014-01-01

    Cryptococcal meningitis is common in sub-Saharan Africa. Given the need for data for a rapid, point-of-care cryptococcal antigen (CRAG) lateral flow immunochromatographic assay (LFA), we assessed diagnostic performance of cerebrospinal fluid (CSF) culture, CRAG latex agglutination, India ink microscopy, and CRAG LFA for 832 HIV-infected persons with suspected meningitis during 2006–2009 (n = 299) in Uganda and during 2010–2012 (n = 533) in Uganda and South Africa. CRAG LFA had the best performance (sensitivity 99.3%, specificity 99.1%). Culture sensitivity was dependent on CSF volume (82.4% for 10 μL, 94.2% for 100 μL). CRAG latex agglutination test sensitivity (97.0%–97.8%) and specificity (85.9%–100%) varied between manufacturers. India ink microscopy was 86% sensitive. Laser thermal contrast had 92% accuracy (R = 0.91, p<0.001) in quantifying CRAG titers from 1 LFA strip to within <1.5 dilutions of actual CRAG titers. CRAG LFA is a major advance for meningitis diagnostics in resource-limited settings. PMID:24378231

  3. Parallel, open-channel lateral flow (immuno) assay substrate based on capillary-channeled polymer films.

    PubMed

    Zhang, Lynn X; Jiang, Liuwei; Willett, Daniel R; Marcus, R Kenneth

    2016-02-07

    Presented here is a novel implementation of polypropylene capillary-channeled polymer (C-CP) films, functionalized for bioaffinity separations and implemented as a platform for lateral flow (immuno) assays. The parallel ∼80 μm × 80 μm channels pass test solutions down the 30 mm film length via spontaneous wicking action, setting up the possibility for immobilizing different capture agents in the respective channels. The base-film modification process is divided into two steps: ultraviolet light treatment to improve hydrophillicity of the polypropylene substrate and the physical adsorption of a functionalized lipid tethered ligand (LTL) as a selective capture agent. The entire modification procedure is performed under ambient conditions in an aqueous solution without extreme pH conditions. In this demonstration, physical adsorption of a biotinylated-LTL onto the UV-treated PP surface selectively captures Texas Red-labeled streptavidin (SAv-TR) in the presence of enhanced green fluorescence protein (EGFP), which passes without retention in less than 5 s. In addition to the fluorescence imaging of the protein solutes, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to confirm the formation of the LTL-SAv conjugates on the channel surface as well as to demonstrate an alternative means of probing the capture step. The present effort sets the groundwork for further development of C-CP films as a parallel, multi-analyte LFA platform; a format that to-date has not been described.

  4. Sensitive biomolecule detection in lateral flow assay with a portable temperature-humidity control device.

    PubMed

    Choi, Jane Ru; Hu, Jie; Feng, Shangsheng; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda; Xu, Feng

    2016-05-15

    Lateral flow assays (LFAs) have currently attracted broad interest for point-of-care (POC) diagnostics, but their application has been restricted by poor quantification and limited sensitivity. While the former has been currently solved to some extent by the development of handheld or smartphone-based readers, the latter has not been addressed fully, particularly the potential influences of environmental conditions (e.g., temperature and relative humidity (RH)), which have not yet received serious attention. The present study reports the use of a portable temperature-humidity control device to provide an optimum environmental requirement for sensitivity improvement in LFAs, followed by quantification by using a smartphone. We found that a RH beyond 60% with temperatures of 55-60°C and 37-40°C produced optimum nucleic acid hybridization and antigen-antibody interaction in LFAs, respectively representing a 10-fold and 3-fold signal enhancement over ambient conditions (25°C, 60% RH). We envision that in the future the portable device could be coupled with a fully integrated paper-based sample-to-answer biosensor for sensitive detection of various target analytes in POC settings.

  5. Flow reproducibility of whole blood and other bodily fluids in simplified no reaction lateral flow assay devices.

    PubMed

    Li, H; Han, D; Hegener, M A; Pauletti, G M; Steckl, A J

    2017-03-01

    The "no reaction" lateral flow assay (nrLFA) uses a simplified LFA structure with no conjugate pad and no stored reagents. In the nrLFA, the capillary-based transport time or distance is the key indicator, rather than the outcome of a biochemical reaction. Hence, the calibration and reproducibility of the nrLFA device are critical. The capillary flow properties of several membrane types (nitrocellulose, nylon, cellulose acetate, polyethersulfone, and polyvinylidene difluoride) are evaluated. Flow rate evaluations of MilliporeSigma Hi-Flow™ Plus (HF075, HF135 and HF180) nitrocellulose membranes on nrLFA are performed using bodily fluids (whole blood, blood plasma, and artificial sweat). The results demonstrate that fluids with lower viscosity travel faster, and membranes with slower flow rate exhibit higher capability to distinguish fluids with different viscosities. Reproducibility tests of nrLFA are performed on HF075, demonstrating excellent reproducibility. The coefficient of variation for blood coagulation tests performed with the nrLFA using induced coagulation was 5% for the plasma front and 2% for the RBC front. The effects of variation in blood hematocrit and sample volume are also reported. The overall results indicate that the nrLFA approach has a high potential to be commercially developed as a blood monitoring point-of-care device with simple calibration capability and excellent reproducibility.

  6. Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers.

    PubMed

    Jauset-Rubio, Miriam; Svobodová, Markéta; Mairal, Teresa; McNeil, Calum; Keegan, Neil; El-Shahawi, Mohammad S; Bashammakh, Abdulaziz S; Alyoubi, Abdulrahman O; O'Sullivan, Ciara K

    2016-11-01

    In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which β-conglutin immobilized on the test line of a nitrocellulose membrane and β-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the β-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized β-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

  7. Development of a novel lateral flow assay for detection of African swine fever in blood.

    PubMed

    Sastre, P; Gallardo, C; Monedero, A; Ruiz, T; Arias, M; Sanz, A; Rueda, P

    2016-09-15

    African swine fever (ASF) is a viral infectious disease of domestic and wild suids of all breeds and ages, causing a wide range of hemorrhagic syndromes and frequently characterized by high mortality. The disease is endemic in Sub-Saharan Africa and Sardinia. Since 2007, it has also been present in different countries of Eastern Europe, where control measures have not been effective so far. The continued spread poses a serious threat to the swine industry worldwide. In the absence of vaccine, early detection of infected animals is of paramount importance for control of the outbreak, to prevent the transmission of the virus to healthy animals and subsequent spreading of the disease. Current laboratory diagnosis is mainly based on virological methods (antigen and genome detection) and serodiagnosis. In the present work, a Lateral Flow Assay (LFA) for antigen detection has been developed and evaluated. The test is based on the use of a MAb against VP72 protein of ASFV, the major viral capsid protein and highly immunogenic. First experiments using VP72 viral and recombinant protein or inactivated culture virus showed promising results with a sensitivity similar to that of a commercially available Antigen-ELISA. Moreover, these strips were tested with blood from experimentally infected pigs and field animals and the results compared with those of PCR and Antigen-ELISA. For the experimentally infected samples, there was an excellent correlation between the LFA and the ELISA, while the PCR always showed to be more sensitive (38 % positive samples by PCR versus 27 % by LFA). The LFA was demonstrated to be positive for animals with circulating virus levels exceeding 10(4) HAU. With the field samples, once again, the PCR detected more positives than either the Antigen-ELISA or LFA, although here the number of positive samples scored by the LFA exceeded the values obtained with the Antigen-ELISA, showing 60 % positivity vs 48 % for the ELISA. For the two groups of sera

  8. Cell-phone-based measurement of TSH using Mie scatter optimized lateral flow assays.

    PubMed

    You, David J; Park, Tu San; Yoon, Jeong-Yeol

    2013-02-15

    Semi-quantitative thyr oid stimulating hormone (TSH) lateral flow immunochromatographic assays (LFA) are used to screen for serum TSH concentration >5 mIUL(-1) (hypothyroidism). The LFA format, however, is unable to measure TSH in the normal range or detect suppressed levels of TSH (<0.4 mIU L(-1); hyperthyroidism). In fact, it does not provide quantitative TSH values at all. Obtaining quantitative TSH results, especially in the low concentration range, has until now required the use of centralized clinical laboratories which require specimen transport, specialized equipment and personnel, and result in increased cost and delays in the timely reporting of important clinical results. We have conducted a series of experiments to develop and validate an optical system and image analysis algorithm based upon a cell phone platform. It is able to provide point-of-care quantitative TSH results with a high level of sensitivity and reproducibility comparable to that of a clinical laboratory-based third-generation TSH immunoassay. Our research approach uses the methodology of the optimized Rayleigh/Mie scatter detection by taking into consideration the optical characteristics of a nitrocellulose membrane and gold nanoparticles on an LFA for quantifying TSH levels. Using a miniature spectrometer, LED light source, and optical fibers on a rotating benchtop apparatus, the light intensity from different angles of incident light and angles of detection to the LFA were measured. The optimum angles were found that the minimized Mie scattering from nitrocellulose membrane, consequently maximizes the Rayleigh scatter detection from the gold nanoparticles in the LFA bands. Using the results from the benchtop apparatus, a cell-phone-based apparatus was designed which utilized the embedded flash in the cell phone camera as the light source, piped the light with an optical fiber from the flash through a collimating lens to illuminate the LFA. Quantification of TSH was performed in an i

  9. Lateral flow urine lipoarabinomannan assay for detecting active tuberculosis in Hiv-positive adults

    PubMed Central

    Shah, Maunank; Hanrahan, Colleen; Wang, Zhuo Yu; Dendukuri, Nandini; Lawn, Stephen D; Denkinger, Claudia M; Steingart, Karen R

    2016-01-01

    Background Rapid detection of tuberculosis (TB) among people living with human immunodeficiency virus (HIV) is a global health priority. HIV-associated TB may have different clinical presentations and is challenging to diagnose. Conventional sputum tests have reduced sensitivity in HIV-positive individuals, who have higher rates of extrapulmonary TB compared with HIV-negative individuals. The lateral flow urine lipoarabinomannan assay (LF-LAM) is a new, commercially available point-of-care test that detects lipoarabinomannan (LAM), a lipopolysaccharide present in mycobacterial cell walls, in people with active TB disease. Objectives To assess the accuracy of LF-LAM for the diagnosis of active TB disease in HIV-positive adults who have signs and symptoms suggestive of TB (TB diagnosis).To assess the accuracy of LF-LAM as a screening test for active TB disease in HIV-positive adults irrespective of signs and symptoms suggestive of TB (TB screening). Search methods We searched the following databases without language restriction on 5 February 2015: the Cochrane Infectious Diseases Group Specialized Register; MEDLINE (PubMed,1966); EMBASE (OVID, from 1980); Science Citation Index Expanded (SCI-EXPANDED, from 1900), Conference Proceedings Citation Index-Science (CPCI-S, from 1900), and BIOSIS Previews (from 1926) (all three using the Web of Science platform; MEDION; LILACS (BIREME, from 1982); SCOPUS (from 1995); the metaRegister of Controlled Trials (mRCT); the search portal of the World Health Organization International Clinical Trials Registry Platform (WHO ICTRP); and ProQuest Dissertations & Theses A&l (from 1861). Selection criteria Eligible study types included randomized controlled trials, cross-sectional studies, and cohort studies that determined LF-LAM accuracy for TB against a microbiological reference standard (culture or nucleic acid amplification test from any body site). A higher quality reference standard was one in which two or more specimen types were

  10. Effects of blood sample anticoagulants on lateral flow assays using luminescent photon-upconverting and Eu(III) nanoparticle reporters.

    PubMed

    Juntunen, Etvi; Arppe, Riikka; Kalliomäki, Laura; Salminen, Teppo; Talha, Sheikh M; Myyryläinen, Tiina; Soukka, Tero; Pettersson, Kim

    2016-01-01

    Many quantitative and semiquantitative lateral flow (LF) assays have been introduced for clinical analytes such as biomarkers for cancer or acute myocardial infarction (AMI). Various detection technologies involving quantitative analyzing devices have been reported to have sufficient analytical sensitivity and quantification capability for clinical point-of-care tests. Fluorescence-based detection technologies such as quantum dots, Eu(III) nanoparticles, and photon-upconverting nanoparticles (UCNPs) have been introduced as promising solutions for point-of-care devices because of their high detectability by optical sensors. Lateral flow assays can be used for various sample types, e.g., urine, saliva, cerebrospinal fluid, and blood. This study focuses on the properties of serum and plasma because of their relevance in cancer and AMI diagnostics. The limit of detection was compared in LF assays having Eu(III) nanoparticles or UCNPs as reporters and the antibody configurations for two different analytes (prostate-specific antigen and cardiac troponin I (cTnI)). The results indicate a significant effect of anticoagulants in venipuncture tubes. The samples in K3EDTA tubes resulted in significant interference by decreased reporter particle mobility, and thus the limit of detection was up to eightfold less sensitive compared to serum samples. Despite the matrix interference in the cTnI assay with UCNP reporters, limits of detection of 41 ng/L with serum and 66 ng/L with the Li-heparin sample were obtained.

  11. Development of enzyme-based bar code-style lateral-flow assay for hydrogen peroxide determination.

    PubMed

    Fung, Ka-Kei; Chan, Cangel Pui-Yee; Renneberg, Reinhard

    2009-02-16

    A unique approach of developing a bar code version of lateral-flow enzymatic-based assay for the semi-quantification of hydrogen peroxide is described. The proposed assay system is mainly composed of a goat anti-mouse IgG-horseradish peroxidase conjugate (Gt anti-M IgG-HRP)-coated nitrocellulose (NC) membrane and a peroxidase substrate pad. Unlike the bar code immunochromatographic assay which depends on the stepwise capture of analyte, the principle of enzyme-based bar code lateral-flow assay is based on the different reaction time on successive lines due to the delay in 3,3',5,5'-tetramethylbenzidine (TMB) release. Hydrogen peroxide (H(2)O(2)) acts as a limiting factor which controls the rate of the enzymatic conversion of TMB to blue color complex. The system expresses the concentration of H(2)O(2) in micromole range as three distinct ladder bars in 9 min therefore without the need of any reading device. The major advantages of this assay are its easily readable result, and also its simplicity and low-cost in production offers a cheaper alternative for testing those expensive biosensors might not be available to the third world countries. By incorporating with H(2)O(2)-generating oxidoreductases, the assay can be further extended to detect a variety of analytes with clinical and environmental importance. Glucose was chosen to be the model analyte where the proposed system gave signal response at between 5 microM and 100 microM.

  12. A novel multi-walled carbon nanotube-based antibody conjugate for quantitative and semi-quantitative lateral flow assays.

    PubMed

    Sun, Wenjuan; Hu, Xiaolong; Liu, Jia; Zhang, Yurong; Lu, Jianzhong; Zeng, Libo

    2017-10-01

    In this study, the multi-walled carbon nanotubes (MWCNTs) were applied in lateral flow strips (LFS) for semi-quantitative and quantitative assays. Firstly, the solubility of MWCNTs was improved using various surfactants to enhance their biocompatibility for practical application. The dispersed MWCNTs were conjugated with the methamphetamine (MET) antibody in a non-covalent manner and then manufactured into the LFS for the quantitative detection of MET. The MWCNTs-based lateral flow assay (MWCNTs-LFA) exhibited an excellent linear relationship between the values of test line and MET when its concentration ranges from 62.5 to 1500 ng/mL. The sensitivity of the LFS was evaluated by conjugating MWCNTs with HCG antibody and the MWCNTs conjugated method is 10 times more sensitive than the one conjugated with classical colloidal gold nanoparticles. Taken together, our data demonstrate that MWCNTs-LFA is a more sensitive and reliable assay for semi-quantitative and quantitative detection which can be used in forensic analysis.

  13. A novel nucleic lateral flow assay for screening of PHA-producing haloarchaea.

    PubMed

    Muangsuwan, Wannaporn; Ruangsuj, Pattarawan; Chaichanachaicharn, Pichai; Yasawong, Montri

    2015-09-01

    Polyhydroxyalkanoates (PHAs) are important for biodegradable plastic production, and prokaryotes play a very important role in PHA production. PHA synthase is a key enzyme for the polymerization of PHAs. There are four classes of PHA synthase. The phaC gene is necessary for the production of all classes of PHA synthase, whereas the phaE gene is necessary for the production of class III PHA synthase. This gene is a biomarker for microorganisms that contain class III PHA synthase, such as haloarchaea. Standard techniques for screening of PHA-producing haloarchaea require time for culturing and have poor specificity and sensitivity. Thus, the phaE biosensor was developed to overcome these issues. PCR and DNA lateral flow biosensor techniques were combined for construction of the phaE biosensor. The phaE biosensor has a high specificity for PHA-producing haloarchaea. The lowest amount of genomic DNA of Haloquadratum walsbyi DSM 16854 that the phaE gene could be detected by the biosensor was approximately 250 fg. The phaE biosensor can be applied for screening of PHA-producing haloarchaea from environmental samples. The phaE biosensor is easy to handle and dispose. For screening PHA-producing haloarchaea, the phaE biosensor requires less time and costs less than the standard methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies. PMID:27819063

  15. Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels

    PubMed Central

    CORSTJENS, PAUL L. A. M.; DE DOOD, CLAUDIA J.; KORNELIS, DIEUWKE; FAT, ELISA M. TJON KON; WILSON, R. ALAN; KARIUKI, THOMAS M.; NYAKUNDI, RUTH K.; LOVERDE, PHILIP T.; ABRAMS, WILLIAM R.; TANKE, HANS J.; VAN LIESHOUT, LISETTE; DEELDER, ANDRÉ M.; VAN DAM, GOVERT J.

    2014-01-01

    SUMMARY The potential of various quantitative lateral flow (LF) based assays utilizing up-converting phosphor (UCP) reporters for the diagnosis of schistosomiasis is reviewed including recent developments. Active infections are demonstrated by screening for the presence of regurgitated worm antigens (genus specific polysaccharides), whereas anti-Schistosoma antibodies may indicate ongoing as well as past infections. The circulating anodic antigen (CAA) in serum or urine (and potentially also saliva) is identified as the marker that may allow detection of single-worm infections. Quantitation of antigen levels is a reliable method to study effects of drug administration, worm burden and anti-fecundity mechanisms. Moreover, the ratio of CAA and circulating cathodic antigen (CCA) is postulated to facilitate identification of either Schistosoma mansoni or Schistosoma haematobium infections. The UCP-LF assays allow simultaneous detection of multiple targets on a single strip, a valuable feature for antibody detection assays. Although antibody detection in endemic regions is not a useful tool to diagnose active infections, it gains potential when the ratio of different classes of antibody specific for the parasite/disease can be determined. The UCP-LF antibody assay format allows this type of multiplexing, including testing a linear array of up to 20 different targets. Multiple test spots would allow detection of specific antibodies, e.g. against different Schistosoma species or other pathogens as soil-transmitted helminths. Concluding, the different UCP-LF based assays for diagnosis of schistosomiasis provide a collection of tests with relatively low complexity and high sensitivity, covering the full range of diagnostics needed in control programmes for mapping, screening and monitoring. PMID:24932595

  16. Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels.

    PubMed

    Corstjens, Paul L A M; De Dood, Claudia J; Kornelis, Dieuwke; Fat, Elisa M Tjon Kon; Wilson, R Alan; Kariuki, Thomas M; Nyakundi, Ruth K; Loverde, Philip T; Abrams, William R; Tanke, Hans J; Van Lieshout, Lisette; Deelder, André M; Van Dam, Govert J

    2014-12-01

    The potential of various quantitative lateral flow (LF) based assays utilizing up-converting phosphor (UCP) reporters for the diagnosis of schistosomiasis is reviewed including recent developments. Active infections are demonstrated by screening for the presence of regurgitated worm antigens (genus specific polysaccharides), whereas anti-Schistosoma antibodies may indicate ongoing as well as past infections. The circulating anodic antigen (CAA) in serum or urine (and potentially also saliva) is identified as the marker that may allow detection of single-worm infections. Quantitation of antigen levels is a reliable method to study effects of drug administration, worm burden and anti-fecundity mechanisms. Moreover, the ratio of CAA and circulating cathodic antigen (CCA) is postulated to facilitate identification of either Schistosoma mansoni or Schistosoma haematobium infections. The UCP-LF assays allow simultaneous detection of multiple targets on a single strip, a valuable feature for antibody detection assays. Although antibody detection in endemic regions is not a useful tool to diagnose active infections, it gains potential when the ratio of different classes of antibody specific for the parasite/disease can be determined. The UCP-LF antibody assay format allows this type of multiplexing, including testing a linear array of up to 20 different targets. Multiple test spots would allow detection of specific antibodies, e.g. against different Schistosoma species or other pathogens as soil-transmitted helminths. Concluding, the different UCP-LF based assays for diagnosis of schistosomiasis provide a collection of tests with relatively low complexity and high sensitivity, covering the full range of diagnostics needed in control programmes for mapping, screening and monitoring.

  17. Staphylococcal enterotoxin B-specific electrochemiluminescence and lateral flow device assays cross-react with staphylococcal enterotoxin D.

    PubMed

    Tallent, Sandra M; Hait, Jennifer; Bennett, Reginald W

    2014-01-01

    Guam school children and faculty members experienced symptoms of vomiting, nausea, abdominal cramps, and diarrhea shortly after eating breakfast prepared by contracted caterers. The first illness was reported within an hour after breakfast, affecting 295 students and two faculty members. Local hospitals treated 130 people, and 61 were admitted for further treatment. Reported symptoms were consistent with staphylococcal food poisoning. Initial food testing using a lateral flow device and electrochemiluminescence method incorrectly implicated staphylococcal enterotoxin B as the causative agent, prompting partial activation of Guam's Emergency Response Center. Traditional ELISAs proved that the food poisoning agent was staphylococcal enterotoxin D. More specific and sensitive assays would have alleviated the issues and confusion that surrounded the reporting and investigation of this outbreak.

  18. Development of Lateral Flow Assay Based on Size-Controlled Gold Nanoparticles for Detection of Hepatitis B Surface Antigen

    PubMed Central

    Kim, Dong Seok; Kim, Yong Tae; Hong, Seok Bok; Kim, Jinwoon; Heo, Nam Su; Lee, Moon-Keun; Lee, Seok Jae; Kim, Byeong Il; Kim, In Soo; Huh, Yun Suk; Choi, Bong Gill

    2016-01-01

    In this study, we developed lateral flow assay (LFA) biosensors for the detection of hepatitis B surface antigens using well-controlled gold nanoparticles (AuNPs). To enhance colorimetric signals, a seeded growth method was used for the preparation of size-controlled AuNPs with a narrow size distribution. Different sizes of AuNPs in the range of 342–137.8 nm were conjugated with antibodies and then optimized for the efficient detection of LFA biosensors. The conjugation stability was investigated by UV-vis spectroscopy of AuNP dispersion at various pH values and concentrations of antibody. Based on optimized conjugation conditions, the use of 42.7 ± 0.8 nm AuNPs exhibited superior performance for the detection of LFAs relative to other sizes of AuNPs. PMID:27999291

  19. Rapid detection of abrin in foods with an up-converting phosphor technology-based lateral flow assay

    PubMed Central

    Liu, Xiao; Zhao, Yong; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Zhang, Pingping; Qiu, Jingfu; Yang, Ruifu; Zhou, Lei

    2016-01-01

    Abrin is a natural plant toxin found in the seeds of Abrus precatorius. It may be used for food poisoning or bioterrorism, seriously endangering public health. In this study, a reliable method for the rapid detection of abrin in foods was developed, based on an up-converting phosphor technology-based lateral flow assay (abrin-UPT-LFA). Nine high-affinity monoclonal antibodies (mAbs) against abrin were prepared, and the optimum mAbs (mAb-6F4 and mAb-10E11) were selected for use in the assay in double-antibody-sandwich mode. The assay was confirmed to be specific for abrin, with a detection sensitivity of 0.1 ng mL−1 for standard abrin solutions. Good linearity was observed for abrin quantitation from 0.1 to 1000 ng mL−1 (r = 0.9983). During the analysis of various abrin-spiked food samples, the assay showed strong sample tolerance and a satisfactory limit of detection for abrin (0.5–10 ng g−1 for solid and powdered samples; 0.30–0.43 ng mL−1 for liquid samples). The analysis of suspected food samples, from sample treatment to result feed-back, could be completed by non-professionals within 20 min. Therefore, the abrin-UPT-LFA is a rapid, sensitive, and reliable method for the on-site detection of abrin in foods. PMID:27703269

  20. Advantages of fluorescent microspheres compared with colloidal gold as a label in immunochromatographic lateral flow assays.

    PubMed

    Xie, Quan-Yuan; Wu, Yan-Hua; Xiong, Qi-Rong; Xu, Heng-Yi; Xiong, Yong-Hua; Liu, Kun; Jin, Yong; Lai, Wei-Hua

    2014-04-15

    Label selection is of vital importance for immunochromatographic assays. In this study, the fluorescent microsphere test strip and colloidal gold immunochromatographic test strip (FM-ICTS and CG-ICTS) were developed for the detection of Escherichia coli O157:H7 on the basis of the sandwich format. Two types of labels, namely, colloidal gold particles (CG) and carboxyl-modified fluorescent microspheres (FMs), were compared while coupling with anti-E. coli O157:H7 monoclonal antibody (mAb). The FM-ICTS and CG-ICTS were also compared. Results show that the coupling rate between FMs and mAb was higher than that between CG and mAb. Under optimum conditions, the sensitivity of FM-ICTS was eight times higher than that of CG-ICTS. Approximately 0.1 μg of mAb was used in every FM-ICTS, whereas 0.4 μg of mAb was used in every CG-ICTS. The coefficient of variation of FM-ICTS and CG-ICTS was 4.8% and 16.7%, respectively. The FM-ICTS and CG-ICTS can be stored at room temperature for 12 months and specific to five E. coli O157:H7 strains. Milk sample inoculated with E. coli O157:H7 were tested by the FM-ICTS and CG-ICTS. The FM-ICTS sensitivity was 10(4) CFU/ml while the CG-ICTS sensitivity was 10(5) CFU/ml. The sensitivity, consumption of antibodies, and coefficient of variation of FM-ICTS were better than those of CG-ICTS for the detection of E. coli O157:H7. © 2013 Published by Elsevier B.V.

  1. Simple and rapid lateral-flow assay for the detection of foot-and-mouth disease virus.

    PubMed

    Oem, Jae Ku; Ferris, Nigel P; Lee, Kwang-Nyeong; Joo, Yi-Seok; Hyun, Bang-Hun; Park, Jong-Hyeon

    2009-11-01

    A simple lateral-flow assay (LFA) based on a monoclonal antibody (MAb 70-17) was developed for the detection of foot-and-mouth disease virus (FMDV) under nonlaboratory conditions. The LFA was evaluated with epithelial suspensions (n = 704) prepared from current and historical field samples which had been submitted to the Pirbright Laboratory (United Kingdom) and from negative samples (n = 100) collected from naïve animals in Korea. Four FMDV serotypes (type O, A, Asia 1, and C) were detected in the LFA, but not the remaining three FMDV serotypes (SAT 1, SAT 2, and SAT 3). The diagnostic sensitivity of the LFA for FMDV types O, A, C, and Asia 1 was similar, at approximately 87.3%, to that of 87.7% obtained with antigen enzyme-linked immunosorbent assay (Ag-ELISA). The diagnostic specificity of the LFA was 98.8%, compared to 100% for the Ag-ELISA. These results demonstrate that the LFA using the FMDV MAb 70-17 to detect FMDV is a supportive method for taking rapid measurements at the site of a suspected foot-and-mouth disease outbreak in Asia before diagnosing the disease in the laboratory, thereby offering the possibility of implementing control procedures more rapidly.

  2. Development of a duplex lateral flow assay for simultaneous detection of antibodies against African and Classical swine fever viruses.

    PubMed

    Sastre, Patricia; Pérez, Teresa; Costa, Sofia; Yang, Xiaoping; Räber, Alex; Blome, Sandra; Goller, Katja V; Gallardo, Carmina; Tapia, Istar; García, Julia; Sanz, Antonio; Rueda, Paloma

    2016-09-01

    Classical swine fever (CSF) and African swine fever (ASF) are both highly contagious diseases of domestic pigs and wild boar and are clinically indistinguishable. For both diseases, antibody detection is an integral and crucial part of prevention and control measures. The purpose of our study was to develop and initially validate a duplex pen-side test for simultaneous detection and differentiation of specific antibodies against CSF virus (CSFV) and ASF virus (ASFV). The test was based on the major capsid protein VP72 of ASFV and the structural protein E2 of CSFV, both considered the most immunogenic proteins of these viruses. The performance of the pen-side test was evaluated using a panel of porcine samples consisting of experimental, reference, and field sera, with the latter collected from European farms free of both diseases. The new lateral flow assay was able to detect specific antibodies to ASFV or CSFV, showing good levels of sensitivity and specificity. These preliminary data indicate the potential of the newly developed pen-side test for rapid differential detection of antibodies found in the 2 diseases, which is of particular importance in the field and in front-line laboratories where equipment and skilled personnel are limited and control of ASF and CSF is crucial. © 2016 The Author(s).

  3. Rapid Identification of OXA-48 and OXA-163 Subfamilies in Carbapenem-Resistant Gram-Negative Bacilli with a Novel Immunochromatographic Lateral Flow Assay

    PubMed Central

    Denorme, Laurence; Ote, Isabelle; Gomez, Sonia; De Belder, Denise; Glupczynski, Youri; Bogaerts, Pierre; Ghiglione, Barbara; Power, Pablo; Mertens, Pascal; Corso, Alejandra

    2016-01-01

    We assessed a novel immunochromatographic lateral flow assay for direct identification of OXA-48-like carbapenemases and accurate differentiation of allele variants with distinct substrate profiles (OXA-48 or OXA-163 subfamilies). The assay allowed rapid (less than 4 min) and reliable direct confirmation of OXA-163- and/or OXA-48-like enzymes (with 100% sensitivity and 100% specificity) from cultured colonies that were recovered from both solid medium and spiked blood culture bottles. PMID:27535687

  4. Evaluation of an Immunochromatographic Lateral Flow Assay (OXA-48 K-SeT) for Rapid Detection of OXA-48-Like Carbapenemases in Enterobacteriaceae

    PubMed Central

    Shah, Rishita; Betts, Jonathan W.; Phee, Lynette M.; Momin, Muhd Haziq F. Abdul

    2015-01-01

    We evaluated an immunochromatographic lateral flow assay to detect OXA-48-like carbapenemases (OXA-48 K-SeT) in Enterobacteriaceae (n = 82). One hundred percent sensitivity and specificity were observed using bacteria recovered from both solid medium and spiked blood culture bottles, and the results were obtained in <10 min. PMID:26607983

  5. Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus.

    PubMed

    Yang, Yang; Qin, Xiaodong; Zhang, Xiangle; Zhao, Zhixun; Zhang, Wei; Zhu, Xueliang; Cong, Guozheng; Li, Yanmin; Zhang, Zhidong

    2017-07-17

    Goatpox virus (GTPV) and sheeppox virus (SPPV), which belong to the Capripoxvirus (CaPV), are economically important pathogens of small ruminants. Therefore, a sensitive, specific and rapid diagnostic assay for detection of GTPV and SPPV is necessary to accurately and promptly control these diseases. Recombinase polymerase amplification (RPA) assays combined with a real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the CaPV G-protein-coupled chemokine receptor (GPCR) gene, respectively. The sensitivity of both CaPV real-time RPA assay and CaPV RPA LFD assay were 3 × 10(2) copies per reaction within 20 min at 38 °C. Both assays were highly specific for CaPV, with no cross-reactions with peste des petits ruminants virus, foot-and-mouth disease virus and Orf virus. The evaluation of the performance of these two assays with clinical sample (n = 107) showed that the CaPV real-time RPA assay and CaPV RPA LFD assay were able to specially detect SPPV or GTPV present in samples of ovine in liver, lung, kidney, spleen, skin and blood. This study provided a highly time-efficient and simple alternative for rapid detection of GTPV and SPPV.

  6. Rapid Identification of OXA-48 and OXA-163 Subfamilies in Carbapenem-Resistant Gram-Negative Bacilli with a Novel Immunochromatographic Lateral Flow Assay.

    PubMed

    Pasteran, Fernando; Denorme, Laurence; Ote, Isabelle; Gomez, Sonia; De Belder, Denise; Glupczynski, Youri; Bogaerts, Pierre; Ghiglione, Barbara; Power, Pablo; Mertens, Pascal; Corso, Alejandra

    2016-11-01

    We assessed a novel immunochromatographic lateral flow assay for direct identification of OXA-48-like carbapenemases and accurate differentiation of allele variants with distinct substrate profiles (OXA-48 or OXA-163 subfamilies). The assay allowed rapid (less than 4 min) and reliable direct confirmation of OXA-163- and/or OXA-48-like enzymes (with 100% sensitivity and 100% specificity) from cultured colonies that were recovered from both solid medium and spiked blood culture bottles. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. Development of real-time and lateral flow strip reverse transcription recombinase polymerase Amplification assays for rapid detection of peste des petits ruminants virus.

    PubMed

    Yang, Yang; Qin, Xiaodong; Song, Yiming; Zhang, Wei; Hu, Gaowei; Dou, Yongxi; Li, Yanmin; Zhang, Zhidong

    2017-02-07

    Peste des petits ruminants (PPR) is an economically important, Office International des Epizooties (OIE) notifiable, transboundary viral disease of small ruminants such as sheep and goat. PPR virus (PPRV), a negative-sense single-stranded RNA virus, is the causal agent of PPR. Therefore, sensitive, specific and rapid diagnostic assay for the detection of PPRV are necessary to accurately and promptly diagnose suspected case of PPR. In this study, reverse transcription recombinase polymerase amplification assays using real-time fluorescent detection (real-time RT-RPA assay) and lateral flow strip detection (LFS RT-RPA assay) were developed targeting the N gene of PPRV. The sensitivity of the developed real-time RT-RPA assay was as low as 100 copies per reaction within 7 min at 40 °C with 95% reliability; while the sensitivity of the developed LFS RT-RPA assay was as low as 150 copies per reaction at 39 °C in less than 25 min. In both assays, there were no cross-reactions with sheep and goat pox viruses, foot-and-mouth disease virus and Orf virus. These features make RPA assay promising candidates either in field use or as a point of care diagnostic technique.

  8. Validation of the Dri-Dot Latex agglutination and IgM lateral flow assays for the diagnosis of typhoid fever in an Egyptian population.

    PubMed

    Nakhla, Isabelle; El Mohammady, Hanan; Mansour, Adel; Klena, John D; Hassan, Khaled; Sultan, Yehia; Pastoor, Rob; Abdoel, Theresia H; Smits, Henk

    2011-08-01

    Laboratory confirmation of typhoid fever is essential for appropriate medical treatment. Blood culture is a standard test for diagnosis of typhoid fever, but well-equipped diagnostic facilities to perform culture are seldom available in endemic areas. We retrospectively compared 2 diagnostic field tests, a latex agglutination Dri-Dot assay and an IgM Lateral Flow assay, to blood culture, in patients with clinically diagnosed typhoid fever. Sensitivity of the Dri-Dot was 71.4%, and specificity was 86.3% for samples collected at time of first diagnosis. Sensitivity and specificity of IgM Lateral Flow were 80% and 71.4%, respectively. A major limitation of these serologic tests is the limited sensitivity at the early stage of the disease. Performing both tests in parallel increased sensitivity to 84.3%, but decreased specificity to 70.5%. There was a trend towards improved diagnostic performance using either assay over a longer duration of illness. These rapid, point-of-care assays for typhoid fever provide easy-to-interpret results in typhoid-endemic countries and may be most useful in patients presenting 1 week after symptom onset. Published by Elsevier Inc.

  9. Advantages of time-resolved fluorescent nanobeads compared with fluorescent submicrospheres, quantum dots, and colloidal gold as label in lateral flow assays for detection of ractopamine.

    PubMed

    Hu, Li-Ming; Luo, Kai; Xia, Jun; Xu, Guo-Mao; Wu, Cheng-Hui; Han, Jiao-Jiao; Zhang, Gang-Gang; Liu, Miao; Lai, Wei-Hua

    2017-05-15

    Label selection is a critical factor for improving the sensitivity of lateral flow assay. Time-resolved fluorescent nanobeads, fluorescent submicrospheres, quantum dots, and colloidal gold-based lateral flow assay (TRFN-LFA, FM-LFA, QD-LFA, and CG-LFA) were first systematically compared for the quantitative detection of ractopamine in swine urine based on competitive format. The limits of detection (LOD) of TRFN-LFA, FM-LFA, QD-LFA, and CG-LFA were 7.2, 14.7, 23.6, and 40.1pg/mL in swine urine samples, respectively. The sensitivity of TRFN-LFA was highest. In the quantitative determination of ractopamine (RAC) in swine urine samples, TRFN-LFA exhibited a wide linear range of 5pg/mL to 2500pg/mL with a reliable coefficient of correlation (R(2)=0.9803). Relatively narrow linear ranges of 10-500pg/mL (FM-LFA) and 25-2500pg/mL (QD-LFA and CG-LFA) were acquired. Approximately 0.005µg of anti-RAC poly antibody (pAb) was used in each TRFN-LFA test strip, whereas 0.02, 0.054, and 0.15µg of pAb were used in each of the FM-LFA, QD-LFA, and CG-LFA test strips, respectively. In addition, TRFN-LFA required the least RAC-BSA antigens and exhibited the shortest detection time compared with the other lateral flow assays. Analysis of the RAC in swine urine samples showed that the result of TRFN-LFA was consistent with that of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and a commercial enzyme-linked immunosorbent assay (ELISA) kit.

  10. Combined multiplex loop-mediated isothermal amplification with lateral flow assay to detect sea and seb genes of enterotoxic Staphylococcus aureus.

    PubMed

    Yin, H Y; Fang, T J; Wen, H W

    2016-07-01

    Staphylococcal enterotoxins (SEs) are the most common cause of food poisoning worldwide and can induce symptoms, such as diarrhoea, vomiting and abdominal cramping. Thus, the aim of this study is to develop a multiplex loop-mediated isothermal amplification combined with a lateral flow assay (m-LAMP/LFA) to simultaneously detect the sea and seb genes of Staphylococcus aureus. The amplicons of the sea gene were labelled with digoxigenin (Dig) and biotin while those of seb gene were labelled with fluorescein isothiocyanate (FITC) and biotin. These amplicons were detected using a multiplex LFA with NeutrAvidin-tagged gold nanoparticles as the detection reagent. After optimization, the detection limit of this assay was 10(2)  CFU ml(-1) Staph. aureus, which was one tenth that of a multiplex PCR. This assay did not exhibit any cross-reactivity in detecting other enterotoxic Staph. aureus strains or other food pathogens. After 6 h of enrichment, this developed assay detected 1 CFU ml(-1) of Staph. aureus in milk, apple juice, cheese and rice. The developed m-LAMP/LFA method does not require expensive equipment and can be completely implemented within an 8-h workday. Therefore, this method can provide an effective means of quickly screening staphylococcal enterotoxin A- and/or staphylococcal enterotoxin B-producing Staph. aureus in food samples. Staphylococcus aureus is one of the major foodborne pathogens worldwide, and its staphylococcal enterotoxin A and B are strongly associated with food poisoning. This work developed a multiplex loop-mediated isothermal amplification combined with a lateral flow assay (m-LAMP/LFA) to simultaneously detect the sea and seb genes of Staph. aureus in food samples. The assay has good specificity and sensitivity with ease-of-use features, making it ideal for on-site detection. © 2016 The Society for Applied Microbiology.

  11. Comparison of Lateral Flow Assay, Kidney Inhibition Swab, and Liquid Chromatography-Tandem Mass Spectrometry for the Detection of Penicillin G Residues in Sow Urine.

    PubMed

    Shelver, Weilin L; Chakrabarty, Shubhashis; Smith, David J

    2017-03-01

    Sows (n = 126) were administered penicillin G; urine, collected at slaughter, was screened by kidney inhibition swab (KIS; 4 h testing time) and then stored at -80 °C (∼1200 days) until analysis by lateral flow assay (LF, ∼5 min testing time) and tandem quadrupole LC-MS/MS (TQ) analysis. The stability of penicillin in urine during storage was verified using TQ analyses. Quantitative results were well-correlated (R(2) = 0.98) with only a ∼10% decrease in penicillin concentration during the 3-year storage period. KIS retesting of stored samples returned results consistent with the original analyses. Lateral flow assay results were highly correlated with the KIS and TQ results. A KIS positive sample, which was not confirmed by TQ or LF, was assayed by Triple-TOF LC-MS to determine the cause of the apparent false positive. This study suggests LF can be used to quickly and efficiently screen for penicillin G residues before slaughter.

  12. Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

    SciTech Connect

    Cary,; Bruce, R; Stubben, Christopher J

    2011-03-22

    The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

  13. Development of a loop-mediated isothermal amplification assay combined with a lateral flow dipstick for rapid and simple detection of classical swine fever virus in the field.

    PubMed

    Chowdry, Vinay Kumar; Luo, Yuzi; Widén, Frederik; Qiu, Hua-Ji; Shan, Hu; Belák, Sándor; Liu, Lihong

    2014-03-01

    Classical swine fever (CSF) is a highly contagious viral disease and may cause heavy economic loss to farmers. The rapid, simple and accurate diagnosis of the disease at the frontline, for example on the farms of concern is crucial for disease control. This study describes the development and evaluation of a new loop-mediated isothermal amplification (LAMP) assay coupled with lateral flow dipstick (LFD) for the detection of classical swine fever virus (CSFV). This RT-LAMP-LFD assay combines the efficient one-step isothermal amplification of CSF viral RNA and the simplicity of the LFD to read the results within two to five minutes. Seven genotypes (1.1, 1.2, 1.3, 2.1, 2.2, 2.3 and 3.1), but not genotype 3.4, were successfully detected by the RT-LAMP-LFD assay, indicating that the method has a broad range of detection and can be applied in different geographical areas where CSFV strains belonging to these genotypes are present. The performance of this RT-LAMP-LFD assay was similar to that of the real-time RT-PCR. The analytical sensitivity was about 100copies per reaction when testing two genotypes (1.1 and 2.3). No cross-reactivity to non-CSFV pestiviruses was observed. This RT-LAMP-LFD assay can be a useful novel tool for the rapid, simple and economic diagnosis of classical swine fever in the field.

  14. Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay.

    PubMed

    Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

    2016-02-17

    The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 10(4) CFU mL(-1) or 10(5) CFU mL(-1) for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R(2)) of 0.916-0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥ 80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water.

  15. Rapid Detection of Panton-Valentine Leukocidin in Staphylococcus aureus Cultures by Use of a Lateral Flow Assay Based on Monoclonal Antibodies

    PubMed Central

    Müller, Elke; Buechler, Joseph; Rejman, John; Stieber, Bettina; Akpaka, Patrick Eberechi; Bandt, Dirk; Burris, Rob; Coombs, Geoffrey; Hidalgo-Arroyo, G. Aida; Hughes, Peter; Kearns, Angela; Abós, Sonia Molinos; Pichon, Bruno; Skakni, Leila; Söderquist, Bo; Ehricht, Ralf

    2013-01-01

    Panton-Valentine leukocidin (PVL) is a virulence factor of Staphylococcus aureus, which is associated with skin and soft-tissue infections and necrotizing pneumonia. To develop a rapid phenotypic assay, recombinant PVL F component was used to generate monoclonal antibodies by phage display. These antibodies were spotted on protein microarrays and screened using different lukF-PV preparations and detection antibodies. This led to the identification of the optimal antibody combination that was then used to establish a lateral flow assay. This test was used to detect PVL in S. aureus cultures. The detection limit of the assay with purified native and recombinant antigens was determined to be around 1 ng/ml. Overnight cultures from various solid and liquid media proved suitable for PVL detection. Six hundred strains and clinical isolates from patients from America, Europe, Australia, Africa, and the Middle East were tested. Isolates were genotyped in parallel by DNA microarray hybridization for confirmation of PVL status and assignment to clonal complexes. The sensitivity, specificity, and positive and negative predictive values of the assay in this trial were 99.7, 98.3, 98.4, and 99.7%, respectively. A total of 302 clinical isolates and reference strains were PVL positive and were assigned to 21 different clonal complexes. In summary, the lateral flow test allows rapid and economical detection of PVL in a routine bacteriology laboratory. As the test utilizes cultures from standard media and does not require sophisticated equipment, it can be easily integrated into a laboratory's workflow and might contribute to timely therapy of PVL-associated infections. PMID:23175260

  16. Gold magnetic nanoparticle conjugate-based lateral flow assay for the detection of IgM class antibodies related to TORCH infections.

    PubMed

    Li, Xingxing; Zhang, Qinlu; Hou, Peng; Chen, Mingwei; Hui, Wenli; Vermorken, Alphons; Luo, Zhiyi; Li, Hong; Li, Qin; Cui, Yali

    2015-11-01

    In this study, a lateral flow immunochromatographic assay (LFIA) system for the detection of immunoglobulin M (IgM) antibodies, related to TORCH [(T)oxoplasmosis, (O)ther agents, (R)ubella (also known as German Measles), (C)ytomegalovirus, and (H)erpes simplex virus infections], based on gold magnetic nanoparticles, was established. Following modification with poly(methacrylic acid), the gold magnetic nanoparticles conjugated with an anti‑human IgM antibody (μ‑chain specific) to construct a probe. A lateral flow assay device was constructed based on these conjugates. IgM antibodies to four types of pathogens, notably toxoplasmosis, rubella virus, cytomegalovirus and herpes simplex virus type 2, were detected using this device. Compared with commercial colloidal gold‑based LFIA strips, our method exhibited higher sensitivity. No interference with triglycerides, hemoglobin and bilirubin occurred, and no cross‑reactivity was noted among the four pathogens. The gold magnetic nanoparticle‑LFIA strips were used to assess 41 seropositive and 121 seronegative serum samples. The sensitivity was 100% (162/162) and the specificity was 100% (162/162). This method cannot only be used for the detection of TORCH IgM-specific antibodies, but it can potentially be developed for use in the diagnosis of other acute or recently identified autoimmune diseases.

  17. Development and Evaluation of Up-Converting Phosphor Technology-Based Lateral Flow Assay for Quantitative Detection of NT-proBNP in Blood

    PubMed Central

    Hao, Qingfang; Zou, Deyong; Zhang, Xiaoli; Zhang, Liping; Li, Hongmei; Qiao, Yong; Zhao, Huansheng; Zhou, Lei

    2017-01-01

    A newly assay, up-converting phosphor technology-based lateral flow (UPT-LF) assay, was developed for rapid and quantitative detection of N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP), one of the most important serum molecular maker of heat failure, in plasma samples as a point of care testing (POCT) method for diagnosis of acute heart failure. Human plasma from 197 patients with acute heart failure and 200 healthy controls was assessed using the UPT-LF assay, in a comparison with a Roche Elecsys assay. The limit of detection of the UPT-LF assay, with a coefficient of variation (CV) of less than 15%, was 116 ng/L, which is lower than the clinical diagnosis cutoff (150 ng/mL). The linear range was 50–35,000 ng/L. The CVs were less than 10% for both UPT-LF and Roche Elecsys assays for plasma samples under different storages, demonstrating the good stability and reproducibility. There are certain linear correlations between the results of UPT-LF and Roche Elecsys assay for EDTA-K2 and heparin-anticoagulated plasma, as well as for serum samples. For UPT-LF assay, there is a significant correlation between the values derived from analysis of EDTA-K2 and heparin-anticoagulated plasma samples (R = 0.995). No statistically significant difference was found between serum and plasma samples for UPT-LF assay. Our results demonstrate that NT-proBNP levels in healthy adults are elevated with age and had a relationship with sex, and with the age increase the NT-proBNP levels of females are significantly higher than those of males (p<0.01). The UPT-LF assay has a high reproducibility, stability, sensitivity, specificity, and is consistent with Roche Elecsys assay, and therefore it could be used as a POCT method for the quantitative detection of NT-proBNP in blood for clinical diagnosis and research of acute heart failure. PMID:28151978

  18. Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification of Bacillus anthracis Spores in Suspicious White Powders and Environmental Samples.

    PubMed

    Ramage, Jason G; Prentice, Kristin W; DePalma, Lindsay; Venkateswaran, Kodumudi S; Chivukula, Sruti; Chapman, Carol; Bell, Melissa; Datta, Shomik; Singh, Ajay; Hoffmaster, Alex; Sarwar, Jawad; Parameswaran, Nishanth; Joshi, Mrinmayi; Thirunavkkarasu, Nagarajan; Krishnan, Viswanathan; Morse, Stephen; Avila, Julie R; Sharma, Shashi; Estacio, Peter L; Stanker, Larry; Hodge, David R; Pillai, Segaran P

    2016-01-01

    We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert(®) test strip, a lateral flow immunoassay (LFA) for the rapid detection of Bacillus anthracis spores. The study, conducted at 2 sites, evaluated this assay for the detection of spores from the Ames and Sterne strains of B. anthracis, as well as those from an additional 22 strains. Phylogenetic near neighbors, environmental background organisms, white powders, and environmental samples were also tested. The Anthrax LFA demonstrated a limit of detection of about 10(6) spores/mL (ca. 1.5 × 10(5) spores/assay). In this study, overall sensitivity of the LFA was 99.3%, and the specificity was 98.6%. The results indicated that the specificity, sensitivity, limit of detection, dynamic range, and repeatability of the assay support its use in the field for the purpose of qualitatively evaluating suspicious white powders and environmental samples for the presumptive presence of B. anthracis spores.

  19. Comparison of Fluorescent Microspheres and Colloidal Gold as Labels in Lateral Flow Immunochromatographic Assays for the Detection of T-2 Toxin.

    PubMed

    Zhang, Xiya; Wu, Chao; Wen, Kai; Jiang, Haiyang; Shen, Jianzhong; Zhang, Suxia; Wang, Zhanhui

    2015-12-28

    A new highly specific and sensitive monoclonal antibody (MAb) to T-2 toxin (T-2) was produced, providing an IC50 value of 1.02 ng/mL and negligible cross-reactivity (CR) to other related mycotoxins. Based on the new MAb, a lateral-flow immunochromatographic assay (LFIA) using colloidal gold (CG) and fluorescent microspheres (FMs) as labels was proposed for T-2. Under the optimized conditions, in rapid qualitative assay, the cut-off values of the CG-LFIA were 400 μg/kg in rice and 50 μg/L in fresh milk, and the cut-off values of the FMs-LFIA were 100 μg/kg in both rice and chicken feed. For the quantitative assay with the FMs-LFIA, the limit of detection (LOD) were 0.23 μg/kg and 0.41 μg/kg in rice and chicken feed, respectively, and the average recoveries ranged from 80.2% to 100.8% with the coefficient of variation (CV) below 10.8%. In addition, we found that the CG-LFIA could tolerate the matrix effect of fresh milk better than the FMs-LFIA, while the FMs-LFIA could tolerate the matrix effect of chicken feed better than CG-LFIA under the same experimental conditions. These results provide a certain reference for the selection of appropriate labels to establish a rapid LFIA in various biological samples.

  20. Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia.

    PubMed

    Heller, Martin; Gicheru, Nimmo; Tjipura-Zaire, Georgina; Muriuki, Cecilia; Yu, Mingyan; Botelho, Ana; Naessens, Jan; Jores, Joerg; Liljander, Anne

    2016-06-01

    Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused by Mycoplasma mycoides subsp. mycoides, a bacterium belonging to the Mycoplasma mycoides cluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenic Mycoplasma mycoides subsp. mycoides proteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinant Mycoplasma immunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  1. Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia

    PubMed Central

    Heller, Martin; Gicheru, Nimmo; Tjipura-Zaire, Georgina; Muriuki, Cecilia; Yu, Mingyan; Botelho, Ana; Naessens, Jan; Jores, Joerg

    2016-01-01

    Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused by Mycoplasma mycoides subsp. mycoides, a bacterium belonging to the Mycoplasma mycoides cluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenic Mycoplasma mycoides subsp. mycoides proteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinant Mycoplasma immunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP. PMID:27053669

  2. Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification of Bacillus anthracis Spores in Suspicious White Powders and Environmental Samples

    PubMed Central

    Ramage, Jason G.; Prentice, Kristin W.; DePalma, Lindsay; Venkateswaran, Kodumudi S.; Chivukula, Sruti; Chapman, Carol; Bell, Melissa; Datta, Shomik; Singh, Ajay; Hoffmaster, Alex; Sarwar, Jawad; Parameswaran, Nishanth; Joshi, Mrinmayi; Thirunavkkarasu, Nagarajan; Krishnan, Viswanathan; Morse, Stephen; Avila, Julie R.; Sharma, Shashi; Estacio, Peter L.; Stanker, Larry; Hodge, David R.

    2016-01-01

    We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert® test strip, a lateral flow immunoassay (LFA) for the rapid detection of Bacillus anthracis spores. The study, conducted at 2 sites, evaluated this assay for the detection of spores from the Ames and Sterne strains of B. anthracis, as well as those from an additional 22 strains. Phylogenetic near neighbors, environmental background organisms, white powders, and environmental samples were also tested. The Anthrax LFA demonstrated a limit of detection of about 106 spores/mL (ca. 1.5 × 105 spores/assay). In this study, overall sensitivity of the LFA was 99.3%, and the specificity was 98.6%. The results indicated that the specificity, sensitivity, limit of detection, dynamic range, and repeatability of the assay support its use in the field for the purpose of qualitatively evaluating suspicious white powders and environmental samples for the presumptive presence of B. anthracis spores. PMID:27661796

  3. Comprehensive laboratory evaluation of a highly specific lateral flow assay for the presumptive identification of ricin in suspicious white powders and environmental samples.

    PubMed

    Hodge, David R; Prentice, Kristin Willner; Ramage, Jason G; Prezioso, Samantha; Gauthier, Cheryl; Swanson, Tanya; Hastings, Rebecca; Basavanna, Uma; Datta, Shomik; Sharma, Shashi K; Garber, Eric A E; Staab, Andrea; Pettit, Denise; Drumgoole, Rahsaan; Swaney, Erin; Estacio, Peter L; Elder, Ian A; Kovacs, Gerald; Morse, Brenda S; Kellogg, Richard B; Stanker, Larry; Morse, Stephen A; Pillai, Segaran P

    2013-12-01

    Ricin, a heterodimeric toxin that is present in the seeds of the Ricinus communis plant, is the biothreat agent most frequently encountered by law enforcement agencies in the United States. Even in untrained hands, the easily obtainable seeds can yield a highly toxic product that has been used in various types of threats, including "white-powder" letters. Although the vast majority of these threats are hoaxes, an impediment to accurate hazard assessments by first responders is the unreliability of rapid detection assays for ricin, such as lateral flow assays (LFAs). One of the complicating factors associated with LFAs is the incorporation of antibodies of poor specificity that cross-react with near-neighbors or with plant lectins that are capable of nonspecifically cross-linking the capture and detector antibodies. Because of the compelling and critical need to promote the interests of public safety and public health, the Department of Homeland Security conducted a comprehensive laboratory evaluation study of a commercial LFA for the rapid detection of ricin. This study was conducted using comprehensive inclusivity and exclusivity panels of ricin and near-neighbor plant materials, along with panels of lectins and "white-powders," to determine the specificity, sensitivity, limits of detection, dynamic range, and repeatability of the assay for the specific intended use of evaluating suspicious white powders and environmental samples in the field.

  4. Comprehensive laboratory evaluation of a specific lateral flow assay for the presumptive identification of abrin in suspicious white powders and environmental samples.

    PubMed

    Ramage, Jason G; Prentice, Kristin Willner; Morse, Stephen A; Carter, Andrew J; Datta, Shomik; Drumgoole, Rahsaan; Gargis, Shaw R; Griffin-Thomas, Latoya; Hastings, Rebecca; Masri, Heather P; Reed, Matthew S; Sharma, Shashi K; Singh, Ajay K; Swaney, Erin; Swanson, Tanya; Gauthier, Cheryl; Toney, Denise; Pohl, Jan; Shakamuri, Priyanka; Stuchlik, Olga; Elder, Ian A; Estacio, Peter L; Garber, Eric A E; Hojvat, Sally; Kellogg, Richard B; Kovacs, Gerald; Stanker, Larry; Weigel, Linda; Hodge, David R; Pillai, Segaran P

    2014-01-01

    Abrin is a heterodimeric toxin present in the seeds of the Abrus precatorius plant. The easily obtainable seeds can yield a highly toxic product that can be used in various types of biocrimes and terrorism-related activities, including "white-powder" letters. Although the vast majority of these threats are hoaxes, the lack of rapid and reliable detection assays for abrin, such as lateral flow assays (LFAs), can be an impediment to accurate and rapid hazard assessment. One of the complicating factors associated with LFAs is the use of antibodies of poor affinity and specificity that cross-react with near neighbors or that bind to plant lectins, which are capable of nonspecifically cross-linking the capture and detector antibodies. Because of the critical need to promote public safety and public health, we conducted a comprehensive laboratory evaluation of a commercial LFA for the rapid detection of abrin. This study was conducted using comprehensive inclusivity and exclusivity panels of abrin and near-neighbor plant materials, along with panels of lectins, related proteins, white powders, and environmental background material, to determine the sensitivity, specificity, limit of detection, dynamic range, and repeatability of the assay for the specific intended use of evaluating suspicious white powders and environmental samples for the presumptive presence of abrin.

  5. Lateral Flow Assay Based on Paper-Hydrogel Hybrid Material for Sensitive Point-of-Care Detection of Dengue Virus.

    PubMed

    Choi, Jane Ru; Yong, Kar Wey; Tang, Ruihua; Gong, Yan; Wen, Ting; Yang, Hui; Li, Ang; Chia, Yook Chin; Pingguan-Murphy, Belinda; Xu, Feng

    2017-01-01

    Paper-based devices have been broadly used for the point-of-care detection of dengue viral nucleic acids due to their simplicity, cost-effectiveness, and readily observable colorimetric readout. However, their moderate sensitivity and functionality have limited their applications. Despite the above-mentioned advantages, paper substrates are lacking in their ability to control fluid flow, in contrast to the flow control enabled by polymer substrates (e.g., agarose) with readily tunable pore size and porosity. Herein, taking the benefits from both materials, the authors propose a strategy to create a hybrid substrate by incorporating agarose into the test strip to achieve flow control for optimal biomolecule interactions. As compared to the unmodified test strip, this strategy allows sensitive detection of targets with an approximately tenfold signal improvement. Additionally, the authors showcase the potential of functionality improvement by creating multiple test zones for semi-quantification of targets, suggesting that the number of visible test zones is directly proportional to the target concentration. The authors further demonstrate the potential of their proposed strategy for clinical assessment by applying it to their prototype sample-to-result test strip to sensitively and semi-quantitatively detect dengue viral RNA from the clinical blood samples. This proposed strategy holds significant promise for detecting various targets for diverse future applications.

  6. Rapid pathogen detection by lateral-flow immunochromatographic assay with gold nanoparticle-assisted enzyme signal amplification.

    PubMed

    Cho, Il-Hoon; Bhunia, Arun; Irudayaraj, Joseph

    2015-08-03

    To date most LF-ICA format for pathogen detection is based on generating color signals from gold nanoparticle (AuNP) tracers that are perceivable by naked eye but often these methods exhibit sensitivity lower than those associated with the conventional enzyme-based immunological methods or mandated by the regulatory guidelines. By developing AuNP avidin-biotin constructs in which a number of enzymes can be labeled we report on an enhanced LF-ICA system to detect pathogens at very low levels. With this approach we show that as low as 100 CFU/mL of Escherichia coli O157:H7 can be detected, indicating that the limit of detection can be increased by about 1000-fold due to our signal amplification approach. In addition, extensive cross-reactivity experiments were conducted (19 different organisms were used) to test and successfully validate the specificity of the assay. Semi-quantitative analysis can be performed using signal intensities which were correlated with the target pathogen concentrations for calibration by image processing.

  7. Evaluation of lateral flow assay as a field test for investigation of brucellosis outbreak in an organized buffalo farm: A pilot study

    PubMed Central

    Shome, R.; Filia, G.; Padmashree, B. S.; Krithiga, N.; Sahay, Swati; Triveni, K.; Shome, B. R.; Mahajan, V.; Singh, Amarjit; Rahman, H.

    2015-01-01

    Aim: The aim was to evaluate lateral flow assay (LFA) as a field test for investigation of brucellosis outbreak in organized buffalo farm. Materials and Methods: A total of 153 serum samples were tested to detect the presence of brucella antibodies by LFA and three other serological tests i.e. rose bengal plate test (RBPT), protein G based indirect enzyme-linked immunoassay (iELISA), and competitive ELISA (cELISA). The performances of LFA and other serological tests were evaluated using OIE complaint cELISA as the gold standard. Results: Serological tests revealed 50% of the animals were seropositive for Brucella antibodies and correlated with clinical history of abortions, infertility, and productive failures. The newly developed assay showed 87.1% and 92.6% sensitivity and specificity, which was even higher than the specificity of RBPT. Conclusions: The investigation proved the potential usefulness of LFA for field diagnosis of brucellosis in the regions where laboratory facilities are limited. PMID:27047121

  8. Evaluation of the C.Diff Quik Chek Complete Assay, a new glutamate dehydrogenase and A/B toxin combination lateral flow assay for use in rapid, simple diagnosis of clostridium difficile disease.

    PubMed

    Sharp, Susan E; Ruden, Lila O; Pohl, Julie C; Hatcher, Patricia A; Jayne, Linda M; Ivie, W Michael

    2010-06-01

    The diagnosis of Clostridium difficile infection continues to be a challenge for many clinical microbiology laboratories. A new lateral flow assay, the C.Diff Quik Chek Complete assay, which tests for the presence of both glutamate dehydrogenase (GDH) and C. difficile toxins A and B, was evaluated for its ability to diagnose C. difficile disease. The results of this assay were compared to those of both PCR and toxigenic culture. The results showed that this assay allows 88% of specimens to be accurately screened as either positive (both tests positive) or negative (both tests negative) for the presence of toxigenic C. difficile in less than 30 min and with minimal hands-on time. Use of a random-access PCR for the analysis of specimens with discrepant results (one test positive and the other negative) allows the easy, rapid, and highly sensitive (100%; 95% confidence interval [CI], 89.6 to 100%) and specific (99.6%; 95% CI, 97.3 to 99.9%) diagnosis of C. difficile disease. The use of this algorithm would save institutional costs, curtail unnecessary isolation days, reduce the nosocomial transmission of disease, and increase the quality of care for patients.

  9. LATERAL FLOW ASSAY FOR CRYPTOCOCCAL ANTIGEN: AN IMPORTANT ADVANCE TO IMPROVE THE CONTINUUM OF HIV CARE AND REDUCE CRYPTOCOCCAL MENINGITIS-RELATED MORTALITY.

    PubMed

    Vidal, Jose E; Boulware, David R

    2015-09-01

    AIDS-related cryptococcal meningitis continues to cause a substantial burden of death in low and middle income countries. The diagnostic use for detection of cryptococcal capsular polysaccharide antigen (CrAg) in serum and cerebrospinal fluid by latex agglutination test (CrAg-latex) or enzyme-linked immunoassay (EIA) has been available for over decades. Better diagnostics in asymptomatic and symptomatic phases of cryptococcosis are key components to reduce mortality. Recently, the cryptococcal antigen lateral flow assay (CrAg LFA) was included in the armamentarium for diagnosis. Unlike the other tests, the CrAg LFA is a dipstick immunochromatographic assay, in a format similar to the home pregnancy test, and requires little or no lab infrastructure. This test meets all of the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User friendly, Rapid/robust, Equipment-free, and Delivered). CrAg LFA in serum, plasma, whole blood, or cerebrospinal fluid is useful for the diagnosis of disease caused by Cryptococcus species. The CrAg LFA has better analytical sensitivity for C. gattii than CrAg-latex or EIA. Prevention of cryptococcal disease is new application of CrAg LFA via screening of blood for subclinical infection in asymptomatic HIV-infected persons with CD4 counts < 100 cells/mL who are not receiving effective antiretroviral therapy. CrAg screening of leftover plasma specimens after CD4 testing can identify persons with asymptomatic infection who urgently require pre-emptive fluconazole, who will otherwise progress to symptomatic infection and/or die.

  10. LATERAL FLOW ASSAY FOR CRYPTOCOCCAL ANTIGEN: AN IMPORTANT ADVANCE TO IMPROVE THE CONTINUUM OF HIV CARE AND REDUCE CRYPTOCOCCAL MENINGITIS-RELATED MORTALITY

    PubMed Central

    VIDAL, Jose E.; BOULWARE, David R.

    2015-01-01

    SUMMARY AIDS-related cryptococcal meningitis continues to cause a substantial burden of death in low and middle income countries. The diagnostic use for detection of cryptococcal capsular polysaccharide antigen (CrAg) in serum and cerebrospinal fluid by latex agglutination test (CrAg-latex) or enzyme-linked immunoassay (EIA) has been available for over decades. Better diagnostics in asymptomatic and symptomatic phases of cryptococcosis are key components to reduce mortality. Recently, the cryptococcal antigen lateral flow assay (CrAg LFA) was included in the armamentarium for diagnosis. Unlike the other tests, the CrAg LFA is a dipstick immunochromatographic assay, in a format similar to the home pregnancy test, and requires little or no lab infrastructure. This test meets all of the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User friendly, Rapid/robust, Equipment-free, and Delivered). CrAg LFA in serum, plasma, whole blood, or cerebrospinal fluid is useful for the diagnosis of disease caused by Cryptococcus species. The CrAg LFA has better analytical sensitivity for C. gattii than CrAg-latex or EIA. Prevention of cryptococcal disease is new application of CrAg LFA via screening of blood for subclinical infection in asymptomatic HIV-infected persons with CD4 counts < 100 cells/mL who are not receiving effective antiretroviral therapy. CrAg screening of leftover plasma specimens after CD4 testing can identify persons with asymptomatic infection who urgently require pre-emptive fluconazole, who will otherwise progress to symptomatic infection and/or die. PMID:26465368

  11. Multiplex diagnosis of viral infectious diseases (AIDS, hepatitis C, and hepatitis A) based on point of care lateral flow assay using engineered proteinticles.

    PubMed

    Lee, Jong-Hwan; Seo, Hyuk Seong; Kwon, Jung-Hyuk; Kim, Hee-Tae; Kwon, Koo Chul; Sim, Sang Jun; Cha, Young Joo; Lee, Jeewon

    2015-07-15

    Lateral flow assay (LFA) is an attractive method for rapid, simple, and cost-effective point of care diagnosis. For LFA-based multiplex diagnosis of three viral intractable diseases (acquired immune deficiency syndrome and hepatitis C and A), here we developed proteinticle-based 7 different 3D probes that display different viral antigens on their surface, which were synthesized in Escherichia coli by self-assembly of human ferritin heavy chain that was already engineered by genetically linking viral antigens to its C-terminus. Each of the three test lines on LFA strip contains the proteinticle probes to detect disease-specific anti-viral antibodies. Compared to peptide probes, the proteinticle probes were evidently more sensitive, and the proteinticle probe-based LFA successfully diagnosed all the 20 patient sera per each disease without a false negative signal, whereas the diagnostic sensitivities in the peptide probe-based LFAs were 65-90%. Duplex and triplex assays performed with randomly mixed patient sera gave only true positive signals for all the 20 serum mixtures without any false positive signals, indicating 100% sensitivity and 100% specificity. It seems that on the proteinticle surface the antigenic peptides have homogeneous orientation and conformation without inter-peptide clustering and hence lead to the enhanced diagnostic performance with solving the problems of traditional diagnostic probes. Although the multiplex diagnosis of three viral diseases above was demonstrated as proof-of-concept here, the proposed LFA system can be applied to multiplex point of care diagnosis of other intractable diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. An immuno-chromatographic lateral flow assay (LFA) for rapid on-the-farm detection of classical swine fever virus (CSFV).

    PubMed

    Sambandam, Rathnapraba; Angamuthu, Raja; Kanagaraj, Vijayarani; Kathaperumal, Kumanan; Chothe, Shubhada K; Nissly, Ruth H; Barry, Rhiannon M; Jayarao, Bhushan M; Kuchipudi, Suresh V

    2017-07-06

    Classical swine fever (CSF) is a highly contagious and potentially fatal disease of domestic pigs. Classical swine fever is routinely diagnosed by clinical signs, serology, detection of CSF virus (CSFV) nucleic acid by PCR and virus isolation. Most of the current CSF diagnostic methods are expensive and have an extended turnaround time. In the majority of the CSF endemic countries, lack of easy access to diagnostic facilities is a major problem for swine producers trying to obtain early diagnosis and often results in the entire herd being infected. The acute form of CSF can show non-specific signs of illness, leaving CSF often undiagnosed. Hence there is an urgent need for a rapid and reliable pen side diagnostic assay for the better detection and control of this economically important disease of swine. We developed an immuno-chromatographic lateral flow assay (LFA) for on the farm detection of CSFV. A CSFV isolate [CSFV/AP/TRP2/2009 (TS2)] of genotype 1.1 was used for the production of monoclonal antibodies (mAbs) for the LFA's development. The virus detection level of the LFA device was 36.8 TCID50/ml of CSFV. The sensitivity and specificity of LFA in comparison with PCR were 80.36% and 87.10%, respectively. The positive and negative predictive values of the LFA device were 91.84% and 87.10%, respectively. In conclusion, the CSFV-LFA is a reliable and convenient resource for preliminary on the farm detection of classic swine fever.

  13. Development and evaluation of a new lateral flow assay for simultaneous detection of antibodies against African Horse Sickness and Equine Infectious Anemia viruses.

    PubMed

    Costa, Sofia; Sastre, Patricia; Pérez, Teresa; Tapia, Istar; Barrandeguy, María; Sánchez-Vizcaíno, José M; Sánchez-Matamoros, Almudena; Wigdorovitz, Andrés; Sanz, Antonio; Rueda, Paloma

    2016-11-01

    African horse sickness (AHS) and equine infectious anemia (EIA) are both notifiable equid specific diseases that may present similar clinical signs. Considering the increased global movement of horses and equine products over the past decades, together with the socio-economic impact of previous AHS and EIA outbreaks, there is a clear demand for an early discrimination and a strict control of their transmission between enzootic and AHS/EIA-free regions. Currently, the individual control and prevention of AHS or EIA relies on a series of measures, including the restriction of animal movements, vector control, and the use of several laboratory techniques for viral identification, amongst others. Despite being widely employed in surveillance programmes and in the control of animal movements, the available serological assays can only detect AHS- or EIA-specific antibodies individually. In this work, a duplex lateral flow assay (LFA) for simultaneous detection and differentiation of specific antibodies against AHS virus (AHSV) and EIA virus (EIAV) was developed and evaluated with experimental and field serum samples. The duplex LFA was based on the AHSV-VP7 outer core protein and the EIAV-P26 major core protein. The results indicated that the duplex LFA presented a good analytical performance, detecting simultaneously and specifically antibodies against AHSV and EIAV. The initial diagnostic evaluation revealed a good agreement with results from the AHS and EIA tests prescribed by the OIE, and it highlighted the usefulness of the new AHSV/EIAV duplex LFA for an on-field and point-of-care first diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Rapid detection of Bombyx mori nucleopolyhedrovirus (BmNPV) by loop-mediated isothermal amplification assay combined with a lateral flow dipstick method.

    PubMed

    Zhou, Yang; Wu, Jiege; Lin, Feng; Chen, Naifu; Yuan, Shaofei; Ding, Lina; Gao, Li; Hang, Bangxing

    2015-12-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of the domestic silkworm. The disease often breaks out in sericultural countries and due to its high infectivity; it is difficult to control, resulting in heavy economic loss. In order to develop a rapid, sensitive visual detection and simple-to-use novel technology for detection of BmNPV, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) method was described. In this study, a set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the BmNPV gp41 gene, and the LAMP for the detection of BmNPV was developed by isothermal amplification at 61 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min. The detection limit of LAMP-LFD was 0.2 pg DNA extracted from silkworm infected with BmNPV and was 100 times more sensitive than conventional PCR. No product was generated from silkworm infected with other viruses. Furthermore, we applied the technique to detect BmNPV in the hemolymph and feces at different intervals post infection (pi). In conclusion, the novel LAMP-LFD setup presented here is simple, rapid, reliable, and has the potential for future use in the detection of BmNPV.

  15. A novel lateral flow assay based on GoldMag nanoparticles and its clinical applications for genotyping of MTHFR C677T polymorphisms

    NASA Astrophysics Data System (ADS)

    Hui, Wenli; Zhang, Sinong; Zhang, Chao; Wan, Yinsheng; Zhu, Juanli; Zhao, Gang; Wu, Songdi; Xi, Dujuan; Zhang, Qinlu; Li, Ningning; Cui, Yali

    2016-02-01

    Current techniques for single nucleotide polymorphism (SNP) detection require tedious experimental procedures and expensive and sophisticated instruments. In this study, a visual genotyping method has been successfully established via combining ARMS-PCR with gold magnetic nanoparticle (GoldMag)-based lateral flow assay (LFA) and applied to the genotyping of methylenetetrahydrofolate reductase (MTHFR) C677T. C677T substitution of the gene MTHFR leads to an increased risk of diseases. The genotyping result is easily achievable by visual observation within 5 minutes after loading of the PCR products onto the LFA device. The system is able to accurately assess a broad detection range of initial starting genomic DNA amounts from 5 ng to 1200 ng per test sample. The limit of detection reaches 5 ng. Furthermore, our PCR-LFA system was applied to clinical trials for screening 1721 individuals for the C677T genotypes. The concordance rate of the genotyping results detected by PCR-LFA was up to 99.6% when compared with the sequencing results. Collectively, our PCR-LFA has been proven to be rapid, accurate, sensitive, and inexpensive. This new method is highly applicable for C677T SNP screening in laboratories and clinical practices. More promisingly, it could also be extended to the detection of SNPs of other genes.

  16. Bisphenol A determination in baby bottles by chemiluminescence enzyme-linked immunosorbent assay, lateral flow immunoassay and liquid chromatography tandem mass spectrometry.

    PubMed

    Maiolini, Elisabetta; Ferri, Elida; Pitasi, Agata Laura; Montoya, Angel; Di Giovanni, Manuela; Errani, Ermanno; Girotti, Stefano

    2014-01-07

    Two immunoassays, a Lateral Flow ImmunoAssay (LFIA) based on colloidal gold nanoparticle labels and an indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA), were developed and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was optimized to assess the possible release of bisphenol A (BPA, 4,4'-isopropylidenediphenol) from different plastic baby bottles treated with simulating solutions. Coating conjugate concentration, anti-BPA antibody dilution, incubation time of the primary and secondary antibodies, and tolerance to different organic solvents were optimized to obtain the best performance of the ELISA with chemiluminescent end-point detection. The influence of different buffers on LFIA performance was also evaluated. Both methods showed good repeatability (mean CV value around 13%) and sensitivity. Reproducibility tests for CL-ELISA gave a mean CV value of about 25%. The IC50 and Limit of Detection (LOD) values of CL-ELISA were 0.2 and 0.02 ng mL(-1), respectively. The LOD of LFIA was 0.1 μg mL(-1). A LC-MS/MS method was also optimized. The separation was performed in a C18 column with a triple-quadrupole mass spectrometer with electrospray ionisation interface. The method showed a good linearity in the range 2 to 500 ng mL(-1), with a regression coefficient of 0.998. In the simulating solutions the detection and quantification limits, calculated by the signal to noise level of 3 (S/N = 3), were 5.8 ng mL(-1) and 17.4 ng mL(-1), respectively. This limit of quantification was about 3 and 35 times lower than the permitted limits set by the official method CEN/TS 13130-13 (0.05 μg mL(-1)) and by the Directive 2004/19/EC (0.6 μg mL(-1)), respectively. The methods were applied to determine BPA release from baby bottles, performing repeated procedures according to EU and national regulations. The results demonstrated that no BPA migration from the tested plastic materials occurred with only one

  17. Rapid determination of phenylethanolamine A in biological samples by enzyme-linked immunosorbent assay and lateral-flow immunoassay.

    PubMed

    Li, Xiangmei; Wang, Wenjun; Wang, Limiao; Wang, Qi; Pei, Xingyao; Jiang, Haiyang

    2015-10-01

    Phenylethanolamine A (PA) is a β-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL(-1). The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL(-1) and 0.39 ng g(-1), respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0-107.4 % and 81.8-113.3%, respectively, with the coefficients of variation in the range 4.1-16.2% and 1.2-6.3%, respectively. The mAbs had negligible cross reactivity with 10 other β-agonists. In contrast, the LFA had a cut-off level of 5 ng mL(-1) (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography-tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples.

  18. Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products.

    PubMed

    Jin, Wanchun; Yamada, Keiko; Ikami, Mai; Kaji, Noritada; Tokeshi, Manabu; Atsumi, Yusuke; Mizutani, Makoto; Murai, Atsushi; Okamoto, Akira; Namikawa, Takao; Baba, Yoshinobu; Ohta, Michio

    2013-03-01

    Staphylococcal enterotoxins (SEs), produced by Staphylococcus aureus, are a major cause of staphylococcal food poisoning. Traditionally, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse passive latex agglutination with rabbit antibody IgG have been used to detect SEs. However, most of these kits require a long processing time and there is a risk of false-positive results since IgG reacts nonspecifically with protein A produced by S. aureus. In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2 ng/ml within 15 min in milk. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1 ng/ml in milk; the SEs were detected within 12 min and specialized skills were not required. The ELISA and LFD detected SEA in dairy products artificially contaminated with S. aureus, including ice cream, yogurt, and café au lait, in a dose-dependent manner. In conclusion, IgY allows highly specific detection of SEs, and ELISAs, LFDs, and immunopillar chips should be useful tools for screening SEs in milk and dairy products. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Early morning urine collection to improve urinary lateral flow LAM assay sensitivity in hospitalised patients with HIV-TB co-infection.

    PubMed

    Gina, Phindile; Randall, Philippa J; Muchinga, Tapuwa E; Pooran, Anil; Meldau, Richard; Peter, Jonny G; Dheda, Keertan

    2017-05-12

    Urine LAM testing has been approved by the WHO for use in hospitalised patients with advanced immunosuppression. However, sensitivity remains suboptimal. We therefore examined the incremental diagnostic sensitivity of early morning urine (EMU) versus random urine sampling using the Determine® lateral flow lipoarabinomannan assay (LF-LAM) in HIV-TB co-infected patients. Consenting HIV-infected inpatients, screened as part of a larger prospective randomized controlled trial, that were treated for TB, and could donate matched random and EMU samples were included. Thus paired sample were collected from the same patient, LF-LAM was graded using the pre-January 2014, with grade 1 and 2 manufacturer-designated cut-points (the latter designated grade 1 after January 2014). Single sputum Xpert-MTB/RIF and/or TB culture positivity served as the reference standard (definite TB). Those treated for TB but not meeting this standard were designated probable TB. 123 HIV-infected patients commenced anti-TB treatment and provided matched random and EMU samples. 33% (41/123) and 67% (82/123) had definite and probable TB, respectively. Amongst those with definite TB LF-LAM sensitivity (95%CI), using the grade 2 cut-point, increased from 12% (5-24; 5/43) to 39% (26-54; 16/41) with random versus EMU, respectively (p = 0.005). Similarly, amongst probable TB, LF-LAM sensitivity increased from 10% (5-17; 8/83) to 24% (16-34; 20/82) (p = 0.001). LF-LAM specificity was not determined. This proof of concept study indicates that EMU could improve the sensitivity of LF-LAM in hospitalised TB-HIV co-infected patients. These data have implications for clinical practice.

  20. Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

    PubMed

    Kulabhusan, Prabir Kumar; Rajwade, Jyutika M; Sahul Hameed, A S; Paknikar, Kishore M

    2017-03-27

    White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (Kd,app) was calculated to be 810 nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5 μg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20 min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.

  1. Asymptomatic cryptococcal antigen prevalence detected by lateral flow assay in hospitalised HIV-infected patients in São Paulo, Brazil.

    PubMed

    Vidal, José E; Toniolo, Carolina; Paulino, Adriana; Colombo, Arnaldo; Dos Anjos Martins, Marilena; da Silva Meira, Cristina; Pereira-Chioccola, Vera Lucia; Figueiredo-Mello, Claudia; Barros, Tiago; Duarte, Jequelie; Fonseca, Fernanda; Alves Cunha, Mirella; Mendes, Clara; Ribero, Taiana; Dos Santos Lazera, Marcia; Rajasingham, Radha; Boulware, David R

    2016-12-01

    To determine the prevalence of asymptomatic cryptococcal antigen (CRAG) using lateral flow assay (LFA) in hospitalised HIV-infected patients with CD4 counts <200 cells/μl. Hospitalised HIV-infected patients were prospectively recruited at Instituto de Infectologia Emilio Ribas, a tertiary referral hospital to HIV-infected patients serving the São Paulo State, Brazil. All patients were >18 years old without prior cryptococcal meningitis, without clinical suspicion of cryptococcal meningitis, regardless of antiretroviral (ART) status, and with CD4 counts <200 cells/μl. Serum CRAG was tested by LFA in all patients, and whole blood CRAG was tested by LFA in positive cases. We enrolled 163 participants of whom 61% were men. The duration of HIV diagnosis was a median of 8 (range, 1-29) years. 26% were antiretroviral (ART)-naïve, and 74% were ART-experienced. The median CD4 cell count was 25 (range, 1-192) cells/μl. Five patients (3.1%; 95%CI, 1.0-7.0%) were asymptomatic CRAG-positive. Positive results cases were cross-verified by performing LFA in whole blood. 3.1% of HIV-infected inpatients with CD4 <200 cells/μl without symptomatic meningitis had cryptococcal antigenemia in São Paulo, suggesting that routine CRAG screening may be beneficial in similar settings in South America. Our study reveals another targeted population for CRAG screening: hospitalised HIV-infected patients with CD4 <200 cells/μl, regardless of ART status. Whole blood CRAG LFA screening seems to be a simple strategy to prevention of symptomatic meningitis. © 2016 John Wiley & Sons Ltd.

  2. Assessment of a new lateral flow immunochromatographic (LFIC) assay for the okadaic acid group of toxins using naturally contaminated bivalve shellfish from the Portuguese coast.

    PubMed

    Vale, P; Gomes, S S; Lameiras, J; Rodrigues, S M; Botelho, M J; Laycock, M V

    2009-02-01

    A new rapid assay for the okadaic acid group of toxins, based on lateral flow immunochromatographic (LFIC) test strips developed by Jellett Rapid Testing Ltd., was assessed on naturally contaminated bivalves from the Portuguese coast. One prototype was evaluated using samples harvested during 2005, extracted with 80% methanol, followed by dilution with the running buffer of a methanolic extract after alkaline hydrolysis for esters. The second prototype was assessed using samples harvested during 2006, extracted with 100% methanol and, after alkaline hydrolysis, the methanol was evaporated by a nitrogen stream followed by re-suspension with the running buffer. The first prototype failed to detect 20% of samples that were positive by LC-MS in the range 160-480 microg kg(-1), and were classified as negative or trace level by LFIC. The presence of methanol in the extracts made correct detection of toxins more difficult. The second prototype classified as positive all samples above 160 microg kg(-1), as confirmed by LC-MS. However, in the second prototype, matrix effects were a major drawback and led to 45% false positives, particularly for mussels, due to compounds in shellfish extracts interfering with the antibodies and reducing the test line intensity. Extraction with a higher percentage of methanol was thought responsible for these matrix effects. Regarding sample migration, both prototypes needed one hour before reading. In an attempt to speed-up sample preparation, a direct digestion of bivalve tissues with sodium hydroxide was evaluated. Low recoveries for esters were found by LC-MS with this hydrolysis technique compared to conventional hydrolysis of methanolic extracts. While prototype A was not sensitive enough, prototype B had too many false positives to be of use to the shellfish industry or in a monitoring program.

  3. Comparison of a novel Aspergillus lateral-flow device and the Platelia® galactomannan assay for the diagnosis of invasive aspergillosis following haematopoietic stem cell transplantation.

    PubMed

    Held, J; Schmidt, T; Thornton, C R; Kotter, E; Bertz, H

    2013-12-01

    The detection of galactomannan in serum is a cornerstone for the diagnosis of invasive fungal disease (IFD). Because a delay in treatment initiation is associated with a poor outcome, the results have to be available promptly. However, due to methodological and economic reasons, the test frequencies of the commonly used galactomannan assays vary between daily to weekly, meaning that results may be available too late to be clinically useful. The novel Aspergillus lateral-flow device (Aspergillus-LFD) is a rapid test that may overcome these limitations. We compared the diagnostic performance of the Aspergillus-LFD and the Platelia® Aspergillus EIA (GM-EIA) in serum from 101 patients during and after allogeneic haematopoietic stem cell transplantation (HSCT). Clinical data and sera were collected prospectively and patients classified according to the European Organisation for Research and Treatment of Cancer (EORTC)/Mycoses Study Group (MSG) 2008 guidelines. By the end of hospitalisation, one proven, nine probable and 20 possible cases of IFD were identified. Depending on the number of positive serum samples required for test positivity, the sensitivities, specificities and diagnostic odds ratios in patients with proven and probable IFD were as follows. One positive serum required: Aspergillus-LFD 40.0 %, 86.8 % and 3.03; GM-EIA 40.0 %, 89.0 % and 3.64. Two positive sera required: Aspergillus-LFD 20.0 %, 97.8 % and 11.13; GM-EIA 30.0 %, 98.9 % and 38.57. Although the GM-EIA was positive in a higher percentage of samples, this did not result in an earlier diagnosis. If used as a screening test (one positive serum required for test positivity) or to rule out IFD, the Aspergillus-LFD has shown a comparable diagnostic performance to the GM-EIA. However, if the results have to be confirmed by a second positive serum, the GM-EIA exhibited superior sensitivity. In terms of practicability, the Aspergillus-LFD has demonstrated to be a quick (15 min) and easy-to-use test for

  4. Short communication: A novel method using immunomagnetic separation with a fluorescent nanobeads lateral flow assay for the rapid detection of low-concentration Escherichia coli O157:H7 in raw milk.

    PubMed

    Huang, Zhen; Cui, Xi; Xie, Quan-Yuan; Liu, Dao-Feng; Lai, Wei-Hua

    2016-12-01

    Escherichia coli O157:H7 is an important serotype of enterohemorrhagic E. coli that was first identified as a human pathogen in 1982. This pathogen causes several serious diseases. In this study, immunomagnetic separation was coupled with a fluorescent nanobeads lateral flow assay to establish a sensitive and rapid detection method for Escherichia coli O157:H7 in raw milk. The pathogen was captured from raw milk by immunomagnetic separation with immunomagnetic nanobeads and then detected using a fluorescent nanobeads lateral flow assay. A fluorescent line was formed in the test line of the test strip and quantitatively detected using a fluorescent reader. Screening times, which included immunomagnetic separation and the fluorescent nanobeads lateral flow assay, were 8, 7, 6, and 5h when 1, 5, 25, and 125 cfu of E. coli O157:H7, respectively, were inoculated into 25mL of raw milk. The established method could be widely applied to the rapid onsite detection of other pathogens to ensure food safety.

  5. Lateral flow immunoassay using magnetoresistive sensors

    NASA Astrophysics Data System (ADS)

    Taton, Kristin; Johnson, Diane; Guire, Patrick; Lange, Erik; Tondra, Mark

    2009-05-01

    Magnetic particles have been adapted for use as labels in biochemical lateral flow strip tests. Standard gold particle lateral flow assays are generally qualitative; however, with magnetic particles, quantitative results can be obtained by using electronic detection systems with giant magnetoresistive (GMR) sensors. As described here, these small integrated sensor chips can detect the presence of magnetic labels in capture spots whose volume is approximately 150 μm×150 μm×150 μm. The range of linear detection is better than two orders of magnitude; the total range is up to four orders of magnitude. The system was demonstrated with both indirect and sandwich enzyme-linked immunosorbent assays (ELISAs) for protein detection of rabbit IgG and interferon-γ, respectively, achieving detection of 12 pg/ml protein. Ultimately, the goal is for the detector to be fully integrated into the lateral flow strip backing to form a single consumable item that is interrogated by a handheld electronic reader.

  6. A new method for the rapid detection of Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), Alaska pollock (Gadus chalcogrammus) and ling (Molva molva) using a lateral flow dipstick assay.

    PubMed

    Taboada, Ledicia; Sánchez, Ana; Pérez-Martín, Ricardo I; Sotelo, Carmen G

    2017-10-15

    Species-specific lateral flow dipstick (LFD) assays for the identification of Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), Alaska pollock (Gadus chalcogrammus) and ling (Molva molva) in food products were developed. The method comprises a PCR system with four sets of specific primers, for each target species. This step was also devised to dual-labeling of PCR products with biotin and 6-FAM, which are then easily read on a lateral flow dipstick, upon which these products are immobilized by a fixed biotin-ligand and visualized with anti-FAM antibody-coated gold nanoparticles. Sensitivity and selectivity were determined for each of the developed assays. Validation of the assays was performed with DNA extracted from commercial fish products, the identification of all samples by PCR-LFD was coherent with the results found with DNA sequencing. Target species were successfully detected in analyzed commercial samples, demonstrating the applicability of this method to the rapid analysis of food products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Multi-Laboratory Evaluation of a Novel Lateral Flow Immunochromatographic Assay for Confirming Isolation of Mycobacterium bovis from Veterinary Diagnostic Specimens.

    PubMed

    Stewart, Linda D; McCallan, Lyanne; McNair, James; McGoldrick, Adrian; Morris, Rowan; Moyen, Jean-Louis; De Juan Ferré, Lucía; Romero, Beatriz; Alonso, Elena; Parsons, Sven D C; Van Helden, Paul; Araújo, Flábio R; Grant, Irene R

    2017-09-27

    A novel lateral flow immunochromatographic device (LFD) was evaluated in several veterinary diagnostic laboratories. It was confirmed to be specific for Mycobacterium bovis and M. caprae cells. The performance of the novel LFD was assessed relative to the confirmatory tests routinely applied after culture (spoligotyping or qPCR) in each laboratory; liquid (MGIT or BacT/Alert) and/or solid (Stonebrink, Coletsos or Lowenstein-Jensen) cultures were tested. In comparison to spoligotyping of acid-fast positive MGIT cultures, percentage agreement between positive LFD and spoligotyping results was excellent in two UK laboratories (97.7-100%), but lower in the Spanish context (76%) where spoligotyping was applied to MGIT cultures previously confirmed to be positive for M. tuberculosis complex (MTBC) by qPCR. Certain spoligotypes of M. bovis and M. caprae were not detected by the LFD in Spanish MGIT cultures. Compared to qPCR confirmation, the percentage agreement between positive LFD and qPCR results was 42.3% and 50% for BacT/Alert and MGIT liquid cultures, respectively, and for solid cultures ranged from 11.1-89.2%, depending on solid medium employed (Coletsos 11.1%, Lowenstein-Jensen 55.6%, Stonebrinks 89.2%). Correlation between the novel LFD and BD MGIT TBc ID test results was excellent when 190 MGIT cultures were tested (r = 0.9791; P<0.0001), with the added benefit that M. bovis was differentiated from another MTBC species in one MGIT culture by the novel LFD. This multi-laboratory evaluation has demonstrated the novel LFD's potential utility as a rapid test to confirm isolation of M. bovis and M. caprae from veterinary specimens following culture. Copyright © 2017 American Society for Microbiology.

  8. Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

    SciTech Connect

    Cary, Robert E.

    2015-12-08

    Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

  9. Development and evaluation of an up-converting phosphor technology-based lateral flow assay for the rapid, simultaneous detection of Vibrio cholerae serogroups O1 and O139.

    PubMed

    Hao, Min; Zhang, Pingping; Li, Baisheng; Liu, Xiao; Zhao, Yong; Tan, Hailing; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Qiu, Haiyan; Wang, Duochun; Diao, Baowei; Jing, Huaiqi; Yang, Ruifu; Kan, Biao; Zhou, Lei

    2017-01-01

    Vibrio cholerae serogroups O1 and O139 are etiological agents of cholera, a serious and acute diarrheal disease, and rapid detection of V. cholerae is a key method for preventing and controlling cholera epidemics. Here, a point of care testing (POCT) method called Vch-UPT-LF, which is an up-converting phosphor technology-based lateral flow (UPT-LF) assay with a dual-target detection mode, was developed to detect V. cholerae O1 and O139 simultaneously from one sample loading. Although applying an independent reaction pair made both detection results for the two Vch-UPT-LF detection channels more stable, the sensitivity slightly declined from 104 to 105 colony-forming units (CFU) mL-1 compared with that of the single-target assay, while the quantification ranges covering four orders of magnitude were maintained. The strip showed excellent specificity for seven Vibrio species that are highly related genetically, and nine food-borne species whose transmission routes are similar to those of V. cholerae. The legitimate arrangement of the two adjacent test lines lessened the mutual impact of the quantitation results between the two targets, and the quantification values did not differ by more than one order of magnitude when the samples contained high concentrations of both V. cholerae O1 and O139. Under pre-incubation conditions, 1×101 CFU mL-1 of V. cholerae O1 or O139 could be detected in fewer than 7 h, while the Vch-UPT-LF assay exhibited sensitivity as high as a real-time fluorescent polymerase chain reaction with fewer false-positive results. Therefore, successful development of Vch-UPT-LF as a dual-target assay for quantitative detection makes this assay a good candidate POCT method for the detection and surveillance of epidemic cholera.

  10. Development and evaluation of an up-converting phosphor technology-based lateral flow assay for the rapid, simultaneous detection of Vibrio cholerae serogroups O1 and O139

    PubMed Central

    Li, Baisheng; Liu, Xiao; Zhao, Yong; Tan, Hailing; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Qiu, Haiyan; Wang, Duochun; Diao, Baowei; Jing, Huaiqi; Yang, Ruifu; Kan, Biao

    2017-01-01

    Vibrio cholerae serogroups O1 and O139 are etiological agents of cholera, a serious and acute diarrheal disease, and rapid detection of V. cholerae is a key method for preventing and controlling cholera epidemics. Here, a point of care testing (POCT) method called Vch-UPT-LF, which is an up-converting phosphor technology-based lateral flow (UPT-LF) assay with a dual-target detection mode, was developed to detect V. cholerae O1 and O139 simultaneously from one sample loading. Although applying an independent reaction pair made both detection results for the two Vch-UPT-LF detection channels more stable, the sensitivity slightly declined from 104 to 105 colony-forming units (CFU) mL−1 compared with that of the single-target assay, while the quantification ranges covering four orders of magnitude were maintained. The strip showed excellent specificity for seven Vibrio species that are highly related genetically, and nine food-borne species whose transmission routes are similar to those of V. cholerae. The legitimate arrangement of the two adjacent test lines lessened the mutual impact of the quantitation results between the two targets, and the quantification values did not differ by more than one order of magnitude when the samples contained high concentrations of both V. cholerae O1 and O139. Under pre-incubation conditions, 1×101 CFU mL−1 of V. cholerae O1 or O139 could be detected in fewer than 7 h, while the Vch-UPT-LF assay exhibited sensitivity as high as a real-time fluorescent polymerase chain reaction with fewer false-positive results. Therefore, successful development of Vch-UPT-LF as a dual-target assay for quantitative detection makes this assay a good candidate POCT method for the detection and surveillance of epidemic cholera. PMID:28662147

  11. Enhanced performance of methamphetamine lateral flow cassettes using an electronic lateral flow reader.

    PubMed

    Smith, Jerome P; Sammons, Deborah L; Robertson, Shirley A; Snawder, John E

    2015-01-01

    Surface contamination from methamphetamine in meth labs continues to be a problem. We had previously developed a lateral flow assay cassette for field detection of methamphetamine contamination that is commercially available and has been used by a number of groups to assess contamination. This cassette uses the complete disappearance of the test line as an end point for detection of 50 ng/100 cm2 of methamphetamine contamination for surface sampling with cotton swabs. In the present study, we further evaluate the response of the cassettes using an electronic lateral flow reader to measure the intensities of the test and control lines. The cassettes were capable of detecting 0.25 ng/ml for calibration solutions. For 100 cm2 ceramic tiles that were spiked with methamphetamine and wiped with cotton-tipped wooden swabs wetted in assay/sampling buffer, 1 ng/tile was detected using the reader. Semi-quantitative results can be produced over the range 0-10 ng/ml for calibration solutions and 0-25 ng/tile for spiked tiles using either a 4-parameter logistic fit of test line intensity versus concentration or spiked mass or the ratio of the control line to the test line intensity fit to concentration or spiked mass. Recovery from the tiles was determined to be about 30% using the fitted curves. Comparison of the control line to the test line was also examined as a possible visual detection end point and it was found that the control line became more intense than the test line at 0.5 to 1 ng/ml for calibration solutions or 1 to 2 ng/tile for spiked tiles. Thus the lateral flow cassettes for methamphetamine have the potential to produce more sensitive semi-quantitative results if an electronic lateral flow reader is used and can be more sensitive for detection if the comparison of the control line to the test line is used as the visual end point.

  12. Enhanced performance of methamphetamine lateral flow cassettes using an electronic lateral flow reader

    PubMed Central

    Smith, Jerome P.; Sammons, Deborah L.; Robertson, Shirley A.; Snawder, John E.

    2015-01-01

    Surface contamination from methamphetamine in meth labs continues to be a problem. We had previously developed a lateral flow assay cassette for field detection of methamphetamine contamination that is commercially available and has been used by a number of groups to assess contamination. This cassette uses the complete disappearance of the test line as an end point for detection of 50 ng/100 cm2 of methamphetamine contamination for surface sampling with cotton swabs. In the present study, we further evaluate the response of the cassettes using an electronic lateral flow reader to measure the intensities of the test and control lines. The cassettes were capable of detecting 0.25 ng/ml for calibration solutions. For 100 cm2 ceramic tiles that were spiked with methamphetamine and wiped with cotton tipped wooden swabs wetted in assay/sampling buffer, 1 ng/tile was detected using the reader. Semi-quantitative results can be produced over the range 0–10 ng/ml for calibration solutions and 0–25 ng/tile for spiked tiles using either a 4-parameter logistic fit of test line intensity versus concentration or spiked mass or the ratio of the control line to the test line intensity fit to concentration or spiked mass. Recovery from the tiles was determined to be about 30% using the fitted curves. Comparison of the control line to the test line was also examined as a possible visual detection end point and it was found that the control line became more intense than the test line at 0.5 to 1 ng/ml for calibration solutions or 1 to 2 ng/tile for spiked tiles. Thus the lateral flow cassettes for methamphetamine have the potential to produce more sensitive semi-quantitative results if an electronic lateral flow reader is used and can be more sensitive for detection if the comparison of the control line to the test line is used as the visual end point. PMID:25379615

  13. Lateral flow microarrays: a novel platform for rapid nucleic acid detection based on miniaturized lateral flow chromatography.

    PubMed

    Carter, Darren J; Cary, R Bruce

    2007-01-01

    Widely used nucleic acid assays are poorly suited for field deployment where access to laboratory instrumentation is limited or unavailable. The need for field deployable nucleic acid detection demands inexpensive, facile systems without sacrificing information capacity or sensitivity. Here we describe a novel microarray platform capable of rapid, sensitive nucleic acid detection without specialized instrumentation. The approach is based on a miniaturized lateral flow device that makes use of hybridization-mediated target capture. The miniaturization of lateral flow nucleic acid detection provides multiple advantages over traditional lateral flow devices. Ten-microliter sample volumes reduce reagent consumption and yield analyte detection times, excluding sample preparation and amplification, of <120 s while providing sub-femtomole sensitivity. Moreover, the use of microarray technology increases the potential information capacity of lateral flow. Coupled with a hybridization-based detection scheme, the lateral flow microarray (LFM) enables sequence-specific detection, opening the door to highly multiplexed implementations for broad-range assays well suited for point-of-care and other field applications. The LFM system is demonstrated using an isothermal amplification strategy for detection of Bacillus anthracis, the etiologic agent of anthrax. RNA from as few as two B. anthracis cells was detected without thermocycling hardware or fluorescence detection systems.

  14. Lateral flow microarrays: a novel platform for rapid nucleic acid detection based on miniaturized lateral flow chromatography

    PubMed Central

    Carter, Darren J.; Cary, R. Bruce

    2007-01-01

    Widely used nucleic acid assays are poorly suited for field deployment where access to laboratory instrumentation is limited or unavailable. The need for field deployable nucleic acid detection demands inexpensive, facile systems without sacrificing information capacity or sensitivity. Here we describe a novel microarray platform capable of rapid, sensitive nucleic acid detection without specialized instrumentation. The approach is based on a miniaturized lateral flow device that makes use of hybridization-mediated target capture. The miniaturization of lateral flow nucleic acid detection provides multiple advantages over traditional lateral flow devices. Ten-microliter sample volumes reduce reagent consumption and yield analyte detection times, excluding sample preparation and amplification, of <120 s while providing sub-femtomole sensitivity. Moreover, the use of microarray technology increases the potential information capacity of lateral flow. Coupled with a hybridization-based detection scheme, the lateral flow microarray (LFM) enables sequence-specific detection, opening the door to highly multiplexed implementations for broad-range assays well suited for point-of-care and other field applications. The LFM system is demonstrated using an isothermal amplification strategy for detection of Bacillus anthracis, the etiologic agent of anthrax. RNA from as few as two B. anthracis cells was detected without thermocycling hardware or fluorescence detection systems. PMID:17478499

  15. Aptamers: Universal capture units for lateral flow applications.

    PubMed

    Fischer, Christin; Wessels, Hauke; Paschke-Kratzin, Angelika; Fischer, Markus

    2017-04-01

    The present work demonstrates the implementation of aptamers as capture molecules for a wide range of target classes in lateral flow assay applications. The targets were chosen in order to cover a wide range of target classes (small sized - metabolite, medium sized - protein, and large sized - whole cell/spore). For each target class one target molecule was selected as representative and appropriate aptamers were used for lateral flow assay development. The work points out that the implementation of aptamers as capture molecules in a universal lateral flow test platform was successful independent form target size. Furthermore, the limit of detection for p-aminohippuric acid in urine (200 ppm), lysozyme in white wine (20 ppm), and Alicyclobacillus spores in buffered orange juice (>8 CFU/mL) were determined using aptamers as capture molecules. The whole approach is considered as a proof of concept, regarding the ability of aptamers as an alternative to antibodies (in conjunction with directive 2010/63/EU on the protection of animals used for scientific purposes) in lateral flow applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Replacing antibodies with aptamers in lateral flow immunoassay.

    PubMed

    Chen, Ailiang; Yang, Shuming

    2015-09-15

    Aptamers have been identified against various targets as a type of chemical or nucleic acid ligand by systematic evolution of ligands by exponential enrichment (SELEX) with high sensitivity and specificity. Aptamers show remarkable advantages over antibodies due to the nucleic acid nature and target-induced structure-switching properties and are widely used to design various fluorescent, electrochemical, or colorimetric biosensors. However, the practical applications of aptamer-based sensing and diagnostics are still lagging behind those of antibody-based tests. Lateral flow immunoassay (LFIA) represents a well established and appropriate technology among rapid assays because of its low cost and user-friendliness. The antibody-based platform is utilized to detect numerous targets, but it is always hampered by the antibody preparation time, antibody stability, and effect of modification on the antibody. Seeking alternatives to antibodies is an area of active research and is of tremendous importance. Aptamers are receiving increasing attention in lateral flow applications because of a number of important potential performance advantages. We speculate that aptamer-based LFIA may be one of the first platforms for commercial use of aptamer-based diagnosis. This review first gives an introduction to aptamer including the selection process SELEX with its focus on aptamer advantages over antibodies, and then depicts LFIA with its focus on aptamer opportunities in LFIA over antibodies. Furthermore, we summarize the recent advances in the development of aptamer-based lateral flow biosensing assays with the aim to provide a general guide for the design of aptamer-based lateral flow biosensing assays.

  17. Robust detection of peak signals for lateral flow immunoassays

    NASA Astrophysics Data System (ADS)

    Kim, Jongwon; Kim, Jong Dae; Nahm, Kie Bong; Choi, Eui Yul; Lee, Geumyoung

    2011-02-01

    Template matching method is presented to identify the peaks from the scanned signals of lateral flow immunoassay strips. The template is composed of two pulses separated by the distance of the control and the target ligand line in the assay, and is convolved with the scanned signal to deliver the maximum at the center of the two peaks. The peak regions were identified with the predefined distances from the center. Glycosylated haemoglobin immunoassay strips and fluorescent strip readers from Boditechmed Inc. were tested to estimate the lot and reader variations of the concentration measurands. The results showed the robustness of the propose method.

  18. Flow cytometer measurement of binding assays

    DOEpatents

    Saunders, George C.

    1987-01-01

    A method of measuring the result of a binding assay that does not require separation of fluorescent smaller particles is disclosed. In a competitive binding assay the smaller fluorescent particles coated with antigen compete with antigen in the sample being analyzed for available binding sites on larger particles. In a sandwich assay, the smaller, fluorescent spheres coated with antibody attach themselves to molecules containing antigen that are attached to larger spheres coated with the same antibody. The separation of unattached, fluorescent smaller particles is made unnecessary by only counting the fluorescent events triggered by the laser of a flow cytometer when the event is caused by a particle with a light scatter measurement within a certain range corresponding to the presence of larger particles.

  19. Lateral flow colloidal gold-based immunoassay for pesticide.

    PubMed

    Wang, Shuo; Zhang, Can; Zhang, Yan

    2009-01-01

    In recent years, immunochromatographic lateral flow test strips are used as a popular diagnostic tool. There are two formats (noncompetitive and competitive) in gold-based immunoassay. Noncompetitive gold-based immunoassay also called sandwich assay is applied for the detection of large molecular mass. For small molecular mass such as pesticide, competitive format of lateral flow colloidal gold-based immunoassay is described in this chapter. The preparation of gold colloidal and the conjugation between antibody and gold colloidal are described. Hi-flow plus nitrocellulose membranes are separately coated with goat anti-rabbit IgG (control line) and hapten-OVA conjugate (test line). Thus, the degree of intensity of color of the test line is the reverse of the concentration of pesticide in the sample and the visual result is immediately observable. Colloidal gold-based immunoassay can also be applied for multianalysis in one test strip if the detected targets show different physico-chemical properties and their haptens show great differences in chemical structure.

  20. Multiplex lateral flow immunoassay for mycotoxin determination.

    PubMed

    Song, Suquan; Liu, Na; Zhao, Zhiyong; Njumbe Ediage, Emmanuel; Wu, Songling; Sun, Changpo; De Saeger, Sarah; Wu, Aibo

    2014-05-20

    A new lateral flow immunoassay (LFA) is proposed for qualitative and/or semiquantitative determination of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON), and their analogues (AFs, ZEAs, DONs) in cereal samples. Each of the mycotoxin specific antibody was class specific and there was no cross reactivity to other groups of compounds. The visual limits of detection (vLOD) of the strip were 0.03, 1.6, and 10 μg/kg for AFB1, ZEA and DON, respectively. The calculated limits of detection (cLOD) were 0.05, 1, and 3 μg/kg, respectively. Meanwhile the cutoff values were achieved at 1, 50, and 60 μg/kg for AFB1, ZEA and DON, respectively. Recoveries ranged from 80% to 122% and RSD from 5% to 20%. Both the vLOD and cLOD for the three mycotoxins were lower than the EU maximum levels. Analysis of naturally contaminated maize samples resulted in a good agreement between the multiplex LFA and LC-MS/MS (100% for DONs and AFs, and 81% for ZEAs). Careful analysis of the results further explained the general overestimation of LFA compared to chromatographic methods for quantification of mycotoxins.

  1. Automated Protein Assay Using Flow Injection Analysis

    NASA Astrophysics Data System (ADS)

    Wolfe, Carrie A. C.; Oates, Matthew R.; Hage, David S.

    1998-08-01

    The technique of flow injection analysis (FIA) is a common instrumental method used in detecting a variety of chemical and biological agents. This paper describes an undergraduate laboratory that uses FIA to perform a bicinchoninic acid (BCA) colorimetric assay for quantitating protein samples. The method requires less than 2 min per sample injection and gives a response over a broad range of protein concentrations. This method can be used in instrumental analysis labs to illustrate the principles and use of FIA, or as a means for introducing students to common methods employed in the analysis of biological agents.

  2. An inexpensive, fast and sensitive quantitative lateral flow magneto-immunoassay for total prostate specific antigen.

    PubMed

    Barnett, Jacqueline M; Wraith, Patrick; Kiely, Janice; Persad, Raj; Hurley, Katrina; Hawkins, Peter; Luxton, Richard

    2014-09-01

    We describe the detection characteristics of a device the Resonant Coil Magnetometer (RCM) to quantify paramagnetic particles (PMPs) in immunochromatographic (lateral flow) assays. Lateral flow assays were developed using PMPs for the measurement of total prostate specific antigen (PSA) in serum samples. A detection limit of 0.8 ng/mL was achieved for total PSA using the RCM and is at clinically significant concentrations. Comparison of data obtained in a pilot study from the analysis of serum samples with commercially available immunoassays shows good agreement. The development of a quantitative magneto-immunoassay in lateral flow format for total PSA suggests the potential of the RCM to operate with many immunoassay formats. The RCM has the potential to be modified to quantify multiple analytes in this format. This research shows promise for the development of an inexpensive device capable of quantifying multiple analytes at the point-of-care using a magneto-immunoassay in lateral flow format.

  3. An Inexpensive, Fast and Sensitive Quantitative Lateral Flow Magneto-Immunoassay for Total Prostate Specific Antigen

    PubMed Central

    Barnett, Jacqueline M.; Wraith, Patrick; Kiely, Janice; Persad, Raj; Hurley, Katrina; Hawkins, Peter; Luxton, Richard

    2014-01-01

    We describe the detection characteristics of a device the Resonant Coil Magnetometer (RCM) to quantify paramagnetic particles (PMPs) in immunochromatographic (lateral flow) assays. Lateral flow assays were developed using PMPs for the measurement of total prostate specific antigen (PSA) in serum samples. A detection limit of 0.8 ng/mL was achieved for total PSA using the RCM and is at clinically significant concentrations. Comparison of data obtained in a pilot study from the analysis of serum samples with commercially available immunoassays shows good agreement. The development of a quantitative magneto-immunoassay in lateral flow format for total PSA suggests the potential of the RCM to operate with many immunoassay formats. The RCM has the potential to be modified to quantify multiple analytes in this format. This research shows promise for the development of an inexpensive device capable of quantifying multiple analytes at the point-of-care using a magneto-immunoassay in lateral flow format. PMID:25587419

  4. Improved flow cytometer measurement of binding assays

    DOEpatents

    Saunders, G.C.

    1984-05-30

    The invention relates to a method of measuring binding assays carried out with different size particles wherein the binding assay sample is run through a flow cytometer without separating the sample from the marking agent. The amount of a binding reactant present in a sample is determined by providing particles with a coating of binder and also a known quantity of smaller particles with a coating of binder reactant. The binding reactant is the same as the binding reactant present in the sample. The smaller particles also contain a fluorescent chemical. The particles are combined with the sample and the binding reaction is allowed to occur for a set length of time followed by combining the smaller particles with the mixture of the particles and the sample produced and allowing the binding reactions to proceed to equilibrium. The fluorescence and light scatter of the combined mixture is then measured as the combined mixture passes through a flow cytometer equipped with a laser to bring about fluorescence, and the number and strength of fluorescent events are compared. A similar method is also provided for determining the amount of antigen present in the sample by providing spheres with an antibody coating and some smaller spheres with an antigen coating. (LEW)

  5. Mitigating the Hook Effect in Lateral Flow Sandwich Immunoassays Using Real-Time Reaction Kinetics.

    PubMed

    Rey, Elizabeth G; O'Dell, Dakota; Mehta, Saurabh; Erickson, David

    2017-05-02

    The quantification of analyte concentrations using lateral flow assays is a low-cost and user-friendly alternative to traditional lab-based assays. However, sandwich-type immunoassays are often limited by the high-dose hook effect, which causes falsely low results when analytes are present at very high concentrations. In this paper, we present a reaction kinetics-based technique that solves this problem, significantly increasing the dynamic range of these devices. With the use of a traditional sandwich lateral flow immunoassay, a portable imaging device, and a mobile interface, we demonstrate the technique by quantifying C-reactive protein concentrations in human serum over a large portion of the physiological range. The technique could be applied to any hook effect-limited sandwich lateral flow assay and has a high level of accuracy even in the hook effect range.

  6. Lateral Flow Immunoassays for Ebola Virus Disease Detection in Liberia.

    PubMed

    Phan, Jill C; Pettitt, James; George, Josiah S; Fakoli, Lawrence S; Taweh, Fahn M; Bateman, Stacey L; Bennett, Richard S; Norris, Sarah L; Spinnler, David A; Pimentel, Guillermo; Sahr, Phillip K; Bolay, Fatorma K; Schoepp, Randal J

    2016-10-15

    Lateral flow immunoassays (LFIs) are point-of-care diagnostic assays that are designed for single use outside a formal laboratory, with in-home pregnancy tests the best-known example of these tests. Although the LFI has some limitations over more-complex immunoassay procedures, such as reduced sensitivity and the potential for false-positive results when using complex sample matrices, the assay has the benefits of a rapid time to result and ease of use. These benefits make it an attractive option for obtaining rapid results in an austere environment. In an outbreak of any magnitude, a field-based rapid diagnostic assay would allow proper patient transport and for safe burials to be conducted without the delay caused by transport of samples between remote villages and testing facilities. Use of such point-of-care instruments in the ongoing Ebola virus disease (EVD) outbreak in West Africa would have distinct advantages in control and prevention of local outbreaks, but proper understanding of the technology and interpretation of results are important.  In this study, a LFI, originally developed by the Naval Medical Research Center for Ebola virus environmental testing, was evaluated for its ability to detect the virus in clinical samples in Liberia. Clinical blood and plasma samples and post mortem oral swabs submitted to the Liberian Institute for Biomedical Research, the National Public Health Reference Laboratory for EVD testing, were tested and compared to results of real-time reverse transcription-polymerase chain reaction (rRT-PCR), using assays targeting Ebola virus glycoprotein and nucleoprotein.  The LFI findings correlated well with those of the real-time RT-PCR assays used as benchmarks.  Rapid antigen-detection tests such as LFIs are attractive alternatives to traditional immunoassays but have reduced sensitivity and specificity, resulting in increases in false-positive and false-negative results. An understanding of the strengths, weaknesses

  7. Multiplex lateral-flow test strips fabricated by two-dimensional shaping.

    PubMed

    Fenton, Erin M; Mascarenas, Monica R; López, Gabriel P; Sibbett, Scott S

    2009-01-01

    We have fabricated paper- and nitrocellulose-based lateral-flow devices that are shaped in two dimensions by a computer-controlled knife. The resulting star, candelabra, and other structures are spotted with multiple bioassay reagents to produce multiplex lateral-flow assays. We have also fabricated laminar composites in which porous nitrocellulose media are sandwiched between vinyl and polyester plastic films. This minimizes evaporation, protects assay surfaces from contamination and dehydration, and eliminates the need for the conventional hard plastic cassette holders that are typically used to package commercial lateral-flow diagnostic strips. The reported fabrication method is novel, low-cost, and well-suited to (i) fabrication and adoption in resource-poor areas, (ii) prototype development, (iii) high-volume manufacturing, and (iii) improving rates of operator error.

  8. Novel development of a lateral flow immunoassay for rapid field detection of citrus tristeza virus

    USDA-ARS?s Scientific Manuscript database

    Maintenance of virus-free citrus in nurseries and orchards is essential to control spread of aphid-borne Citrus tristeza virus (CTV) in California. A lateral flow assay (LFA) test strip with a polyclonal antiserum made from virus particles produced in Nicotiana benthamiana plants inoculated with an ...

  9. Evaluation of a Rapid Lateral Flow Point-of-Care Test for Detection of Cryptosporidium

    PubMed Central

    Fleece, Molly E.; Heptinstall, Jack; Khan, Shaila S.; Kabir, Mamum; Herbein, Joel; Haque, Rashidul; Petri, William A.

    2016-01-01

    A new rapid lateral flow fecal antigen detection test for Cryptosporidium was evaluated using diarrheal stool samples from a cohort of children in Bangladesh. The test had a sensitivity of 100% and a specificity of 94% when compared with enzyme-linked immunosorbent assay antigen detection. PMID:27573629

  10. A lateral flow biosensor for detection of single nucleotide polymorphism by circular strand displacement reaction.

    PubMed

    Xiao, Zhuo; Lie, Puchang; Fang, Zhiyuan; Yu, Luxin; Chen, Junhua; Liu, Jie; Ge, Chenchen; Zhou, Xuemeng; Zeng, Lingwen

    2012-09-04

    A lateral flow biosensor for detection of single nucleotide polymorphism based on circular strand displacement reaction (CSDPR) has been developed. Taking advantage of high fidelity of T4 DNA ligase, signal amplification by CSDPR, and the optical properties of gold nanoparticles, this assay has reached a detection limit of 0.01 fM.

  11. Lateral flow based immunobiosensors for detection of food contaminants.

    PubMed

    Raeisossadati, Mohammad Javad; Danesh, Noor Mohammad; Borna, Fazlollah; Gholamzad, Mehrdad; Ramezani, Mohammad; Abnous, Khalil; Taghdisi, Seyed Mohammad

    2016-12-15

    Safety of food is of great concern these days due to various contaminations including toxins, infectious agents and chemical contaminants. Therefore, there is a need to develop promising and user's friendly method to monitor food safety. Lateral flow tests are new, simple and rapid alternative for detection of food-borne pathogens compared with traditional methods. In this review article, we surveyed application of lateral flow biosensors in detection of different food contaminants and labels used to enhance the efficiency of the system. Finally, the unique feature of multi-parametric analysis of analytes by lateral flow device has been reported, proving a lateral flow system is able to be designed in a way to detect multiple targets, simultaneously.

  12. [The use of hydrosol hexacianferrate (II) ferrum (III) for developing diagnostic lateral flow tests].

    PubMed

    Skopinskaia, S N; Iarkov, S P; Zlobin, V N; Valiev, Kh Kh

    2012-01-01

    On the basis of synthesized negatively charged hydrosol hexacianferrate (II) ferrum (III) (HCFF) by diameter 10-20 HM are received stable conjugates with antibodies and antigens glycoprotein and lipopolysaccharide (LPS) nature. Synthesized hydrosol (HCFF) is new type of a disperse phase in the lateral flow assay. Conjugates mentioned above were applied for construction lateral flow tests-systems for revealing cholera toxin, the rabbit antibodies to recombinant glycoproteine complex from Micobacterium tuberculosis H37 Rv, human immunoglobulin, LPS antigens S. typhimurium, S. enteritidis. Developed lateral flow tests-systems had high analysis speed (5-7 min), good specificity and sensitivity: on cholera toxin of 2.0 ug/ml, on LPS antigens S. typhimurium, S. enteritidis 0.5 ug/ml.

  13. Test characteristics and potential impact of the urine LAM lateral flow assay in HIV-infected outpatients under investigation for TB and able to self-expectorate sputum for diagnostic testing.

    PubMed

    Peter, Jonny; Theron, Grant; Chanda, Duncan; Clowes, Petra; Rachow, Andrea; Lesosky, Maia; Hoelscher, Michael; Mwaba, Peter; Pym, Alex; Dheda, Keertan

    2015-07-09

    The commercially available urine LAM strip test, a point-of-care tuberculosis (TB) assay, requires evaluation in a primary care setting where it is most needed. There is currently inadequate data to guide implementation in TB and HIV-endemic settings. Adult HIV-infected outpatients with suspected pulmonary TB able to self-expectorate sputum from four primary clinics in South Africa, Zambia and Tanzania underwent diagnostic evaluation [sputum smear microscopy, Xpert-MTB/RIF, and culture (reference standard)] as part of a prospective parent study. Urine LAM testing (grade-2 cut-point) was performed on archived samples. Performance characteristics of LAM alone or in combination with sputum-based diagnostics were evaluated. Potential impact on 2 and 6-month morbidity (TBscore), patient dropout rates, and prognosis (death/ loss to follow-up) were evaluated. Among 583 participants with suspected TB that were HIV-infected or refused testing, the overall LAM sensitivity (95 % CI; n/N) and in the CD4 ≤ 100 cells/mm(3) sub-group was 22.7 % (16.6-28.7; 41/181) and 30.4 % (17.1-43.7; 14/46), respectively. Overall specificity was 93.0 % (90.5-95.6; 361/388). Amongst culture-positive TB cases, adjunctive LAM testing did not improve the sensitivity of either sputum Xpert-MTB/RIF [78.2 % (69.8-86.7; 72/92) versus 76.1 % (67.4-84.8; 70/92), p = 0.7] or smear-microscopy [56.2 % (45.9-66.5; 50/89) versus 43.8 % (33.5-54.1; 39/89), p = 0.1). Clinic-based LAM, as an adjunct to either smear microscopy or Xpert MTB/RIF same-day testing, would neither have decreased patient dropout, nor increased same-day treatment initiation in this clinical setting where same-day chest radiography was available. LAM positivity was associated with 6-month lost-to-follow-up/death (AOR 4.4; p = 0.002) but not TBscore (at baseline or change in TBscore 2-months post-treatment) (p = 0.17). In African HIV-TB co-infected outpatients able to self-expectorate sputum LAM had limited sensitivity even at low CD4

  14. Lateral flow biosensors for the detection of nucleic acid.

    PubMed

    Zeng, Lingwen; Lie, Puchang; Fang, Zhiyuan; Xiao, Zhuo

    2013-01-01

    The detection of nucleic acid is of central importance for the diagnosis of genetic diseases, infectious agents, and biowarfare agents. Traditional strategies and technologies for nucleic acid detection are time-consuming and labor-intensive. Recently, isothermal strand-displacement reaction-based lateral flow biosensors have attracted a great deal of research interest because they are sensitive, simple, fast, and easy to use. Here, we describe a lateral flow biosensor based on isothermal strand-displacement polymerase reaction and gold nanoparticles for the visual detection of nucleic acid.

  15. Evaluation of Lateral-Flow Clostridium botulinum Neurotoxin Detection Kits for Food Analysis

    PubMed Central

    Sharma, Shashi K.; Eblen, Brian S.; Bull, Robert L.; Burr, Donald H.; Whiting, Richard C.

    2005-01-01

    The suitability and sensitivity of two in vitro lateral-flow assays for detecting Clostridium botulinum neurotoxins (BoNTs) in an assortment of foods were evaluated. Toxin extraction and preparation methods for various liquid, solid, and high-fat-content foods were developed. The lateral-flow assays, one developed by the Naval Medical Research Center (Silver Spring, MD) and the other by Alexeter Technologies (Gaithersburg, MD), are based on the immunodetection of BoNT types A, B, and E. The assays were found to be rapid and easy to perform with minimum requirements for laboratory equipment or skills. They can readily detect 10 ng/ml of BoNT types A and B and 20 ng/ml of BoNT type E. Compared to other in vitro detection methods, these assays are less sensitive, and the assessment of a result is strictly qualitative. However, the assay was found to be simple to use and to require minimal training. The assays successfully detected BoNT types A, B, and E in a wide variety of foods, suggesting their potential usefulness as a preliminary screening system for triaging food samples with elevated BoNT levels in the event of a C. botulinum contamination event. PMID:16000807

  16. Evaluation of lateral-flow Clostridium botulinum neurotoxin detection kits for food analysis.

    PubMed

    Sharma, Shashi K; Eblen, Brian S; Bull, Robert L; Burr, Donald H; Whiting, Richard C

    2005-07-01

    The suitability and sensitivity of two in vitro lateral-flow assays for detecting Clostridium botulinum neurotoxins (BoNTs) in an assortment of foods were evaluated. Toxin extraction and preparation methods for various liquid, solid, and high-fat-content foods were developed. The lateral-flow assays, one developed by the Naval Medical Research Center (Silver Spring, MD) and the other by Alexeter Technologies (Gaithersburg, MD), are based on the immunodetection of BoNT types A, B, and E. The assays were found to be rapid and easy to perform with minimum requirements for laboratory equipment or skills. They can readily detect 10 ng/ml of BoNT types A and B and 20 ng/ml of BoNT type E. Compared to other in vitro detection methods, these assays are less sensitive, and the assessment of a result is strictly qualitative. However, the assay was found to be simple to use and to require minimal training. The assays successfully detected BoNT types A, B, and E in a wide variety of foods, suggesting their potential usefulness as a preliminary screening system for triaging food samples with elevated BoNT levels in the event of a C. botulinum contamination event.

  17. Rapid Simultaneous Detection of Anti-protozoan Drugs Using a Lateral-Flow Immunoassay Format.

    PubMed

    Fitzgerald, Jenny; Leonard, Paul; Danaher, Martin; O'Kennedy, Richard

    2015-05-01

    This research describes the development of a multi-analyte lateral-flow immunoassay intended for the simultaneous detection of three anti-protozoan drugs (coccidiostats). These drugs, namely, halofuginone, toltrazuril and diclazuril, are used in the treatment of Eimeria spp. infections in cattle, pigs, chickens and turkeys. Coloured carboxylated microspheres were coated with each of the detection antibodies and employed in a lateral-flow assay format for detection of these residues in eggs. Using this approach, halofuginone was detectable at a limit of 10 ng/mL or greater, toltrazuril at 100 ng/mL and, similarly, diclazuril had a detection limit of 100 ng/mL, which is below the maximum allowed residue limit for all three as outlined by EU regulation. This simple cost-efficient assay and analysis method could pave the way for more efficient simultaneous monitoring of small-molecule residues in the future.

  18. Serotype sensitivity of a lateral flow immunoassay for cryptococcal antigen.

    PubMed

    Gates-Hollingsworth, Marcellene A; Kozel, Thomas R

    2013-04-01

    To meet the needs of a global community, an immunoassay for cryptococcal antigen (CrAg) must have high sensitivity for CrAg of all major serotypes. A new immunoassay for CrAg in lateral flow format was evaluated and found to have a high sensitivity for detection of serotypes A, B, C, and D.

  19. The effect of lateral interaction on traffic flow

    NASA Astrophysics Data System (ADS)

    Bouadi, M.; Jetto, K.; Benyoussef, A.; Kenz, A.

    2016-10-01

    We propose an extended cellular automaton model for traffic flow, taking into account lateral interactions with defects and between vehicles. The fundamental diagram for a given defects density on the road is studied. It is found that the plateau size increases linearly with the decreasing road width for little defects densities. Furthermore, the capacity increases linearly with the increasing road width. However, for a fixed road width, the capacity decreases exponentially with the increasing defects density. The lateral effects for non-mutual interactions between lanes and for the same maximal velocity is also investigated. It is found that the lateral effects on one lane are meaningful only when the density on the other lane is above the critical density. However, the lateral effects are always present if fast and slow lanes exist. Little differences have been found for the mutual interactions.

  20. Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk.

    PubMed

    Berlina, Anna N; Taranova, Nadezhda A; Zherdev, Anatoly V; Vengerov, Yuri Y; Dzantiev, Boris B

    2013-05-01

    A novel rapid (20 min) fluorescent lateral flow test for chloramphenicol (CAP) detection in milk was developed. The chosen format is a binding-inhibition assay. Water-soluble quantum dots with an emission peak at 625 nm were applied as a label. Milk samples were diluted by 20 % with phosphate buffer to eliminate the matrix effect. The result of the assay could be seen by eye under UV light excitation or registered by a portable power-dependent photometer. The limit of CAP detection by the second approach is 0.2 ng/mL, and the limit of quantitation is 0.3 ng/mL.

  1. Development of a highly sensitive lateral immunochromatographic assay for rapid detection of Vibrio parahaemolyticus.

    PubMed

    Liu, Xinfeng; Guan, Yuyao; Cheng, Shiliang; Huang, Yidan; Yan, Qin; Zhang, Jun; Huang, Guanjun; Zheng, Jian; Liu, Tianqiang

    2016-12-01

    Vibrio parahaemolyticus is widely present in brackish water all over the world, causing infections in certain aquatic animals. It is also a foodborne pathogen that causes diarrhea in humans. The aim of this study is to develop an immunochromatographic lateral flow assay (LFA) for rapid detection of V. parahaemolyticus in both aquatic products and human feces of diarrheal patients. Two monoclonal antibody (MAb) pairs, GA1a-IC9 and IC9-KB4c, were developed and proven to be highly specific and sensitive to V. parahaemolyticus. Based on the two MAb pairs, two types of LFA strips were prepared. Their testing limits for V. parahaemolyticus culture were both 1.2×10(3)CFU/ml. The diagnostic sensitivities and specificities were both 100% for the 32 tested microbial species, including 6 Vibrio species. Subsequently, the LFA strips were used to test Whiteleg shrimps and human feces. The type II strip showed a higher diagnostic sensitivity. Its sensitivity and specificity for hepatopancreas and fecal samples from 13 Whiteleg shrimps and fecal samples from 146 human diarrheal patients were all 100%. In conclusion, our homemade type II LFA is a very promising testing device for rapid and convenient detection of V. parahaemolyticus infection not only in aquatic animals, but also in human diarrheal patients. This sensitive immunochromtographic LFA allows rapid detection of V. parahaemolyticus without requirement of culture enrichment. Copyright © 2016. Published by Elsevier B.V.

  2. A lateral flow immunoassay for the rapid detection of ochratoxin A in wine and grape must.

    PubMed

    Anfossi, Laura; Giovannoli, Cristina; Giraudi, Gianfranco; Biagioli, Flavia; Passini, Cinzia; Baggiani, Claudio

    2012-11-21

    A one-step lateral flow immunoassay was developed for semiquantitatively detecting ochratoxin A (OTA) in wines and grape musts. Matrix-matched calibration curves carried out in blank wines showed a detection limit of 1 μg L(-1) and IC(50) of 3.2 μg L(-1). Relative standard deviations for intra- and interday precision were in the 20-40% range. A simple treatment of samples, which only included dilution with sodium bicarbonate and polyethylene glycol (4% w/v) for red and white wines and the further addition of ethanol (12% v/v) for grape musts, was established. The developed assay allowed OTA detection in 5 min and proved to be accurate and sensitive enough to allow the correct attribution of samples as compliant or noncompliant according to EU legislation. Results agreeing with those of a reference chromatographic method were obtained on 38 wines and 16 musts. Although some lateral flow devices aimed at detecting OTA have been previously described, this is the first assay capable of measuring the toxin in wine and grape must, which represent a major source of OTA dietary intake. Analytical performances of the method are comparable to or better than previously reported assays showed. In addition, the assay, including sample treatments, is extremely simple and rapid and can be effectively regarded as a one-step assay usable virtually anywhere.

  3. Flow visualization of lateral jet injection into swirling crossflow

    NASA Technical Reports Server (NTRS)

    Ferrell, G. B.; Aoki, K.; Lilley, D. G.

    1985-01-01

    Flow visualization experiments have been conducted to characterize the time-mean flowfield of a deflected turbulent jet in a confining cylindrical crossflow. Jet-to-crossflow velocity ratios of 2, 4, and 6 were investigated, under crossflow inlet swirler vane angles of 0 (swirler removed), 45 and 70 degrees. Smoke, neutrally-buoyant helium-filled soap bubbles, and multi-spark flow visualization were employed to highlight interesting features of the deflected jet, as well as the trajectory and spread pattern of the jet. Gross flowfield characterization was obtained for a range of lateral jet-to-crossflow velocity ratios and a range of inlet swirl strengths in the main flow. The flow visualization results agree well with the measurements obtained elsewhere with the six-orientation single hot-wire method.

  4. Flow visualization of lateral jet injection into swirling crossflow

    NASA Technical Reports Server (NTRS)

    Ferrell, G. B.; Aoki, K.; Lilley, D. G.

    1985-01-01

    Flow visualization experiments have been conducted to characterize the time-mean flowfield of a deflected turbulent jet in a confining cylindrical crossflow. Jet-to-crossflow velocity ratios of 2, 4, and 6 were investigated, under crossflow inlet swirler vane angles of 0 (swirler removed), 45 and 70 degrees. Smoke, neutrally-buoyant helium-filled soap bubbles, and multi-spark flow visualization were employed to highlight interesting features of the deflected jet, as well as the trajectory and spread pattern of the jet. Gross flowfield characterization was obtained for a range of lateral jet-to-crossflow velocity ratios and a range of inlet swirl strengths in the main flow. The flow visualization results agree well with the measurements obtained elsewhere with the six-orientation single hot-wire method.

  5. Silver and gold enhancement methods for lateral flow immunoassays.

    PubMed

    Rodríguez, Myriam Oliveira; Covián, Lucía Blanco; García, Agustín Costa; Blanco-López, Maria Carmen

    2016-01-01

    Sensitivity is the main concern at the development of rapid test by lateral flow immunoassays. On the other hand, low limits of detection are often required at medical diagnostics and other field of analysis. To overcome this drawback, several enhancement protocols have been described. In this paper, we have selected different silver enhancement methods and one dual gold conjugation, and we critically compared the amplification produced when applied to a gold-nanoparticle based lateral flow immunoassay for the detection of prostate specific antigen (PSA). The highest amplification was obtained by using an immersion method based on a solution of silver nitrate and hydroquinone/citrate buffer in proportion 1:1. Under these conditions, the system is capable of detecting PSA within 20 min at levels as low as 0.1 ng/mL, with a 3-fold sensitivity improvement. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Rapid prototyping of electrochemical lateral flow devices: stencilled electrodes.

    PubMed

    Aller Pellitero, Miguel; Kitsara, Maria; Eibensteiner, Friedrich; del Campo, F Javier

    2016-04-21

    A straightforward and very cost effective method is proposed to prototype electrodes using pressure sensitive adhesives (PSA) and a simple cutting technique. Two cutting methods, namely blade cutting and CO2 laser ablation, are compared and their respective merits are discussed. The proposed method consists of turning the protective liner on the adhesive into a stencil to apply screen-printing pastes. After the electrodes have been printed, the liner is removed and the PSA can be used as a backing material for standard lateral flow membranes. We present the fabrication of band electrodes down to 250 μm wide, and their characterization using microscopy techniques and cyclic voltammetry. The prototyping approach presented here facilitates the development of new electrochemical devices even if very limited fabrication resources are available. Here we demonstrate the fabrication of a simple lateral-flow device capable of determining glucose in blood. The prototyping approach presented here is highly suitable for the development of novel electroanalytical tools.

  7. Multiplexed lateral flow biosensors: Technological advances for radically improving point-of-care diagnoses.

    PubMed

    Li, Jia; Macdonald, Joanne

    2016-09-15

    Lateral flow biosensors are a leading technology in point-of-care diagnostics due to their simplicity, rapidness and low cost. Their primacy in this arena continues through technological breakthroughs such as multiplexing: the detection of more than one biomarker in a single assay. Multiplexing capacity is critical for improving diagnostic efficiency, enhancing the diagnostic precision for specific diseases and reducing diagnostic cost. Here we review, for the first time, the various types and strategies employed for creating multiplexed lateral flow biosensors. These are classified into four main categories in terms of specific application or multiplexing level, namely linear, parameter, spatial and conceptual. We describe the practical applications and implications for each approach and compare their advantages and disadvantages. Importantly, multiplexing is still subject to limitations of the traditional lateral flow biosensor, such as sensitivity and specificity. However, by pushing the limitations of the traditional medium into the multiplex arena, several technological breakthroughs are emerging with novel solutions that further expand the utility of lateral flow biosensing for point-of-care applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Isothermal Recombinase Polymerase amplification (RPA) of Schistosoma haematobium DNA and oligochromatographic lateral flow detection.

    PubMed

    Rosser, A; Rollinson, D; Forrest, M; Webster, B L

    2015-09-04

    Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs. Amplification of schistosome DNA in urine by PCR is sensitive and specific but requires infrastructure, financial resources and skilled personnel, often not available in endemic areas. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology that is simple, rapid, portable and needs few resources. Here a Schistosoma haematobium RPA assay was developed and adapted so that DNA amplicons could be detected using oligochromatographic Lateral Flow (LF) strips. The assay successfully amplified S. haematobium DNA at 30-45 °C in 10 mins and was sensitive to a lower limit of 100 fg of DNA. The assay was also successful with the addition of crude urine, up to 5% of the total reaction volume. Cross amplification occurred with other schistosome species but not with other common urine microorganisms. The LF-RPA assay developed here can amplify and detect low levels of S. haematobium DNA. Reactions are rapid, require low temperatures and positive reactions are interpreted using lateral flow strips, reducing the need for infrastructure and resources. This together with an ability to withstand inhibitors within urine makes RPA a promising technology for further development as a molecular diagnostic tool for urogenital schistosomiasis.

  9. Decreased intramuscular blood flow in patients with lateral epicondylitis.

    PubMed

    Oskarsson, E; Gustafsson, B-E; Pettersson, K; Aulin, K Piehl

    2007-06-01

    The purpose of this pilot study was to investigate intramuscular microcirculation in extensor carpi radialis brevis (ECRB) in patients with lateral epicondylitis. Ten patients with unilateral epicondylitis, mean duration of symptoms of 39 (12-96) months participated. The diagnosis was based on clinical examination and none was under treatment for the last 6 months. Isometric handgrip strength, 2-pinch grip strength and muscle strength during radial deviation and dorsal extension were determined. Functional perceived pain was evaluated by a modified behaviour rating scale and perceived pain during contraction by visual analogue scale. Intramuscular and skin blood flow was recorded by a laser-Doppler flowmetry system technique (LDF) during stable temperature condition. Intramuscular blood flow was significantly lower in the affected side, 22.7+/-9.8 perfusion units (PU), as compared with 35.2+/-11.9 PU in the control side (P=0.01). There was no difference in skin blood flow or temperature between the affected and the control side. A positive correlation was found between the duration of symptoms and the difference in intramuscular blood flow between the affected and the control arm (r=0.65, P=0.06). The present data indicate that decreased microcirculation and anaerobic metabolism in ECRB may contribute to the lateral epicondylitis symptoms.

  10. New flow cytometric assays for monitoring cell-mediated cytotoxicity

    PubMed Central

    Zaritskaya, Liubov; Shurin, Michael R; Sayers, Thomas J; Malyguine, Anatoli M

    2010-01-01

    The exact immunologic responses after vaccination that result in effective antitumor immunity have not yet been fully elucidated and the data from ex vivo T-cell assays have not yet defined adequate surrogate markers for clinical efficacy. A more detailed knowledge of the specific immune responses that correlate with positive clinical outcomes should help to develop better or novel strategies to effectively activate the immune system against tumors. Furthermore, clinically relevant material is often limited and, thus, precludes the ability to perform multiple assays. The two main assays currently used to monitor lymphocyte-mediated cytoxicity in cancer patients are the 51Cr-release assay and IFN-γ ELISpot assay. The former has a number of disadvantages, including low sensitivity, poor labeling and high spontaneous release of isotope from some tumor target cells. Additional problems with the 51Cr-release assay include difficulty in obtaining autologous tumor targets, and biohazard and disposal problems for the isotope. The ELISpot assays do not directly measure cytotoxic activity and are, therefore, a surrogate marker of cyotoxic capacity of effector T cells. Furthermore, they do not assess cytotoxicity mediated by the production of the TNF family of death ligands by the cytotoxic cells. Therefore, assays that allow for the simultaneous measurement of several parameters may be more advantageous for clinical monitoring. In this respect, multifactor flow cytometry-based assays are a valid addition to the currently available immunologic monitoring assays. Use of these assays will enable detection and enumeration of tumor-specific cytotoxic T lymphocytes and their specific effector functions and any correlations with clinical responses. Comprehensive, multifactor analysis of effector cell responses after vaccination may help to detect factors that determine the success or failure of a vaccine and its immunological potency. PMID:20518716

  11. New flow cytometric assays for monitoring cell-mediated cytotoxicity.

    PubMed

    Zaritskaya, Liubov; Shurin, Michael R; Sayers, Thomas J; Malyguine, Anatoli M

    2010-06-01

    The exact immunologic responses after vaccination that result in effective antitumor immunity have not yet been fully elucidated and the data from ex vivo T-cell assays have not yet defined adequate surrogate markers for clinical efficacy. A more detailed knowledge of the specific immune responses that correlate with positive clinical outcomes should help to develop better or novel strategies to effectively activate the immune system against tumors. Furthermore, clinically relevant material is often limited and, thus, precludes the ability to perform multiple assays. The two main assays currently used to monitor lymphocyte-mediated cytoxicity in cancer patients are the (51)Cr-release assay and IFN-gamma ELISpot assay. The former has a number of disadvantages, including low sensitivity, poor labeling and high spontaneous release of isotope from some tumor target cells. Additional problems with the (51)Cr-release assay include difficulty in obtaining autologous tumor targets, and biohazard and disposal problems for the isotope. The ELISpot assays do not directly measure cytotoxic activity and are, therefore, a surrogate marker of cyotoxic capacity of effector T cells. Furthermore, they do not assess cytotoxicity mediated by the production of the TNF family of death ligands by the cytotoxic cells. Therefore, assays that allow for the simultaneous measurement of several parameters may be more advantageous for clinical monitoring. In this respect, multifactor flow cytometry-based assays are a valid addition to the currently available immunologic monitoring assays. Use of these assays will enable detection and enumeration of tumor-specific cytotoxic T lymphocytes and their specific effector functions and any correlations with clinical responses. Comprehensive, multifactor analysis of effector cell responses after vaccination may help to detect factors that determine the success or failure of a vaccine and its immunological potency.

  12. Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus.

    PubMed

    Doerflinger, Sylvie Y; Tabatabai, Julia; Schnitzler, Paul; Farah, Carlo; Rameil, Steffen; Sander, Peter; Koromyslova, Anna; Hansman, Grant S

    2016-01-01

    Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. These viruses are usually detected by molecular methods, including reverse transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Human noroviruses are genetically and antigenically diverse, with two main genogroups that are further subdivided into over 40 different genotypes. During the past decade, genogroup 2 genotype 4 (GII.4) has dominated in most countries, but recently, viruses belonging to GII.17 have increased in prevalence in a number of countries. A number of commercially available ELISAs and lateral flow immunoassays were found to have lower sensitivities to the GII.17 viruses, indicating that the antibodies used in these methods may not have a high level of cross-reactivity. In this study, we developed a rapid Nanobody-based lateral flow immunoassay (Nano-immunochromatography [Nano-IC]) for the detection of human norovirus in clinical specimens. The Nano-IC assay detected virions from two GII.4 norovirus clusters, which included the current dominant strain and a novel variant strain. The Nano-IC method had a sensitivity of 80% and specificity of 86% for outbreak specimens. Norovirus virus-like particles (VLPs) representing four genotypes (GII.4, GII.10, GII.12, and GII.17) could be detected by this method, demonstrating the potential in clinical screening. However, further modifications to the Nano-IC method are needed in order to improve this sensitivity, which may be achieved by the addition of other broadly reactive Nanobodies to the system. IMPORTANCE We previously identified a Nanobody (termed Nano-85) that bound to a highly conserved region on the norovirus capsid. In this study, the Nanobody was biotinylated and gold conjugated for a lateral flow immunoassay (termed Nano-IC). We showed that the Nano-IC assay was capable of detecting at least four antigenically distinct GII genotypes, including the newly emerging GII.17. In the clinical setting, the

  13. Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus

    PubMed Central

    Doerflinger, Sylvie Y.; Tabatabai, Julia; Schnitzler, Paul; Farah, Carlo; Rameil, Steffen; Sander, Peter; Koromyslova, Anna

    2016-01-01

    ABSTRACT Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. These viruses are usually detected by molecular methods, including reverse transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Human noroviruses are genetically and antigenically diverse, with two main genogroups that are further subdivided into over 40 different genotypes. During the past decade, genogroup 2 genotype 4 (GII.4) has dominated in most countries, but recently, viruses belonging to GII.17 have increased in prevalence in a number of countries. A number of commercially available ELISAs and lateral flow immunoassays were found to have lower sensitivities to the GII.17 viruses, indicating that the antibodies used in these methods may not have a high level of cross-reactivity. In this study, we developed a rapid Nanobody-based lateral flow immunoassay (Nano-immunochromatography [Nano-IC]) for the detection of human norovirus in clinical specimens. The Nano-IC assay detected virions from two GII.4 norovirus clusters, which included the current dominant strain and a novel variant strain. The Nano-IC method had a sensitivity of 80% and specificity of 86% for outbreak specimens. Norovirus virus-like particles (VLPs) representing four genotypes (GII.4, GII.10, GII.12, and GII.17) could be detected by this method, demonstrating the potential in clinical screening. However, further modifications to the Nano-IC method are needed in order to improve this sensitivity, which may be achieved by the addition of other broadly reactive Nanobodies to the system. IMPORTANCE We previously identified a Nanobody (termed Nano-85) that bound to a highly conserved region on the norovirus capsid. In this study, the Nanobody was biotinylated and gold conjugated for a lateral flow immunoassay (termed Nano-IC). We showed that the Nano-IC assay was capable of detecting at least four antigenically distinct GII genotypes, including the newly emerging GII.17. In the clinical

  14. Rapid and sensitive lateral flow immunoassay method for determining alpha fetoprotein in serum using europium (III) chelate microparticles-based lateral flow test strips.

    PubMed

    Liang, Rong-Liang; Xu, Xu-Ping; Liu, Tian-Cai; Zhou, Jian-Wei; Wang, Xian-Guo; Ren, Zhi-Qi; Hao, Fen; Wu, Ying-Song

    2015-09-03

    Alpha-fetoprotein (AFP), a primary marker for many diseases including various cancers, is important in clinical tumor diagnosis and antenatal screening. Most immunoassays provide high sensitivity and accuracy for determining AFP, but they are expensive, often complex, time-consuming procedures. A simple and rapid point-of-care system that integrates Eu (III) chelate microparticles with lateral flow immunoassay (LFIA) has been developed to determine AFP in serum with an assay time of 15 min. The approach is based on a sandwich immunoassay performed on lateral flow test strips. A fluorescence strip reader was used to measure the fluorescence peak heights of the test line (HT) and the control line (HC); the HT/HC ratio was used for quantitation. The Eu (III) chelate microparticles-based LFIA assay exhibited a wide linear range (1.0-1000 IU mL(-1)) for AFP with a low limit of detection (0.1 IU mL(-1)) based on 5ul of serum. Satisfactory specificity and accuracy were demonstrated and the intra- and inter-assay coefficients of variation (CV) for AFP were both <10%. Furthermore, in the analysis of human serum samples, excellent correlation (n = 284, r = 0.9860, p < 0.0001) was obtained between the proposed method and a commercially available CLIA kit. Results indicated that the Eu (III) chelate microparticles-based LFIA system provided a rapid, sensitive and reliable method for determining AFP in serum, indicating that it would be suitable for development in point-of-care testing.

  15. Alkaline unwinding flow cytometry assay to measure nucleotide excision repair.

    PubMed

    Thyagarajan, Bharat; Anderson, Kristin E; Lessard, Christopher J; Veltri, Gregory; Jacobs, David R; Folsom, Aaron R; Gross, Myron D

    2007-03-01

    Nucleotide excision repair (NER), one of the DNA repair pathways, is the primary mechanism for repair of bulky adducts caused by physical and chemical agents, such as UV radiation, cisplatin and 4-nitroquinolones. Variations in DNA repair may be a significant risk factor for several cancers, but its measurement in epidemiological studies has been hindered by the high variability, complexity and laborious nature of currently available assays. An alkaline unwinding flow cytometric assay using UV-C radiation as a DNA-damaging agent was adapted for measurement of NER-mediated breaks. This assay was based on the principle of alkaline unwinding of strand breaks in double-stranded DNA to yield single-stranded DNA with the number of strand breaks being proportional to the amount of DNA damage. This assay measured 50,000 events per sample with several samples being analyzed per specimen, thereby providing very reliable measurements, which can be performed on a large-scale basis. Using area under the curve (AUC) to quantitate amount of NER-mediated breaks, this assay was able to detect increased NER-mediated breaks with increasing doses of UV-C radiation. The assay detected NER-mediated breaks in lymphocytes from normal donors and not in xeroderma pigmentosum lymphoblastoid cell lines indicating specificity for the detection of NER-mediated breaks. The assay measured NER-mediated breaks within G(1), S and G(2)/M phases of the cell cycle; thereby decreasing variability in measurements of NER-mediated breaks due to differences in cell cycle phases. Intraindividual variability for AUC after 120 min of repair was 15% with interindividual variability being approximately 43% for cells in the G(1) phase, indicating substantial between-subject variation and relatively low within-subject variation. Thus, the alkaline unwinding flow cytometry-based assay provides a high-throughput method for the specific measurement of NER-mediated breaks in lymphocytes.

  16. A new nonlinear Muskingum flood routing model incorporating lateral flow

    NASA Astrophysics Data System (ADS)

    Karahan, Halil; Gurarslan, Gurhan; Geem, Zong Woo

    2015-06-01

    A new nonlinear Muskingum flood routing model taking the contribution from lateral flow into consideration was developed in the present study. The cuckoo search algorithm, a quite novel and robust algorithm, was used in the calibration and verification of the model parameters. The success and the dependability of the proposed model were tested on five different sets of synthetic and real flood data. The optimal solutions for the test cases were determined by the currently proposed model rather than by different models taken from the literature, indicating that this model could be suitable for use in flood routing problems.

  17. Reagentless bidirectional lateral flow bioactive paper sensors for detection of pesticides in beverage and food samples.

    PubMed

    Hossain, S M Zakir; Luckham, Roger E; McFadden, Meghan J; Brennan, John D

    2009-11-01

    A reagentless bioactive paper-based solid-phase biosensor was developed for detection of acetylcholinesterase (AChE) inhibitors, including organophosphate pesticides. The assay strip is composed of a paper support (1 x 10 cm), onto which AChE and a chromogenic substrate, indophenyl acetate (IPA), were entrapped using biocompatible sol-gel derived silica inks in two different zones (e.g., sensing and substrate zones). The assay protocol involves first introducing the sample to the sensing zone via lateral flow of a pesticide-containing solution. Following an incubation period, the opposite end of the paper support is placed into distilled deionized water (ddH(2)O) to allow lateral flow in the opposite direction to move paper-bound IPA to the sensing area to initiate enzyme catalyzed hydrolysis of the substrate, causing a yellow-to-blue color change. The modified sensor is able to detect pesticides without the use of any external reagents with excellent detection limits (bendiocarb approximately 1 nM; carbaryl approximately 10 nM; paraoxon approximately 1 nM; malathion approximately 10 nM) and rapid response times (approximately 5 min). The sensor strip showed negligible matrix effects in detection of pesticides in spiked milk and apple juice samples. Bioactive paper-based assays on pesticide residues collected from food samples showed good agreement with a conventional mass spectrometric assay method. The bioactive paper assay should, therefore, be suitable for rapid screening of trace levels of organophosphate and carbamate pesticides in environmental and food samples.

  18. Graphene oxide and carboxylated graphene oxide: Viable two-dimensional nanolabels for lateral flow immunoassays.

    PubMed

    Yu, Li; Li, Peiwu; Ding, Xiaoxia; Zhang, Qi

    2017-04-01

    Graphene oxide (GO) and carboxylated GO were used as labels for lateral flow immunoassays, instead of the conventionally used colloidal gold and colored latex labels. A sensor is demonstrated that enables fast screening for aflatoxin B1 (AFB1) as a model analyte using the antibody-GO complex as the recognition element. The visual limit of detection and cut-off value for AFB1 are 0.3 and 1ng/mL, respectively. It is shown that GO and carboxylated GO are viable black labels for use in lateral flow assays, one typical advantage being the saving cost (compared to the use of colloidal gold). Qualitative results are achieved within 15min, and the analytical results were in good agreement with the reference LC MS/MS method. The method was successfully applied to the on-site determination of AFB1 in agricultural products. In our perception, it opens new possibilities for the screening of other toxins by lateral flow immunoassays using GO and carboxylated GO as labels.

  19. Comparing nucleic acid lateral flow and electrochemical genosensing for the simultaneous detection of foodborne pathogens.

    PubMed

    Ben Aissa, A; Jara, J J; Sebastián, R M; Vallribera, A; Campoy, S; Pividori, M I

    2017-02-15

    Due to the increasing need of rapid tests for application in low resource settings, WHO summarized their ideal features under the acronym ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, Delivered to those who need it). In this work, two different platforms for the rapid and simultaneous testing of the foodborne pathogens E. coli O157:H7 and Salmonella enterica, in detail a nucleic acid lateral flow and an electrochemical magneto-genosensor are presented and compared in terms of their analytical performance. The DNA of the bacteria was amplified by polymerase chain reaction using a quadruple-tagging set of primers specific for E. coli eaeA (151bp) and Salmonella enterica yfiR (375bp) genes. During the amplification, the amplicons were labelled at the same time with biotin/digoxigenin or biotin/fluorescein tags, respectively. The nucleic acid lateral flow assay was based on the use of streptavidin gold nanoparticles for the labelling of the tagged amplicon from E. coli and Salmonella. The visual readout was achieved when the gold-modified amplicons were captured by the specific antibodies. The features of this approach are discussed and compared with an electrochemical magneto-genosensor. Although nucleic acid lateral flow showed higher limit of detection, this strategy was able to clearly distinguish positive and negative samples of both bacteria being considered as a rapid and promising detection tool for bacteria screening.

  20. MWCNTs based high sensitive lateral flow strip biosensor for rapid determination of aqueous mercury ions.

    PubMed

    Yao, Li; Teng, Jun; Zhu, Mengya; Zheng, Lei; Zhong, Youhao; Liu, Guodong; Xue, Feng; Chen, Wei

    2016-11-15

    Here, we describe a disposable multi-walled carbon nanotubes (MWCNTs) labeled nucleic acid lateral flow strip biosensor for rapid and sensitive detection of aqueous mercury ions (Hg(2+)). Unlike the conventional colloidal gold nanoparticle based strip biosensors, the carboxylated MWCNTs were selected as the labeling substrate because of its high specific surface area for immobilization of recognition probes, improved stability and enhanced detection sensitivity of the strip biosensor. Combining the sandwich-type of T-Hg(2+)-T recognition mechanism with the optical properties of MWCNTs on lateral flow strip, optical black bands were observed on the lateral flow strips. Parameters (such as membrane category, the MWCNTs concentration, the amount of MWCNT-DNA probe, and the volume of the test probe) that govern the sensitivity and reproducibility of the sensor were optimized. The response of the optimized biosensor was highly linear over the range of 0.05-1ppb target Hg(2+), and the detection threshold was estimated at 0.05 ppb within a 15-min assay time. The sensitivity was 10-fold higher than the conventional colloidal gold based strip biosensor. More importantly, the stability of the sensor was also greatly improved with the usage of MWCNTs as the labeling.

  1. An Ultrasensitive Gold Nanoparticle-based Lateral Flow Test for the Detection of Active Botulinum Neurotoxin Type A.

    PubMed

    Liu, Jing; Gao, Shan; Kang, Lin; Ji, Bin; Xin, Wenwen; Kang, Jingjing; Li, Ping; Gao, Jie; Wang, Hanbin; Wang, Jinglin; Yang, Hao

    2017-12-01

    Botulism is a severe and potentially lethal paralytic disease caused by several botulinum neurotoxin-producing Clostridia spp. In China, the majority of the cases caused by botulism were from less-developed rural areas. Here, we designed specific substrate peptides and reconfigured gold nanoparticle-based lateral flow test strip (LFTS) to develop an endopeptidase-based lateral flow assay for the diagnosis of botulism. We performed this lateral flow assay on botulinum neurotoxin-spiked human serum samples. The as-prepared LFTS had excellent performance in the detection of botulinum neurotoxin using only 1 μL of simulated serum, and its sensitivity and specificity were comparable to that of mouse lethality assay. Moreover, the assay takes only half a day and does not require highly trained laboratory staff, specialized facility, or equipment. Finally, our LFTS can be potentially extended to other serotypes of BoNTs by designing specific substrate peptides against the different types of BoNTs. Overall, we demonstrate a strategy by which LFTS and endopeptidase activity assays can be integrated to achieve facile and economic diagnosis of botulism in resource-limited settings.

  2. An Ultrasensitive Gold Nanoparticle-based Lateral Flow Test for the Detection of Active Botulinum Neurotoxin Type A

    NASA Astrophysics Data System (ADS)

    Liu, Jing; Gao, Shan; Kang, Lin; Ji, Bin; Xin, Wenwen; Kang, Jingjing; Li, Ping; Gao, Jie; Wang, Hanbin; Wang, Jinglin; Yang, Hao

    2017-03-01

    Botulism is a severe and potentially lethal paralytic disease caused by several botulinum neurotoxin-producing Clostridia spp. In China, the majority of the cases caused by botulism were from less-developed rural areas. Here, we designed specific substrate peptides and reconfigured gold nanoparticle-based lateral flow test strip (LFTS) to develop an endopeptidase-based lateral flow assay for the diagnosis of botulism. We performed this lateral flow assay on botulinum neurotoxin-spiked human serum samples. The as-prepared LFTS had excellent performance in the detection of botulinum neurotoxin using only 1 μL of simulated serum, and its sensitivity and specificity were comparable to that of mouse lethality assay. Moreover, the assay takes only half a day and does not require highly trained laboratory staff, specialized facility, or equipment. Finally, our LFTS can be potentially extended to other serotypes of BoNTs by designing specific substrate peptides against the different types of BoNTs. Overall, we demonstrate a strategy by which LFTS and endopeptidase activity assays can be integrated to achieve facile and economic diagnosis of botulism in resource-limited settings.

  3. Lateral flow immunoassay for quantitative detection of ractopamine in swine urine.

    PubMed

    Ren, Mei Ling; Chen, Xue Lan; Li, Chao Hui; Xu, Bo; Liu, Wen Juan; Xu, Heng Yi; Xiong, Yong Hua

    2014-02-01

    A strip reader based lateral flow immunoassay (LFIA) was established for the rapid and quantitative detection of ractopamine (RAC) in swine urine. The ratio of the optical densities (ODs) of the test line (AT) to that of the control line (AC) was used to effectively minimize interference among strips and sample variations. The linear range for the quantitative detection of RAC was 0.2 ng/mL to 3.5 ng/mL with a median inhibitory concentration (IC50) of 0.59 ± 0.06 ng/mL. The limit of detection (LOD) of the LFIA was 0.13 ng/mL. The intra-assay recovery rates were 92.97%, 97.25%, and 107.41%, whereas the inter-assay rates were 80.07%, 108.17%, and 93.7%, respectively.

  4. Enhanced lateral flow immunoassay using gold nanoparticles loaded with enzymes.

    PubMed

    Parolo, Claudio; de la Escosura-Muñiz, Alfredo; Merkoçi, Arben

    2013-02-15

    The use of gold nanoparticles (AuNPs) as labeling carriers in combination with the enzymatic activity of the horseradish peroxidase (HRP) in order to achieve an improved optical lateral flow immunoassay (LFIA) performance is presented here. Briefly in a LFIA with an immune-sandwich format AuNPs are functionalized with a detection antibody already modified with HRP, obtaining an 'enhanced' label. Two different detection strategies have been tested: the first one following just the red color of the AuNPs and the second one using a substrate for the HRP (3 different substrates are evaluated), which produces a darker color that enhances the intensity of the previous red color of the unmodified AuNPs. In such very simple way it is gaining sensitivity (up to 1 order of magnitude) without losing the simplicity of the LFIA format, opening the way to other LFIA applications including their on-demand performance tuning according to the analytical scenario.

  5. Lateral Diffusion of Bedload Transport under Laminar Flow

    NASA Astrophysics Data System (ADS)

    Ortiz, C. P.; Houssais, M.; Purohit, P. K.; Durian, D. J.; Jerolmack, D. J.

    2014-12-01

    Lateral sediment transport is a key momentum-exchange mechanism to model equilibrium channel geometry and channel bar evolution. We study sediment transport from a statistical mechanical point of view akin to Furbish et al. 2012. This approach holds promise for linking grain-scale motion to macroscopic transport, but there are few data to definitively develop and test such models. We study an experimental model river, composed of monodisperse acrylic spheres dispersed in silicon oil, driven by a layer of fluid under steady shear. We choose to drive fluid flow in the laminar regime (Re < 1) to suppress fluid turbulence and isolate granular and bed structure controls. We use a refractive-index-matched laser scanning technique to observe the motion of particles at the surface of the bed as well as the particle dynamics below the surface. We study how the probability distribution of displacements varies as a function of distance from the bed surface and as a function of distance to the channel center. In the streamwise direction, in agreement with Furbish et al. 2012, we find that the dynamics can be decomposed into an advection and a diffusion term. In the lateral direction, we find a competition between diffusion and an elastic-like interaction with the bed. We study this lateral stochastic process and find a need to introduce two parameters to quantify this competition. The first parameter describes the tendency for particles to reside near the center of the channel and the second parameter describes the kinetic energy distribution of the particles. We study how the requisite averaging scales and ensemble sizes to achieve statistically convergent parameters, and we explore how these parameters depend on the driving rate.

  6. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis

    PubMed Central

    2014-01-01

    Background Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. Methods A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. Results The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. Conclusions Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in

  7. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis.

    PubMed

    Kersting, Sebastian; Rausch, Valentina; Bier, Frank Fabian; von Nickisch-Rosenegk, Markus

    2014-03-15

    Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n=77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or

  8. Establishing a cost-per-result of laboratory-based, reflex Cryptococcal antigenaemia screening (CrAg) in HIV+ patients with CD4 counts less than 100 cells/μl using a Lateral Flow Assay (LFA) at a typical busy CD4 laboratory in South Africa

    PubMed Central

    Cassim, Naseem; Schnippel, Kathryn; Coetzee, Lindi Marie

    2017-01-01

    Introduction Cryptococcal meningitis is a major cause of mortality and morbidity in countries with high HIV prevalence, primarily affecting patients whose CD4 are < = 100 cells/μl. Routine Cryptococcal Antigen (CrAg) screening is thus recommended in the South African HIV treatment guidelines for all patients with CD4 counts < = 100 cells/μl, followed by pre-emptive anti-fungal therapy where CrAg results are positive. A laboratory-based reflexed CrAg screening approach, using a Lateral Flow Assay (LFA) on remnant EDTA CD4 blood samples, was piloted at three CD4 laboratories. Objectives This study aimed to assess the cost-per-result of laboratory-based reflexed CrAg screening at one pilot CD4 referral laboratory. Methods CD4 test volumes from 2014 were extracted to estimate percentage of CD4 < = 100 cells/μl. Daily average volumes were derived, assuming 12 months per/year and 21.73 working days per/month. Costing analyses were undertaken using Microsoft Excel and Stata with a provider prospective. The cost-per-result was estimated using a bottom-up method, inclusive of test kits and consumables (reagents), laboratory equipment and technical effort costs. The ZAR/$ exchange of 14.696/$1 was used, where applicable. One-way sensitivity analyses on the cost-per-result were conducted for possible error rates (3%– 8%, reductions or increases in reagent costs as well as test volumes (ranging from -60% to +60%). Results The pilot CD4 laboratory performed 267000 CD4 tests in 2014; ~ 9.3% (27500) reported CD4< = 100 cells/μl, equivalent to 106 CrAg tests performed daily. A batch of 30-tests could be performed in 1.6 hours, including preparation and analysis time. A cost-per-result of $4.28 was reported, with reagents contributing $3.11 (72.8%), while technical effort and laboratory equipment overheads contributed $1.17 (27.2%) and $0.03 (<1%) respectively. One-way sensitivity analyses including increasing or decreasing test volumes by 60% revealed a cost-per-result range

  9. Establishing a cost-per-result of laboratory-based, reflex Cryptococcal antigenaemia screening (CrAg) in HIV+ patients with CD4 counts less than 100 cells/μl using a Lateral Flow Assay (LFA) at a typical busy CD4 laboratory in South Africa.

    PubMed

    Cassim, Naseem; Schnippel, Kathryn; Coetzee, Lindi Marie; Glencross, Deborah Kim

    2017-01-01

    Cryptococcal meningitis is a major cause of mortality and morbidity in countries with high HIV prevalence, primarily affecting patients whose CD4 are < = 100 cells/μl. Routine Cryptococcal Antigen (CrAg) screening is thus recommended in the South African HIV treatment guidelines for all patients with CD4 counts < = 100 cells/μl, followed by pre-emptive anti-fungal therapy where CrAg results are positive. A laboratory-based reflexed CrAg screening approach, using a Lateral Flow Assay (LFA) on remnant EDTA CD4 blood samples, was piloted at three CD4 laboratories. This study aimed to assess the cost-per-result of laboratory-based reflexed CrAg screening at one pilot CD4 referral laboratory. CD4 test volumes from 2014 were extracted to estimate percentage of CD4 < = 100 cells/μl. Daily average volumes were derived, assuming 12 months per/year and 21.73 working days per/month. Costing analyses were undertaken using Microsoft Excel and Stata with a provider prospective. The cost-per-result was estimated using a bottom-up method, inclusive of test kits and consumables (reagents), laboratory equipment and technical effort costs. The ZAR/$ exchange of 14.696/$1 was used, where applicable. One-way sensitivity analyses on the cost-per-result were conducted for possible error rates (3%- 8%, reductions or increases in reagent costs as well as test volumes (ranging from -60% to +60%). The pilot CD4 laboratory performed 267000 CD4 tests in 2014; ~ 9.3% (27500) reported CD4< = 100 cells/μl, equivalent to 106 CrAg tests performed daily. A batch of 30-tests could be performed in 1.6 hours, including preparation and analysis time. A cost-per-result of $4.28 was reported, with reagents contributing $3.11 (72.8%), while technical effort and laboratory equipment overheads contributed $1.17 (27.2%) and $0.03 (<1%) respectively. One-way sensitivity analyses including increasing or decreasing test volumes by 60% revealed a cost-per-result range of $3.84 to $6.03. A cost-per-result of

  10. Developing rapid, point-of-care, multiplex detection for use in lateral flow devices

    NASA Astrophysics Data System (ADS)

    Rao, R. S.; Albala, J. S.; Lane, S. L.; Matthews, D. L.; Fisher, A. M.; Lambert, J. L.; Coleman, M. A.

    2005-11-01

    Immunoassays have been widely used in commercial, scientific and medical research for detection and quantification of analytes in complex mixtures. There is however a need for a point-of-care, multiplex diagnostic assays capable of providing rapid and quantitative measurements of analytes present in samples that are sufficiently simple to carry out without use of a laboratory or individuals trained in chemical analysis. We are developing a fluorescent lateral flow immunoassay platform to perform simultaneous, multiplexed detection of analytes in a complex fluid mixture along with instrumentation to optically quantitate the analytes in the sample. Our prototype imaging system is based on conventional 16-bit CCD optics, which enables the development of a rugged diagnostic instrument that can be further scaled down for point-of-care applications. We have compared protein microarrays with lateral flow assays (LFAs) to determine the sensitivity of each system for the measurement of distinct proteins in complex samples. We are pursuing the LFA platform such that it can easily be scaled to meet the requirements of any given screening application, and be implemented for use in a medical or surgical setting.

  11. Rapid and Sensitive Lateral Flow Immunoassay Method for Procalcitonin (PCT) Based on Time-Resolved Immunochromatography

    PubMed Central

    Shao, Xiang-Yang; Wang, Cong-Rong; Xie, Chun-Mei; Wang, Xian-Guo; Liang, Rong-Liang; Xu, Wei-Wen

    2017-01-01

    Procalcitonin (PCT) is a current, frequently-used marker for severe bacterial infection. The aim of this study was to develop a cost-effective detection kit for rapid quantitative and on-site detection of PCT. To develop the new PCT quantitative detecting kit, a double-antibody sandwich immunofluorescent assay was employed based on time-resolved immunofluorescent assay (TRFIA) combined with lateral flow immunoassay (LFIA). The performance of the new developed kit was evaluated in the aspects of linearity, precision, accuracy, and specificity. Two-hundred thirty-four serum samples were enrolled to carry out the comparison test. The new PCT quantitative detecting kit exhibited a higher sensitivity (0.08 ng/mL). The inter-assay coefficient of variation (CV) and the intra-assay CV were 5.4%–7.7% and 5.7%–13.4%, respectively. The recovery rates ranged from 93% to 105%. Furthermore, a high correlation (n = 234, r = 0.977, p < 0.0001) and consistency (Kappa = 0.875) were obtained when compared with the PCT kit from Roche Elecsys BRAHMS. Thus, the new quantitative method for detecting PCT has been successfully established. The results indicated that the newly-developed system based on TRFIA combined with LFIA was suitable for rapid and on-site detection for PCT, which might be a useful platform for other biomarkers in point-of-care tests. PMID:28264502

  12. Rapid authentication of Cordyceps by lateral flow dipstick.

    PubMed

    Wong, Yuk-Lau; Wong, Ka-Lok; Shaw, Pang-Chui

    2015-01-01

    Cordyceps (Dongchongxiacao), a valuable traditional Chinese medicine, is composed of the fruiting body of Ophiocordyceps sinensis (Family: Ophiocordycipitaceae) on a caterpillar of ghost-moth species (Family: Hepialidae). Owing to its multiple potential functions, Cordyceps are in great demand and represent significant economic value. Adulterants or substitutes named Cordyceps or Chongcao from related fungi have been reported. In this study, polymerase chain reaction (PCR) coupled with a lateral flow dipstick (LFD) system was developed to distinguish genuine herb O. sinensis from its common adulterant Cordyceps gunnii and Cordyceps militaris. Specific primers (EF-CS-F1-Biotin, EF-CG-F1-Biotin and EF-CM-F1- Biotin) were designed to differentiate the three Cordyceps species. Internal control (EF-F1-b-DIG and EF-R1-FITC) was included to minimize the false signal due to PCR inhibitors or DNA degradation. LFD was then successfully employed for speedy and accurate detection of the respective PCR products. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Nitrate removal with lateral flow sulphur autotrophic denitrification reactor.

    PubMed

    Lv, Xiaomei; Shao, Mingfei; Li, Ji; Xie, Chuanbo

    2014-01-01

    An innovative lateral flow sulphur autotrophic denitrification (LFSAD) reactor was developed in this study; the treatment performance was evaluated and compared with traditional sulphur/limestone autotrophic denitrification (SLAD) reactor. Results showed that nitrite accumulation in the LFSAD reactor was less than 1.0 mg/L during the whole operation. Denitrification rate increased with the increased initial alkalinity and was approaching saturation when initial alkalinity exceeded 2.5 times the theoretical value. Higher influent nitrate concentration could facilitate nitrate removal capacity. In addition, denitrification efficiency could be promoted under an appropriate reflux ratio, and the highest nitrate removal percentage was achieved under reflux ratio of 200%, increased by 23.8% than that without reflux. Running resistance was only about 1/9 of that in SLAD reactor with equal amount of nitrate removed, which was the prominent excellence of the new reactor. In short, this study indicated that the developed reactor was feasible for nitrate removal from waters with lower concentrations, including contaminated surface water, groundwater or secondary effluent of municipal wastewater treatment with fairly low running resistance. The innovation in reactor design in this study may bring forth new ideas of reactor development of sulphur autotrophic denitrification for nitrate-contaminated water treatment.

  14. Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay.

    PubMed

    Liu, Yuan; Wu, Aihua; Hu, Jing; Lin, Manman; Wen, Mengtang; Zhang, Xiao; Xu, Chongxin; Hu, Xiaodan; Zhong, Jianfeng; Jiao, Lingxia; Xie, Yajing; Zhang, Cunzhen; Yu, Xiangyang; Liang, Ying; Liu, Xianjin

    2015-08-15

    3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA-bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I50 and I10 values of purified mAbs were 0.63 and 0.13 μg/ml, respectively, with a dynamic range between 0.19 and 2.04 μg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 μg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.

  15. Optimization of a lateral flow immunoassay for the ultrasensitive detection of aflatoxin M1 in milk.

    PubMed

    Anfossi, Laura; Baggiani, Claudio; Giovannoli, Cristina; Biagioli, Flavia; D'Arco, Gilda; Giraudi, Gianfranco

    2013-04-15

    A high sensitive immunoassay-based lateral flow device for semi-quantitatively determine aflatoxin M1 (AFM1) in milk was developed. Investigation and optimization of the competitor design and of the gold-labelling strategy allowed the attainment of the ultra-sensitive assessment of AFM1 contamination at nanograms per litre level (LOD 20 ng L(-1), IC50 99 ng L(-1)), as requested by European regulations. A one order of magnitude detectability enhancement in comparison to previously reported gold colloid immunochromatographic assays for this toxin was obtained. Direct detection of the target toxin in milk could be obtained by acquiring images of the strips and correlating intensities of the coloured lines with analyte concentrations. The one-step assay can be completed in 17 min, including a very simple and rapid sample preparation, which allowed the application of the assay to milk samples which differ in fat and protein contents. Although imprecise (mean RSD about 30%), the method proved to be accurate and sensitive enough to allow the correct attribution of sample as compliant or non-compliant according to EU legislation in force. Agreeing results to those of a reference ELISA were obtained on 40 milk samples by matrix-matched calibration in pasteurized milk.

  16. Development of a lateral flow fluorescent microsphere immunoassay for the determination of sulfamethazine in milk.

    PubMed

    Chen, Rui; Li, Heng; Zhang, Han; Zhang, Suxia; Shi, Weimin; Shen, Jianzhong; Wang, Zhanhui

    2013-08-01

    The fluorescent microsphere has been increasingly used as detecting label in immunoassay because of its stable configuration, high fluorescence intensity, and photostability. In this paper, we developed a novel lateral flow fluorescent microsphere immunoassay (FMIA) for the determination of sulfamethazine (SMZ) in milk in a quantitative manner with high sensitivity, selectivity, and rapidity. A monoclonal antibody to SMZ was covalently conjugated with the carboxylate-modified fluorescent microsphere, which is polystyrene with a diameter of 200 nm. Quantitative detection of SMZ in milk was accomplished by recording the fluorescence intensity of microspheres captured on the test line after the milk samples were diluted five times. Under optimal conditions, the FMIA displays a rapid response for SMZ with a limit of detection of as low as 0.025 ng mL(-1) in buffer and 0.11 μg L(-1) in milk samples. The FMIA was then successfully applied on spiked milk samples and the recoveries ranged from 101.1 to 113.6% in the inter-batch assay with coefficient of variations of 6.0 to 14.3%. We demonstrate here that the fluorescent microsphere-based lateral flow immunoassay (LFIA) is capable of rapid, sensitive, and quantitative detection of SMZ in milk.

  17. Plasma surface modification of cyclo-olefin polymers and its application to lateral flow bioassays.

    PubMed

    Dudek, Magdalena M; Gandhiraman, R P; Volcke, C; Cafolla, Attilio A; Daniels, Stephen; Killard, Anthony J

    2009-09-15

    The modification of cyclo-olefin polymer Zeonor by plasma-enhanced chemical vapor deposition to form a silica-like surface and evaluation of its application for lateral flow bioassays applications are discussed in this study. The SiOx layer was extensively characterized using contact angle measurements, atomic force microscopy, and Fourier transform infrared spectroscopy in attenuated total internal reflectance mode where the presence of a uniform SiOx film was clearly identified. The SiOx modification resulted in a surface with enhanced wettability and excellent fluidic properties when combined with a hot-embossed micropillar capillary fill-based substrate. The SiOx surface also had the ability to accelerate the clotting of human plasma, which may have application in certain types of blood coagulation assays.

  18. Unchannelized dam-break flows: Effects of the lateral spreading on the flow dynamics

    NASA Astrophysics Data System (ADS)

    Girolami, L.; Wachs, A.; Vinay, G.

    2013-04-01

    In this paper, we used a three-dimensional discrete-grain model (Grains3D) to explore the flow and deposit characteristics of unchannelized dam-break collapses. A series of numerical experiments was performed to predict the behaviour of different granular columns (characterized by different initial aspect ratio a, varying from 0.5 to 18). As observed previously in similar channelized dam-break flows and axisymmetric slumps, the phenomenology of the collapse depends strongly on a, revealing different flow regimes. Small collapsing heaps (a ≲ 3) develop shallow cascades that form deposits characterized by a tail extended by a rounded frontal region. Instead, tall avalanching columns (a ≳ 3) generate dense, fast-moving currents that form a circular final deposit that resembles to those obtained from axisymmetric columns. The conversion from vertical to horizontal momentum was observed to be more efficient in these flows. The aggrading inner tapering region of static grains developed during the flow increases the number of final cascades necessary to adjust the slope of the final deposit, extending the total flow duration from 3 τc in dam-break flows to 7 τc in unchannelized ones. Surprisingly, mean aggradation velocities measured at different locations were observed to be independent of the lateral position, depending primarily on the longitudinal distance from reservoir and the initial aspect ratio. Scaled deposit widths and runouts revealed different power-law dependences on a, exposing a non-isotropic behaviour which tends to form a smooth transition from channelized dam-break flows to axisymmetric slumps.

  19. Development of a lateral flow immunoassay for rapid diagnosis of potato blackleg caused by Dickeya species.

    PubMed

    Safenkova, Irina V; Zaitsev, Ilya A; Varitsev, Yuri A; Byzova, Nadezhda A; Drenova, Natalia V; Zherdev, Anatoly V; Dzantiev, Boris B

    2017-03-01

    Early detection of potato infections is essential for effective disease management. The aim of this study was to develop a lateral flow immunoassay (LFIA) for rapid detection of a serious potato disease, potato blackleg, caused by Dickeya dianthicola and Dickeya solani. Polyclonal antibodies specific to different strains of Dickeya were obtained from rabbits after immunization with bacterial cells of D. dianthicola and D. solani. Enzyme-linked immunosorbent assay testing with use of a wide range of bacterial species showed that the polyclonal antibodies detect closely related strains of D. dianthicola and D. solani. Cross-reactivity with widespread pathogenic bacteria (nine species) and saprophytes of healthy potato plants was not detected. The LFIA based on the obtained antibodies and gold nanoparticles with average diameter of 20 nm was developed. Under optimized conditions, the LFIA method enabled the analysis of potato extracts within 10 min, with a visual limit of detection of 1 × 10(5) CFU/ml for leaves and 4 × 10(5) CFU/ml for tubers. The assay was tested on potato stem and tuber extracts, and the results of the LFIA were confirmed in 92.1% of samples using the real-time polymerase chain reaction. The findings confirmed that the developed LFIA could be used for monitoring blackleg infection without the need for special equipment or skills. Graphical Abstract The developed lateral flow immunoassay is an efficient tool for rapid detection of a serious potato disease, potato blackleg, caused by Dickeya dianthicola and Dickeya solani.

  20. Analytical study of lateral-circulation-induced exchange flow in tidally dominated well-mixed estuaries

    NASA Astrophysics Data System (ADS)

    Cheng, Peng; Wang, Aijun; Jia, Jianju

    2017-05-01

    In straight estuary channels, differential advection and the Coriolis force are the major driving mechanisms for lateral circulation. An analytical model was developed to explore the roles of the two mechanisms in the dynamics of tidally dominated well-mixed estuaries. The model provided a nondimensional parameter, Keh, a type of Kelvin number (considered as horizontal Kelvin number) to elucidate the relative importance of the two mechanisms. Differential advection is effective under small Keh, while the Coriolis force is effective under larger Keh. The critical value of Keh has an order of magnitude of 0.1 in well-mixed estuaries. Lateral circulations generate residual currents through the lateral advection term in the along-estuary momentum equation. When differential advection is effective, the lateral-advection-induced flow has a laterally sheared structure with the landward flow in the channel and seaward flows over shoals. When the Coriolis force is effective, it has a laterally sheared structure with the landward flow in the left part of the cross-section and the seaward flow in the right (facing ocean). When the two mechanisms are equally important, it has an asymmetric laterally sheared structure with a stronger seaward flow over the right shoal. Those lateral structures indicate that the lateral-circulation-induced flow generally reinforces the estuarine gravitational circulation.

  1. An evaluation of the hydrologic relevance of lateral flow in snow at hillslope and catchment scales

    Treesearch

    David Eiriksson; Michael Whitson; Charles H. Luce; Hans Peter Marshall; John Bradford; Shawn G. Benner; Thomas Black; Hank Hetrick; James P. McNamara

    2013-01-01

    Lateral downslope flow in snow during snowmelt and rain-on-snow (ROS) events is a well-known phenomenon, yet its relevance to water redistribution at hillslope and catchment scales is not well understood. We used dye tracers, geophysical methods, and hydrometric measurements to describe the snow properties that promote lateral flow, assess the relative velocities of...

  2. Species Specific Bacterial Spore Detection Using Lateral-Flow Immunoassay with DPA-Triggered Tb Luminescence

    NASA Technical Reports Server (NTRS)

    Ponce, Adrian

    2003-01-01

    A method of detecting bacterial spores incorporates (1) A method of lateral-flow immunoassay in combination with (2) A method based on the luminescence of Tb3+ ions to which molecules of dipicolinic acid (DPA) released from the spores have become bound. The present combination of lateral-flow immunoassay and DPA-triggered Tb luminescence was developed as a superior alternative to a prior lateral-flow immunoassay method in which detection involves the visual observation and/or measurement of red light scattered from colloidal gold nanoparticles. The advantage of the present combination method is that it affords both (1) High selectivity for spores of the species of bacteria that one seeks to detect (a characteristic of lateral-flow immunoassay in general) and (2) Detection sensitivity much greater (by virtue of the use of DPA-triggered Tb luminescence instead of gold nanoparticles) than that of the prior lateral-flow immunoassay method

  3. Novel strategies to enhance lateral flow immunoassay sensitivity for detecting foodborne pathogens.

    PubMed

    Shan, Shan; Lai, Weihua; Xiong, Yonghua; Wei, Hua; Xu, Hengyi

    2015-01-28

    Food contaminated by foodborne pathogens causes diseases, affects individuals, and even kills those affected individuals. As such, rapid and sensitive detection methods should be developed to screen pathogens in food. One current detection method is lateral flow immunoassay, an efficient technique because of several advantages, including rapidity, simplicity, stability, portability, and sensitivity. This review presents the format and principle of lateral flow immunoassay strip and the development of conventional lateral flow immunoassay for detecting foodborne pathogens. Furthermore, novel strategies that can be applied to enhance the sensitivity of lateral flow immunoassay to detect foodborne pathogens are presented; these strategies include innovating new label application, designing new formats of lateral flow immunoassay, combining with other methods, and developing signal amplification systems. With these advancements, detection sensitivity and detection time can be greatly improved.

  4. Automated Low-Cost Smartphone-Based Lateral Flow Saliva Test Reader for Drugs-of-Abuse Detection

    PubMed Central

    Carrio, Adrian; Sampedro, Carlos; Sanchez-Lopez, Jose Luis; Pimienta, Miguel; Campoy, Pascual

    2015-01-01

    Lateral flow assay tests are nowadays becoming powerful, low-cost diagnostic tools. Obtaining a result is usually subject to visual interpretation of colored areas on the test by a human operator, introducing subjectivity and the possibility of errors in the extraction of the results. While automated test readers providing a result-consistent solution are widely available, they usually lack portability. In this paper, we present a smartphone-based automated reader for drug-of-abuse lateral flow assay tests, consisting of an inexpensive light box and a smartphone device. Test images captured with the smartphone camera are processed in the device using computer vision and machine learning techniques to perform automatic extraction of the results. A deep validation of the system has been carried out showing the high accuracy of the system. The proposed approach, applicable to any line-based or color-based lateral flow test in the market, effectively reduces the manufacturing costs of the reader and makes it portable and massively available while providing accurate, reliable results. PMID:26610513

  5. Automated Low-Cost Smartphone-Based Lateral Flow Saliva Test Reader for Drugs-of-Abuse Detection.

    PubMed

    Carrio, Adrian; Sampedro, Carlos; Sanchez-Lopez, Jose Luis; Pimienta, Miguel; Campoy, Pascual

    2015-11-24

    Lateral flow assay tests are nowadays becoming powerful, low-cost diagnostic tools. Obtaining a result is usually subject to visual interpretation of colored areas on the test by a human operator, introducing subjectivity and the possibility of errors in the extraction of the results. While automated test readers providing a result-consistent solution are widely available, they usually lack portability. In this paper, we present a smartphone-based automated reader for drug-of-abuse lateral flow assay tests, consisting of an inexpensive light box and a smartphone device. Test images captured with the smartphone camera are processed in the device using computer vision and machine learning techniques to perform automatic extraction of the results. A deep validation of the system has been carried out showing the high accuracy of the system. The proposed approach, applicable to any line-based or color-based lateral flow test in the market, effectively reduces the manufacturing costs of the reader and makes it portable and massively available while providing accurate, reliable results.

  6. Pretreatment-free lateral flow enzyme immunoassay for progesterone detection in whole cows' milk.

    PubMed

    Samsonova, J V; Safronova, V A; Osipov, A P

    2015-01-01

    New rapid method of lateral flow enzyme immunoassay (LFEIA) for progesterone detection in whole cows' milk was developed. The test system utilized horseradish peroxidase as a label along with the substrate solution containing 3,3',5,5'-tetramethylbenzidine and dextran sulfate to obtain an insoluble blue colored product of the enzyme reaction on a surface of analytical membrane (test and control lines). Several aspects of LFEIA were optimized: time of the signal detection, membrane materials and assay conditions. Resulting competitive LFEIA can be performed within 15 minutes with the limit of progesterone detection of 0.8 ng/ml. Progesterone concentration in whole milk samples was determined by LFEIA and enzyme-linked immunosorbent assay (ELISA). The results obtained were in good correlation (R=0.97, n=46). Thus new sensitive LFEIA can be successfully used for on-site monitoring of oestrus status of cows' reproductive system and for early none-pregnancy detection. The method is fast, easy to perform and needs no preliminary sample preparation.

  7. [Development of a lateral flow dipstick immunoassay for rapid detection of ginsenoside Re].

    PubMed

    Nan, Tie-Gui; Cao, Zhen; He, Li-Shan; Yuan, Yuan; Huang, Lu-Qi; Wang, Bao-Min

    2013-08-01

    A sensitive antibody-based lateral flow dipstick was developed for ginsenoside Re (GRe) detection. The stick consisted of a sample pad, a conjugate pad, membrane and an absorbent pad. The membrane was coated with two capture reagents, GRe-BSA conjugate and goat anti-mouse antibodies, forming a test line and a control line, respectively. The conjugate pad was saturated with colloidal gold particles coated with affinity purified monoclonal anti-GRe antibody. The visual detection limit was 200 microg x L(-1) of GRe and the reaction time was 10 min. The Panax ginseng roots were identified after these samples (10 mg) were extracted with 5 mL tap water for 30 min at room temperature, and the extracts were tested by the dipsticks and ELISA kit. The true and false P. ginseng could be distinguished with dipsticks. The dipstick could be used to detect the quality of the P. ginseng samples when the extract was diluted 100-folds. The results were compared with those obtained using an indirect competitive enzyme-linked immunosorbent assay (icELISA). The dipstick assay proved to be a sensitive and rapid tool for quality control of P. ginseng.

  8. Rapid detection of Bacillus anthracis by γ phage amplification and lateral flow immunochromatography.

    PubMed

    Cox, Christopher R; Jensen, Kirk R; Mondesire, Roy R; Voorhees, Kent J

    2015-11-01

    New, rapid point-of-need diagnostic methods for Bacillus anthracis detection can enhance civil and military responses to accidental or deliberate dispersal of anthrax as a biological weapon. Current laboratory-based methods for clinical identification of B. anthracis require 12 to 120h, and are confirmed by plaque assay using the well-characterized γ typing phage, which requires an additional minimum of 24h for bacterial culture. To reduce testing time, the natural specificity of γ phage amplification was investigated in combination with lateral flow immunochromatography (LFI) for rapid, point-of-need B. anthracis detection. Phage-based LFI detection of B. anthracis Sterne was validated over a range of bacterial and phage concentrations with optimal detection achieved in as little as 2h from the onset of amplification with a threshold sensitivity of 2.5×10(4)cfu/mL. The novel use of γ phage amplification detected with a simple, inexpensive LFI assay provides a rapid, sensitive, highly accurate, and field-deployable method for diagnostic ID of B. anthracis in a fraction of the time required by conventional techniques, and without the need for extensive laboratory culture.

  9. Dual-Quantum-Dots-Labeled Lateral Flow Strip Rapidly Quantifies Procalcitonin and C-reactive Protein

    NASA Astrophysics Data System (ADS)

    Qi, XiaoPing; Huang, YunYe; Lin, ZhongShi; Xu, Liang; Yu, Hao

    2016-03-01

    In the article, a dual-quantum-dots-labeled (dual-QDs-labeled) lateral flow strip (LFS) method was developed for the simultaneous and rapid quantitative detection of procalcitonin (PCT) and C-reactive protein (CRP) in the blood. Two QD-antibody conjugates with different fluorescence emission spectra were produced and sprayed on the LFS to capture PCT and CRP in the blood. Furthermore, a double antibody sandwich method for PCT and, meanwhile, a competitive inhibition method for CRP were employed in the LFS. For PCT and CRP in serum assayed by the dual-QDs-labeled LFS, their detection sensitivities reached 0.1 and 1 ng/mL, respectively, and their linear quantitative detection ranges were from 0.3 to 200 ng/mL and from 50 to 250 μg/mL, respectively. There was little evidence that the PCT and CRP assays would be interfered with each other. The correlations for testing CRP and PCT in clinical samples were 99.75 and 97.02 %, respectively, between the dual-QDs-labeled LFS we developed and commercial methods. The rapid quantification of PCT and CRP on dual-QDs-labeled LFS is of great clinical value to distinguish inflammation, bacterial infection, or viral infection and to provide guidance for the use of antibiotics or other medicines.

  10. Lateral Flow Field Behavior Downstream of Mixing Vanes In a Simulated Nuclear Fuel Rod Bundle

    SciTech Connect

    Conner, Michael E.; Smith, L. David III; Holloway, Mary V.; Beasley, Donald E.

    2004-07-01

    To assess the fuel assembly performance of PWR nuclear fuel assemblies, average subchannel flow values are used in design analyses. However, for this highly complex flow, it is known that local conditions around fuel rods vary dependent upon the location of the fuel rod in the fuel assembly and upon the support grid design that maintains the fuel rod pitch. To investigate the local flow in a simulated nuclear fuel rod bundle, a testing technique has been employed to measure the lateral flow field in a 5 x 5 rod bundle. Particle Image Velocimetry was used to measure the lateral flow field downstream of a support grid with mixing vanes for four unique subchannels in the 5 x 5 bundle. The dominant lateral flow structures for each subchannel are compared in this paper including the decay of these flow structures. (authors)

  11. Development of Dual Quantitative Lateral Flow Immunoassay for the Detection of Mycotoxins.

    PubMed

    Wang, Yuan-Kai; Yan, Ya-Xian; Sun, Jian-He

    2017-01-01

    Lateral flow immunoassays have been widely used in recent years for detection of toxins, heavy metals, and biomarkers. To improve the efficiency of individual lateral flow immunoassays, multiplex analytical strips play an important role in the detection of several important analytes. In this chapter, development of a dual lateral flow immunoassay is presented for detection of a variety of low molecular weight molecules. Various buffers, additives, and materials are introduced and evaluated. Depending on the analyte to be tested, the technique allows for selection of optimum buffers, additives, and other materials.

  12. Integrating Vegetation, Soil and Topography to Assess the Impact of Lateral Flow on Plant Solute Uptake

    NASA Astrophysics Data System (ADS)

    Rebel, K. T.; Riha, S. J.; Stedinger, J. R.

    2005-05-01

    Simulation of solute uptake by vegetation in complex terrain typically fails to account for subsurface lateral movement of solutes. This study uses a spatially explicit plant-soil-water simulation model to investigate whether subsurface lateral flow at the sand-clay interface impacts tritium uptake by mixed forest vegetation. Ten hectares of a mixed pine - laurel oak forest on Coastal Plain soils periodically received irrigation with tritium-enriched water (activity ranged from 5,000 to 20,000 pCi/ml) over a three year time period. To simulate water and tritium fluxes we developed a spatially explicit water balance model. Tritium was completely mixed daily with water in each soil layer. Vertical flow of water was simulated using a capacitance model with lateral flow dependent on head development and the local slope of the impeding clay layer. The model was evaluated by comparing biweekly measurements of tritium activity (measured to 3 meter depth) and soil water content (measured to 2 meter depth) in 18 measurement clusters distributed over the catchment. We evaluated the importance of including subsurface flow in model simulations. Lateral flow was locally important (mean distance tritium traveled laterally was 1.35 m). However, after three years of simulation, the maximum predicted lateral movement of tritium did not exceed 70 meters. On the catchment scale, the average simulated amount of tritium taken up by vegetation was not impacted by lateral flow, but smaller scale spatial variability in tritium uptake increased with the inclusion of lateral flow. Simulated tritium uptake was most sensitive to changes in vegetation cover, and was less sensitive to differences in soil properties (e.g. field capacity, hydraulic conductivity and root distribution). When integrated over the study area, the simulation of solute uptake by a mixed forest in Coastal Pain soils was not sensitive to inclusion of subsurface lateral flow of water.

  13. A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification.

    PubMed

    Wu, Wei; Zhao, Shiming; Mao, Yiping; Fang, Zhiyuan; Lu, Xuewen; Zeng, Lingwen

    2015-02-25

    Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.

  14. An aptamer based lateral flow strip for on-site rapid detection of ochratoxin A in Astragalus membranaceus.

    PubMed

    Zhou, Weilu; Kong, Weijun; Dou, Xiaowen; Zhao, Ming; Ouyang, Zhen; Yang, Meihua

    2016-06-01

    An aptamer based lateral flow strip based on competitive format was developed for on-site rapid detection of ochratoxin A (OTA) in Astragalus membranaceus. Some crucial parameters that might influence the sensitive detection, such as the characterization of the colloidal gold, size and shape of gold nanoparticles (AuNPs), amount of AuNPs-aptamer conjugate, migration rate and the addition amount of methanol, were investigated to provide the optimum assay performance. To perform the test, 1g sample was extracted with 2.5mL of methanol-water (80:20, v/v) and diluted by 4-fold running buffer to eliminate the matrix and methanol interferences. Under optimized conditions, the aptamer-based assay showed a visual limit of detection (LOD) of 1ngmL(-1), and with no significant cross-reactivity with several homologous toxins. The whole detection could be completed within 15min without special equipment because of available visual results. One out of nine A. membranaceus samples was found to be positive of OTA, which was in a good agreement with those obtained from LC-MS/MS analysis. The results demonstrated that the aptamer-based lateral flow assay could be used as a rapid, reliable, cost-effective and robust on-site screening technique for mycotoxins at trace level in complex matrices without special instrumentation. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Development of a barcode-style lateral flow immunoassay for the rapid semi-quantification of gliadin in foods.

    PubMed

    Yin, Hsin-Yi; Chu, Pei-Tzu; Tsai, Wen-Che; Wen, Hsiao-Wei

    2016-02-01

    In this work, a barcode-style lateral flow immunoassay is developed using two cut-off values (10 and 50 mg kg(-1) gliadin) to provide a semi-quantification for identifying "gluten-free" and "very low gluten" foods, based on the international Codex Alimentarius Standard. This developed assay exhibits favorable specificity in differentiating wheat from seven commonly used grains, with only a slight cross-reaction with barely. The intra-assay and inter-assay CV values of this assay were 1.5-1.7% and 2.5-4.5%, respectively, revealing high reproducibility. In the analysis of 48 food samples, the results of this assay closely agreed with those obtained using AOAC-approved ELISA or strip kits, as the Cohen's kappa coefficients for both comparisons exceeded 0.8. Thus, this developed assay can be used to quickly estimate the gliadin content in foods in order to protect people with wheat allergy or celiac disease from the accidental ingestion of gliadin.

  16. [Detection of fish protein in food products by lateral flow immunoassay].

    PubMed

    Shibahara, Yusuke; Ii, Toshihiro; Wang, Jun; Yamada, Shoichi; Shiomi, Kazuo

    2014-01-01

    The major fish allergen is parvalbumin, a sarcoplasmic protein. In this study, a novel lateral flow immunoassay for the detection of fish protein in food products was developed using a polyclonal antibody raised against Pacific mackerel Scomber japonicus parvalbumin. The proposed lateral flow immunoassay showed high reactivity to various fish parvalbumins, but the reactivity to bullfrog parvalbumin was very low. The detection limit of the immunoassay for fish parvalbumin was estimated to be 2.0 μg protein/g, which matches the sensitivity required in the current Japanese food labeling system. Furthermore, the lateral flow immunoassay could detect fish parvalbumin without being affected by food matrices and was applicable even to heat-denatured parvalbumin. These results showed that the lateral flow immunoassay developed in this study is specific to fish parvalbumin, and should be useful as a rapid detection method for fish protein in processed food products.

  17. Effects of anthropogenic water regulation and groundwater lateral flow on land processes

    NASA Astrophysics Data System (ADS)

    Zeng, Yujin; Xie, Zhenghui; Yu, Yan; Liu, Shuang; Wang, Linying; Zou, Jing; Qin, Peihua; Jia, Binghao

    2016-09-01

    Both anthropogenic water regulation and groundwater lateral flow essentially affect groundwater table patterns. Their relationship is close because lateral flow recharges the groundwater depletion cone, which is induced by over-exploitation. In this study, schemes describing groundwater lateral flow and human water regulation were developed and incorporated into the Community Land Model 4.5. To investigate the effects of human water regulation and groundwater lateral flow on land processes as well as the relationship between the two processes, three simulations using the model were conducted for the years 2003-2013 over the Heihe River Basin in northwestern China. Simulations showed that groundwater lateral flow driven by changes in water heads can essentially change the groundwater table pattern with the deeper water table appearing in the hillslope regions and shallower water table appearing in valley bottom regions and plains. Over the last decade, anthropogenic groundwater exploitation deepened the water table by approximately 2 m in the middle reaches of the Heihe River Basin and rapidly reduced the terrestrial water storage, while irrigation increased soil moisture by approximately 0.1 m3 m-3. The water stored in the mainstream of the Heihe River was also reduced by human surface water withdrawal. The latent heat flux was increased by 30 W m-2 over the irrigated region, with an identical decrease in sensible heat flux. The simulated groundwater lateral flow was shown to effectively recharge the groundwater depletion cone caused by over-exploitation. The offset rate is higher in plains than mountainous regions.

  18. Rapid detection of Bacillus anthracis spores using a super-paramagnetic lateral-flow immunological detection system.

    PubMed

    Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Deng, Jiao-Yu; Cui, Zong-Qiang; Yang, Rui-Fu; Wang, Xu-Ying; Wei, Hong-Ping; Zhang, Xian-En

    2013-04-15

    There is an urgent need for convenient, sensitive, and specific methods to detect the spores of Bacillus anthracis, the causative agent of anthrax, because of the bioterrorism threat posed by this bacterium. In this study, we firstly develop a super-paramagnetic lateral-flow immunological detection system for B. anthracis spores. This system involves the use of a portable magnetic assay reader, super-paramagnetic iron oxide particles, lateral-flow strips and two different monoclonal antibodies directed against B. anthracis spores. This detection system specifically recognises as few as 400 pure B. anthracis spores in 30 min. This system has a linear range of 4×10³-10⁶ CFU ml⁻¹ and reproducible detection limits of 200 spores mg⁻¹ milk powder and 130 spores mg⁻¹ soil for simulated samples. In addition, this approach shows no obvious cross-reaction with other related Bacillus spores, even at high concentrations, and has no significant dependence on the duration of the storage of the immunological strips. Therefore, this super-paramagnetic lateral-flow immunological detection system is a promising tool for the rapid and sensitive detection of Bacillus anthracis spores under field conditions.

  19. Variable parameter McCarthy-Muskingum routing method considering lateral flow

    NASA Astrophysics Data System (ADS)

    Yadav, Basant; Perumal, Muthiah; Bardossy, Andras

    2015-04-01

    The fully mass conservative variable parameter McCarthy-Muskingum (VPMM) method recently proposed by Perumal and Price (2013) for routing floods in channels and rivers without considering lateral flow is extended herein for accounting uniformly distributed lateral flow contribution along the reach. The proposed procedure is applied for studying flood wave movement in a 24.2 km river stretch between Rottweil and Oberndorf gauging stations of Neckar River in Germany wherein significant lateral flow contribution by intermediate catchment rainfall prevails during flood wave movement. The geometrical elements of the cross-sectional information of the considered routing river stretch without considering lateral flow are estimated using the Robust Parameter Estimation (ROPE) algorithm that allows for arriving at the best performing set of bed width and side slope of a trapezoidal section. The performance of the VPMM method is evaluated using the Nash-Sutcliffe model efficiency criterion as the objective function to be maximized using the ROPE algorithm. The twenty-seven flood events in the calibration set are considered to identify the relationship between 'total rainfall' and 'total losses' as well as to optimize the geometric characteristics of the prismatic channel (width and slope of the trapezoidal section). Based on this analysis, a relationship between total rainfall and total loss of the intermediate catchment is obtained and then used to estimate the lateral flow in the reach. Assuming the lateral flow hydrograph is of the form of inflow hydrograph and using the total intervening catchment runoff estimated from the relationship, the uniformly distributed lateral flow rate qL at any instant of time is estimated for its use in the VPMM routing method. All the 27 flood events are simulated using this routing approach considering lateral flow along the reach. Many of these simulations are able to simulate the observed hydrographs very closely. The proposed approach

  20. An automatic enzyme immunoassay based on a chemiluminescent lateral flow immunosensor.

    PubMed

    Joung, Hyou-Arm; Oh, Young Kyoung; Kim, Min-Gon

    2014-03-15

    Microfluidic integrated enzyme immunosorbent assay (EIA) sensors are efficient systems for point-of-care testing (POCT). However, such systems are not only relatively expensive but also require a complicated manufacturing process. Therefore, additional fluidic control systems are required for the implementation of EIAs in a lateral flow immunosensor (LFI) strip sensor. In this study, we describe a novel LFI for EIA, the use of which does not require additional steps such as mechanical fluidic control, washing, or injecting. The key concept relies on a delayed-release effect of chemiluminescence substrates (luminol enhancer and hydrogen peroxide generator) by an asymmetric polysulfone membrane (ASPM). When the ASPM was placed between the nitrocellulose (NC) membrane and the substrate pad, substrates encapsulated in the substrate pad were released after 5.3 ± 0.3 min. Using this delayed-release effect, we designed and implemented the chemiluminescent LFI-based automatic EIA system, which sequentially performed the immunoreaction, pH change, substrate release, hydrogen peroxide generation, and chemiluminescent reaction with only 1 sample injection. In a model study, implementation of the sensor was validated by measuring the high sensitivity C-reactive protein (hs-CRP) level in human serum.

  1. Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays

    PubMed Central

    Wang, Jinliang; Katani, Robab; Li, Lingling; Hegde, Narasimha; Roberts, Elisabeth L.; Kapur, Vivek; DebRoy, Chitrita

    2016-01-01

    Shiga toxin-producing Escherichia coli O157:H7 (STEC) cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2) that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA); one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 105 CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment. PMID:27023604

  2. A lateral flow biosensor for rapid detection of DNA-binding protein c-jun.

    PubMed

    Fang, Zhiyuan; Ge, Chenchen; Zhang, Wenjuan; Lie, Puchang; Zeng, Lingwen

    2011-09-15

    A lateral flow biosensor based on an immuno-chromatographic assay has been developed for the detection of DNA-binding proteins. The biosensor is composed of four parts: a sample pad, a conjugate pad, a strip of nitrocellulose membrane and an absorbent pad. A DNA probe containing a specific protein binding consensus sequence is coated onto gold nanoparticles, while an antibody against the DNA-binding protein is immobilized onto a test zone of the nitrocellulose membrane. The target protein binds to the protein binding DNA sequence that is coated on the gold nanoparticles to form nanoparticle-DNA-protein complexes, and the complexes are then captured by the antibody immobilized on the test zone to form a red line for visual detection of the target protein. This biosensor was successfully applied to a DNA-binding protein, c-jun, and the developed biosensor allows for the rapid detection of down to 0.2 footprint unit of c-jun protein within 10 min. This biosensor was verified using HeLa cells and it visually detected c-jun activity in 100 μg of crude cell lysate protein. The antibody against c-jun used in the biosensor can distinguish c-jun from other nonspecific proteins, with high specificity. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Left-right organizer flow dynamics: how much cilia activity reliably yields laterality?

    PubMed

    Sampaio, Pedro; Ferreira, Rita R; Guerrero, Adán; Pintado, Petra; Tavares, Bárbara; Amaro, Joana; Smith, Andrew A; Montenegro-Johnson, Thomas; Smith, David J; Lopes, Susana S

    2014-06-23

    Internal organs are asymmetrically positioned inside the body. Embryonic motile cilia play an essential role in this process by generating a directional fluid flow inside the vertebrate left-right organizer. Detailed characterization of how fluid flow dynamics modulates laterality is lacking. We used zebrafish genetics to experimentally generate a range of flow dynamics. By following the development of each embryo, we show that fluid flow in the left-right organizer is asymmetric and provides a good predictor of organ laterality. This was tested in mosaic organizers composed of motile and immotile cilia generated by dnah7 knockdowns. In parallel, we used simulations of fluid dynamics to analyze our experimental data. These revealed that fluid flow generated by 30 or more cilia predicts 90% situs solitus, similar to experimental observations. We conclude that cilia number, dorsal anterior motile cilia clustering, and left flow are critical to situs solitus via robust asymmetric charon expression.

  4. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor

    PubMed Central

    Cedeño-Arias, Mercedes; Sánchez-Ramírez, Javier; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique

    2011-01-01

    An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use. PMID:21886904

  5. Detection of circulating immune complexes by Raji cell assay: comparison of flow cytometric and radiometric methods

    SciTech Connect

    Kingsmore, S.F.; Crockard, A.D.; Fay, A.C.; McNeill, T.A.; Roberts, S.D.; Thompson, J.M.

    1988-01-01

    Several flow cytometric methods for the measurement of circulating immune complexes (CIC) have recently become available. We report a Raji cell flow cytometric assay (FCMA) that uses aggregated human globulin (AHG) as primary calibrator. Technical advantages of the Raji cell flow cytometric assay are discussed, and its clinical usefulness is evaluated in a method comparison study with the widely used Raji cell immunoradiometric assay. FCMA is more precise and has greater analytic sensitivity for AHG. Diagnostic sensitivity by the flow cytometric method is superior in systemic lupus erythematosus (SLE), rheumatoid arthritis, and vasculitis patients: however, diagnostic specificity is similar for both assays, but the reference interval of FCMA is narrower. Significant correlations were found between CIC levels obtained with both methods in SLE, rheumatoid arthritis, and vasculitis patients and in longitudinal studies of two patients with cerebral SLE. The Raji cell FCMA is recommended for measurement of CIC levels to clinical laboratories with access to a flow cytometer.

  6. Detection of artificial water flows by the lateral line system of a benthic feeding cichlid fish.

    PubMed

    Schwalbe, Margot A B; Sevey, Benjamin J; Webb, Jacqueline F

    2016-04-01

    The mechanosensory lateral line system of fishes detects water motions within a few body lengths of the source. Several types of artificial stimuli have been used to probe lateral line function in the laboratory, but few studies have investigated the role of flow sensing in benthic feeding teleosts. In this study, we used artificial flows emerging from a sandy substrate to assess the contribution of flow sensing to prey detection in the peacock cichlid, Aulonocara stuartgranti, which feeds on benthic invertebrates in Lake Malawi. Using a positive reinforcement protocol, we trained fish to respond to flows lacking the visual and chemical cues generated by tethered prey in prior studies with A. stuartgranti Fish successfully responded to artificial flows at all five rates presented (characterized using digital particle image velocimetry), and showed a range of flow-sensing behaviors, including an unconditioned bite response. Immediately after lateral line inactivation, fish rarely responded to flows and the loss of vital fluorescent staining of hair cells (with 4-di-2-ASP) verified lateral line inactivation. Within 2 days post-treatment, some aspects of flow-sensing behavior returned and after 7 days, flow-sensing behavior and hair cell fluorescence both returned to pre-treatment levels, which is consistent with the reported timing of hair cell regeneration in other vertebrates. The presentation of ecologically relevant water flows to assess flow-sensing behaviors and the use of a positive reinforcement protocol are methods that present new opportunities to study the role of flow sensing in the feeding ecology of benthic feeding fishes.

  7. Variable parameter McCarthy-Muskingum flow transport model for compound channels accounting for distributed non-uniform lateral flow

    NASA Astrophysics Data System (ADS)

    Swain, Ratnakar; Sahoo, Bhabagrahi

    2015-11-01

    In this study, the fully volume conservative simplified hydrodynamic-based variable parameter McCarthy-Muskingum (VPMM) flow transport model advocated by Perumal and Price in 2013 is extended to exclusively incorporate the distributed non-uniform lateral flow in the routing scheme accounting for compound river channel flows. The revised VPMM formulation is exclusively derived from the combined form of the de Saint-Venant's continuity and momentum equations with the spatiotemporally distributed lateral flow which is solved using the finite difference box scheme. This revised model could address the earlier model limitations of: (i) non-accounting non-uniformly distributed lateral flow, (ii) ignoring floodplain flow, and (iii) non-consideration of catchment dynamics of lateral flow generation restricting its real-time application. The efficacy of the revised formulation is tested to simulate 16 years (1980-1995) river runoff from real-time storm events under scarce morpho-hydrological data conditions in a tropical monsoon-type 48 km Bolani-Gomlai reach of the Brahmani River in eastern India. The spatiotemporally distributed lateral flows generated in real-time is computed by water balance approach accounting for catchment characteristics of normalized network area function, land use land cover classes, and soil textural classes; and hydro-meteorological variables of precipitation, soil moisture, minimum and maximum temperatures, wind speed, relative humidity, and solar radiation. The multiple error measures used in this study and the simulation results reveal that the revised VPMM model has a greater practical utility in estimating the event-based and long-term meso-scale river runoff (both discharge and its stage) at any ungauged site, enhancing its application for real-time flood estimation.

  8. Multiplexed Lateral Flow Test for Detection and Differentiation of Cronobacter sakazakii Serotypes O1 and O2.

    PubMed

    Scharinger, Eva J; Dietrich, Richard; Wittwer, Tobias; Märtlbauer, Erwin; Schauer, Kristina

    2017-01-01

    The ubiquitous and opportunistic pathogen Cronobacter sakazakii is responsible for severe meningitis, sepsis, and necrotizing enterocolitis in neonates and infants associated with ingestion of contaminated powdered infant formula (PIF). The current ISO method for isolation and detection of Cronobacter spp. is laborious, time-consuming and expensive. In this study, a multiplexed lateral flow test strip was developed to rapidly detect and simultaneously serotype O1 and O2 C. sakazakii serotypes. The assay is based on two monoclonal antibodies (MAb) that specifically bind to the lipopolysaccharides (LPS) of these pathogens. The test strip provides results very quickly; C. sakazakii could be detected in pure culture within 15 min with a sensitivity of 10(7) CFU/ml. After non-selective enrichment for 18 h as low as one Cronobacter cell per g PIF could be detected. Moreover, the established lateral flow assay (LFA) offers excellent specificity showing no cross-reactivity with other C. sakazakii serotypes, Cronobacter species or Enterobacteriaceae tested. These characteristics, together with several advantages such as speed, simplicity in performance, low analysis cost, and no requirement of specialized skills or sophisticated equipment make the developed multiplexed LFA suitable for reliable detection and serotyping of C. sakazakii serotypes O1 and O2.

  9. Dual Immunomagnetic Nanobeads-Based Lateral Flow Test Strip for Simultaneous Quantitative Detection of Carcinoembryonic Antigen and Neuron Specific Enolase

    PubMed Central

    Lu, Wenting; Wang, Kan; Xiao, Kun; Qin, Weijian; Hou, Yafei; Xu, Hao; Yan, Xinyu; Chen, Yanrong; Cui, Daxiang; He, Jinghua

    2017-01-01

    A novel immunomagnetic nanobeads -based lateral flow test strip was developed for the simultaneous quantitative detection of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA), which are sensitive and specific in the clinical diagnosis of small cell lung cancer. Using this nanoscale method, high saturation magnetization, carboxyl-modified magnetic nanobeads were successfully synthesized. To obtain the immunomagnetic probes, a covalent bioconjugation of the magnetic nanobeads with the antibody of NSE and CEA was carried out. The detection area contained test line 1 and test line 2 which captured the immune complexes sensitively and formed sandwich complexes. In this assay, cross-reactivity results were negative and both NSE and CEA were detected simultaneously with no obvious influence on each other. The magnetic signal intensity of the nitrocellulose membrane was measured by a magnetic assay reader. For quantitative analysis, the calculated limit of detection was 0.094 ng/mL for NSE and 0.045 ng/mL for CEA. One hundred thirty clinical samples were used to validate the test strip which exhibited high sensitivity and specificity. This dual lateral flow test strip not only provided an easy, rapid, simultaneous quantitative detection strategy for NSE and CEA, but may also be valuable in automated and portable diagnostic applications. PMID:28186176

  10. A fish perspective: detecting flow features while moving using an artificial lateral line in steady and unsteady flow

    PubMed Central

    Chambers, L. D.; Akanyeti, O.; Venturelli, R.; Ježov, J.; Brown, J.; Kruusmaa, M.; Fiorini, P.; Megill, W. M.

    2014-01-01

    For underwater vehicles to successfully detect and navigate turbulent flows, sensing the fluid interactions that occur is required. Fish possess a unique sensory organ called the lateral line. Sensory units called neuromasts are distributed over their body, and provide fish with flow-related information. In this study, a three-dimensional fish-shaped head, instrumented with pressure sensors, was used to investigate the pressure signals for relevant hydrodynamic stimuli to an artificial lateral line system. Unsteady wakes were sensed with the objective to detect the edges of the hydrodynamic trail and then explore and characterize the periodicity of the vorticity. The investigated wakes (Kármán vortex streets) were formed behind a range of cylinder diameter sizes (2.5, 4.5 and 10 cm) and flow velocities (9.9, 19.6 and 26.1 cm s−1). Results highlight that moving in the flow is advantageous to characterize the flow environment when compared with static analysis. The pressure difference from foremost to side sensors in the frontal plane provides us a useful measure of transition from steady to unsteady flow. The vortex shedding frequency (VSF) and its magnitude can be used to differentiate the source size and flow speed. Moreover, the distribution of the sensing array vertically as well as the laterally allows the Kármán vortex paired vortices to be detected in the pressure signal as twice the VSF. PMID:25079867

  11. An enzyme-amplified lateral flow strip biosensor for visual detection of microRNA-224.

    PubMed

    Gao, Xuefei; Xu, Li-Ping; Wu, Tingting; Wen, Yongqiang; Ma, Xinlei; Zhang, Xueji

    2016-01-01

    An enzyme-based dual-labeled nanoprobe is designed to fabricate a sensitive enzyme-amplified lateral flow biosensor for visual detection of mircoRNA-224 (miRNA-224). The recognition DNA probe (detection probe) and signal amplification enzyme (Horseradish peroxidase, HRP) are immobilized on gold nanoparticle (GNPs) surface, simultaneously. The capture DNA probes are immobilized on the test zone of the lateral flow biosensor. When miRNA-224 is present, the enzyme-based dual-labeled nanoprobes will be captured by forming the "sandwich structure" on the test zone of the lateral flow biosensor, enabling the visual detection for miRNA-224. Sensitivity is amplified by applying the 3,3,5,5-tetramethylbenzidine enzymatic substrate (TMB/H2O2 enzymatic substrate) onto the test zone. The enzymatic reactions between the HRP and the TMB/H2O2 enzymatic substrate will produce blue products, which deposit on the nanoprobe surface to enhance the visual effect and the corresponding response intensities of the test zone. This enzyme-amplified lateral flow biosensor shows a low limit of detection (LOD) (7.5 pM) toward miRNA-224 in the buffer solution, which is improved by 10-fold than that of the single-labeled lateral flow biosensor. This biosensor has been successfully used for the detection of the target miRNA-224 detection in A549 cell lysate.

  12. Investigation of particle lateral migration in sample-sheath flow of viscoelastic fluid and Newtonian fluid.

    PubMed

    Yuan, Dan; Zhang, Jun; Yan, Sheng; Peng, Gangrou; Zhao, Qianbin; Alici, Gursel; Du, Hejun; Li, Weihua

    2016-08-01

    In this work, particle lateral migration in sample-sheath flow of viscoelastic fluid and Newtonian fluid was experimentally investigated. The 4.8-μm micro-particles were dispersed in a polyethylene oxide (PEO) viscoelastic solution, and then the solution was injected into a straight rectangular channel with a deionised (DI) water Newtonian sheath flow. Micro-particles suspended in PEO solution migrated laterally to a DI water stream, but migration in the opposite direction from a DI water stream to a PEO solution stream or from one DI water stream to another DI water stream could not be achieved. The lateral migration of particles depends on the viscoelastic properties of the sample fluids. Furthermore, the effects of channel length, flow rate, and PEO concentration were studied. By using viscoelastic sample flow and Newtonian sheath flow, a selective particle lateral migration can be achieved in a simple straight channel, without any external force fields. This particle lateral migration technique could be potentially used in solution exchange fields such as automated cell staining and washing in microfluidic platforms, and holds numerous biomedical applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Lateral migration of an elastic capsule by optical force in a uniform flow.

    PubMed

    Chang, Cheong Bong; Huang, Wei-Xi; Sung, Hyung Jin

    2012-12-01

    The lateral migration of an elastic capsule under an optical force in a uniform flow was studied to show the separation characteristics according to the elastic properties in the cross-type optical separator. The initially spherical capsule was moved through the fluid flow using a laser beam with a Gaussian distribution focused along the direction normal to the flow device surface. To simulate such a system, a penalty immersed boundary method was adopted to enable fluid-membrane coupling, and a dynamic ray tracing method was applied to the optical force calculation. The effects of the elastic properties of the capsule membrane (the surface Young's modulus and the bending modulus) on the lateral migration were studied. By increasing the surface Young's modulus, the capsule deformed less and the migration distance increased; however, buckling occurred in the capsule with a high surface Young's modulus. Buckling could be suppressed by increasing the bending rigidity. The effects of the flow velocity and the laser beam power were also examined. In the simulation, the S number, i.e., the ratio of the optical force to the viscous force, was adjusted by decreasing the flow velocity or increasing the laser beam power. The migration distance increased as the S number increased, and a constant lateral migration distance was obtained for a rigid particle for a given S number. An elastic capsule under conditions intermediate between a fixed flow velocity and a fixed laser beam power, however, did not yield a constant lateral migration distance due to the extent of the deformation in the different situations. To predict the lateral migration distance of an elastic capsule, a nondimensional parameter, S_{e}, was defined to include the effects of the optical force, the elastic force, and the fluid viscous force. A unified tendency of the lateral migration distance with S_{e} was obtained for capsules with intermediate elasticity, by varying either the flow velocity or the laser

  14. A universal flow cytometry assay for screening carbohydrate-active enzymes using glycan microspheres.

    PubMed

    Chandrasekaran, Aarthi; Deng, Kai; Koh, Chung-Yan; Takasuka, Taichi; Bergeman, Lai F; Fox, Brian G; Adams, Paul D; Singh, Anup K

    2013-06-18

    We describe a simple, multiplexed assay that integrates glycan synthesis, bioconjugation to microspheres, fluorescent chemical/biochemical detection and multiparameter flow cytometric analysis to screen activities of different families of carbohydrate-active enzymes.

  15. Feasibility of a Lateral Flow Test for Neurocysticercosis Using Novel Up-Converting Nanomaterials and a Lightweight Strip Analyzer

    PubMed Central

    Corstjens, Paul L. A. M.; de Dood, Claudia J.; Priest, Jeffrey W.; Tanke, Hans J.; Handali, Sukwan

    2014-01-01

    Neurocysticercosis is a frequent parasitic infection of the human brain, occurring in most of the world, and requires imaging of the brain to diagnose. To determine the burden of disease and to simplify diagnosis, a field-friendly rapid lateral flow (LF) based antibody screening test was developed. The assay utilizes novel nano-sized up-converting phosphor (UCP) reporter particles in combination with a portable lightweight analyzer and detects antibodies in serum samples reactive with bacterial-expressed recombinant (r) T24H, a marker for detecting neurocysticercosis cases. Three sequential flow steps allow enrichment of antibodies on the Test (T) line and consecutive binding of protein-A coated UCP reporter particles. Antibody binding was determined by measuring 550 nm emission after excitation of the UCP label with a 980 nm infrared (IR) diode. Clinical sensitivity and specificity of the assay to detect cases of human neurocysticercosis with 2 or more viable brain cysts were 96% and 98%, respectively, using a sample set comprised of sera from 63 confirmed cases and 170 healthy parasite-naïve non-endemic controls. In conclusion: Proof-of-principle, of a rapid UCP-LF screening assay for neurocysticercosis was demonstrated. The assay utilized bacterial-expressed rT24H as a potential alternative for baculovirus-expressed rT24H. Performance of the UCP-LF assay was excellent, although further studies need to confirm that bacterial expressed antigen can entirely replace previously used baculovirus antigen. In addition, the increasing availability of commercial sources for UCP reporter materials as well as the accessibility of affordable semi-handheld scanners may allow UCP-based bioanalytical systems for point-of-care to evolve at an even faster pace. PMID:24992686

  16. Experimental investigation of lateral forces induced by flow through model labyrinth glands

    NASA Technical Reports Server (NTRS)

    Leong, Y. M. M. S.; Brown, R. D.

    1984-01-01

    The lateral forces induced by flow through model labyrinth glands were investigated. Circumferential pressure distributions, lateral forces and stiffness coefficients data obtained are discussed. The force system is represented as a negative spring and a tangential force orthogonal to eccentricity. The magnitude of these forces are dependent on eccentricity, entry swirl, rotor peripheral velocity and seal size. A pressure equalization chamber at midgland tests should in significantly reduced forces and stiffness coefficients.

  17. Rapid detection of measles virus using reverse transcription loop-mediated isothermal amplification coupled with a disposable lateral flow device.

    PubMed

    Xu, Changping; Feng, Yan; Chen, Yin; Gao, Jian; Lu, Yiyu

    2016-06-01

    The measles virus (MeV) causes a highly contagious disease and efforts to reduce its spread are critical. A reverse transcription loop-mediated isothermal amplification assay coupled with a disposable lateral flow device (RT-LAMP-LFD) was developed for the rapid detection of MeV. The assay was performed in 40 min at an optimal temperature of 58 °C, with endpoint results visualized directly. A probe that was complementary to the RT-LAMP amplicon was designed to enhance assay specificity. Detection limit of the assay was 8.8 copies/μL synthetic RNA, which equals the sensitivity of real-time RT-PCR. Clinical specimens were used to validate the RT-LAMP-LFD in provincial Center for Disease Control and Prevention (CDC) (n = 245) and six municipal CDCs (n = 249). The results obtained using RT-LAMP-LFD and real-time RT-PCR were highly concordant. The RT-LAMP-LFD is rapid, stable, and does not require expensive equipment, which can be used for routine MeV monitoring in CDC laboratories.

  18. A flow cytometry-based dopamine transporter binding assay using antagonist-conjugated quantum dots

    SciTech Connect

    Kovtun, Oleg; Ross, Emily J.; Tomlinson, Ian D.; Rosenthal, Sandra J.

    2012-04-05

    Here we present the development and validation of a flow cytometry-based dopamine transporter (DAT) binding assay that uses antagonist-conjugated quantum dots (QDs). Our anticipation is that our QD-based assay is of immediate value to the high throughput screening of novel DAT modulators.

  19. Recharge and Lateral Groundwater Flow Boundary Conditions for the Saturated Zone Site-Scale Flow and Transport Model

    SciTech Connect

    S. James

    2004-10-06

    This analysis is designed to use existing modeling and analysis results as the basis for estimated groundwater flow rates into the saturated zone (SZ) site-scale model domains, both as recharge (infiltration) at the upper boundary (water table), and as underflow at the lateral boundaries. Specifically, this work compiles information on the recharge boundary conditions supplied to the base-case and alternate SZ site-scale flow models taken from (1) distributed recharge from the 1997 (D'Agnese et al. 1997 [DIRS 100131]) or 2001 (D'Agnese et al. 2002 [DIRS 158876]) SZ regional-scale (Death Valley Regional Flow System [DVRFS]) model; (2) recharge below the area of the 1997 (Wu et al. 1997 [DIRS 156453]) or 2003 (BSC 2004 [DIRS 169861]) unsaturated zone (UZ) site-scale flow model; and (3) focused recharge along Fortymile Wash. In addition, this analysis includes extraction of the groundwater flow rates simulated by the 1997 and 2001 DVRFS models coincident with the lateral boundaries of the SZ site-scale flow models. The fluxes from the 1997 DVRFS were used to calibrate the base-case SZ site-scale flow model. The 2001 DVRFS fluxes are used in the alternate SZ site-scale flow model.

  20. Determination of object position, vortex shedding frequency and flow velocity using artificial lateral line canals

    PubMed Central

    Bleckmann, Horst

    2011-01-01

    Summary The lateral line system of fish consists of superficial neuromasts, and neuromasts embedded in lateral line canals. Lateral line neuromasts allow fish to sense both minute water motions and pressure gradients, thereby enabling them to detect predators and prey or to recognize and discriminate stationary objects while passing them. With the aid of the lateral line, fish can also sense vortices caused by an upstream object or by undulatory swimming movements of fish. We show here that artificial lateral line canals equipped with optical flow sensors can be used to detect the water motions generated by a stationary vibrating sphere, the vortices caused by an upstream cylinder or the water (air) movements caused by a passing object. The hydrodynamic information retrieved from optical flow sensors can be used to calculate bulk flow velocity and thus the size of the cylinder that shed the vortices. Even a bilateral sensor platform equipped with only one artificial lateral line canal on each side is sufficient to determine the position of an upstream cylinder. PMID:21977440

  1. Lateral Mixing Mechanisms in Vertical and Horizontal Interconnected Subchannel Two-Phase Flows

    SciTech Connect

    Gencay, Sarman; Teyssedou, Alberto; Tye, Peter

    2002-05-15

    A lateral mixing model based on equal volume exchange between two laterally interconnected subchannels is presented. The following mixing mechanisms are taken into account in this model: (a) diversion cross flow, caused by the lateral pressure difference between adjacent subchannels; (b) turbulent void diffusion, which is governed by the lateral void fraction difference between the subchannels; (c) void drift, responsible for the tendency of the vapor phase to drift toward unobstructed regions; and (d) buoyancy drift, which takes into account the effect of gravity in horizontal flows. Experimental two-phase air-water data obtained using two test sections having different geometries and orientations are used to determine the diffusion coefficients required by the mixing model. Under the absence of diversion crossflow, i.e., negligible lateral pressure difference between the subchannels, it is observed that the diffusion coefficient increases with increasing average void fraction in the subchannels. Moreover, for vertical flows turbulent void diffusion seems to be considerably affected by the geometry of the subchannels. For horizontal flows under nonsymmetric inlet void fraction conditions, even though the interconnected subchannels have the same geometry, different turbulent void diffusion and void drift coefficients are required to satisfy the conditions of hydrodynamic equilibrium. In the present study this condition is achieved by introducing a new void drift coefficient expressed as a correction term applied to the turbulent void drift term.

  2. Disposable lateral flow-through strip for smartphone-camera to quantitatively detect alkaline phosphatase activity in milk.

    PubMed

    Yu, Ling; Shi, ZhuanZhuan; Fang, Can; Zhang, YuanYuan; Liu, YingShuai; Li, ChangMing

    2015-07-15

    A disposable lateral flow-through strip was developed for smartphone to fast one-step quantitatively detect alkaline phosphatase (ALP) activity in raw milk. The strip comprises two functional components, a conjugation pad loaded with phosphotyrosine-coated gold nanoparticles (AuNPs@Cys-Try-p) and a testing line coated with anti-phosphotryosine antibody (anti-Tyr-p mAb). The dephosphorylation activity of ALP at the testing zone can be quantitatively assayed by monitoring the accumulated AuNPs-induced color changes by smartphone camera, thus providing a highly convenient portable detection method. A trace amount of ALP as low as 0.1UL(-1) with a linear dynamic range of 0.1-150UL(-1) (R(2)=0.999) in pasteurized milk and raw milk can be one-step detected by the developed flow-through strip within 10min, demonstrating the potential of smartphone-based portable sensing device for pathogen detection. This bio-hazards free lateral flow-through testing strip can be also used to fabricate rapid, sensitive and inexpensive enzyme or immunosensors for broad portable clinic diagnosis and food contamination analysis, particularly in point-of-care and daily food quality inspection. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Magnetic lateral flow immunoassay test strip development - Considerations for proof of concept evaluation.

    PubMed

    Connolly, R; O' Kennedy, R

    2017-03-01

    Lateral flow immunoassays (LFIA) have grown to become the predominant test device format for the diagnostics and point-of-care industries. The demand for robust and reproducible LFIAs has been facilitated through scale-up production methods using specialized and automated instruments. However, the feasibility of a LFIA device can still be evaluated in a small-scale laboratory setting through controlled manual preparation methods. The advent of super-paramagnetic (SPMP) labels for use in lateral flow has heralded the possibility of highly sensitive and stable LFIAs. The methods used for the preparation of a magnetic LFIA prototype device using a reserved suite of laboratory equipment are described.

  4. Evaluation of early conception factor lateral flow test to determine nonpregnancy in dairy cattle

    PubMed Central

    Ambrose, Divakar J.; Radke, Brian; Pitney, Phyllis A.; Goonewardene, Laksiri A.

    2007-01-01

    The early conception factor (ECF) lateral flow test was evaluated for its ability to accurately determine nonpregnant status in dairy cattle. Results of 2 field trials involving 191 cows and 832 tests indicated the probability that a cow can be correctly diagnosed as nonpregnant by using the ECF test is only about 50%. Agreement of test results between milk and serum obtained from the same cow was 57.5%. The ECF test was not consistent in identifying nonpregnancy when the same cows were tested repeatedly over a period of 4 weeks. We conclude that the ECF lateral flow test does not accurately identify nonpregnancy in dairy cattle. PMID:17824326

  5. Electrochemical lateral flow immunosensor for detection and quantification of dengue NS1 protein.

    PubMed

    Sinawang, Prima Dewi; Rai, Varun; Ionescu, Rodica E; Marks, Robert S

    2016-03-15

    An Electrochemical Lateral Flow Immunosensor (ELFI) is developed combining screen-printed gold electrodes (SPGE) enabling quantification together with the convenience of a lateral flow test strip. A cellulose glassy fiber paper conjugate pad retains the marker immunoelectroactive nanobeads which will bind to the target analyte of interest. The specific immunorecognition event continues to occur along the lateral flow bed until reaching the SPGE-capture antibodies at the end of the cellulosic lateral flow strip. The rationale of the immunoassay consists in the analyte antigen NS1 protein being captured selectively and specifically by the dengue NS1 antibody conjugated onto the immunonanobeads thus forming an immunocomplex. With the aid of a running buffer, the immunocomplexes flow and reach the immuno-conjugated electrode surface and form specific sandwich-type detection due to specific, molecular recognition, while unbound beads move along past the electrodes. The successful sandwich immunocomplex formation is then recorded electrochemically. Specific detection of NS1 is translated into an electrochemical signal contributed by a redox label present on the bead-immobilized detection dengue NS1 antibody while a proportional increase of faradic current is observed with increase in analyte NS1 protein concentration. The first generation ELFI prototype is simply assembled in a cassette and successfully demonstrates wide linear range over a concentration range of 1-25 ng/mL with an ultrasensitive detection limit of 0.5 ng/mL for the qualitative and quantitative detection of analyte dengue NS1 protein.

  6. Integrated assessment of lateral flow, density effects and dispersion in aquifer storage and recovery

    NASA Astrophysics Data System (ADS)

    Ward, James D.; Simmons, Craig T.; Dillon, Peter J.; Pavelic, Paul

    2009-05-01

    SummaryAquifer storage and recovery (ASR) involves the injection of freshwater into an aquifer for later recovery and use. This paper investigates three major factors leading to reduction in performance of ASR systems in brackish or saline aquifers: lateral flow, density-driven flow and dispersive mixing. Previous analyses of aquifer storage and recovery (ASR) have considered at most two of the above processes, but never all three together, and none have considered lateral flow and density effects together. In this analysis, four dimensionless parameters are defined to give an approximate characterisation of lateral flow, dispersive mixing, mixed convection (density effects during pumping) and free convection (density effects during storage). An extensive set of numerical models spanning a wide parameter range is then used to develop a predictive framework using the dimensionless numbers. If the sum of the four dimensionless numbers (denoted RASR) exceeds 10, the ASR operation is likely to fail with no recoverable freshwater, while if RASR < 0.1, the ASR operation is likely to provide at least some recovery of freshwater. The predictive framework is tested using limited data available from ASR field sites, broadly lending support to the framework. This study has several important implications. Firstly, the lack of completeness of field data sets in the literature must be rectified if we are to properly characterise mixed-convective flow processes in ASR operations. Once data are available, the dimensionless numbers can be used to identify suitable ASR sites and the desirable operational conditions that maximise recovery efficiencies.

  7. Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.

    PubMed

    Rohrman, Brittany; Richards-Kortum, Rebecca

    2015-02-03

    Recombinase polymerase amplification (RPA) may be used to detect a variety of pathogens, often after minimal sample preparation. However, previous work has shown that whole blood inhibits RPA. In this paper, we show that the concentrations of background DNA found in whole blood prevent the amplification of target DNA by RPA. First, using an HIV-1 RPA assay with known concentrations of nonspecific background DNA, we show that RPA tolerates more background DNA when higher HIV-1 target concentrations are present. Then, using three additional assays, we demonstrate that the maximum amount of background DNA that may be tolerated in RPA reactions depends on the DNA sequences used in the assay. We also show that changing the RPA reaction conditions, such as incubation time and primer concentration, has little effect on the ability of RPA to function when high concentrations of background DNA are present. Finally, we develop and characterize a lateral flow-based method for enriching the target DNA concentration relative to the background DNA concentration. This sample processing method enables RPA of 10(4) copies of HIV-1 DNA in a background of 0-14 μg of background DNA. Without lateral flow sample enrichment, the maximum amount of background DNA tolerated is 2 μg when 10(6) copies of HIV-1 DNA are present. This method requires no heating or other external equipment, may be integrated with upstream DNA extraction and purification processes, is compatible with the components of lysed blood, and has the potential to detect HIV-1 DNA in infant whole blood with high proviral loads.

  8. Rapid fluorescent lateral-flow immunoassay for hepatitis B virus genotyping.

    PubMed

    Song, Liu-Wei; Wang, Ying-Bin; Fang, Lin-Lin; Wu, Yong; Yang, Lin; Chen, Jie-Yu; Ge, Sheng-Xiang; Zhang, Jing; Xiong, You-Zheng; Deng, Xiu-Mei; Min, Xiao-Ping; Zhang, Jun; Chen, Pei-Jer; Yuan, Quan; Xia, Ning-Shao

    2015-01-01

    Hepatitis B virus (HBV) genotyping plays an important role in the clinical management of chronic hepatitis B (CHB) patients. However, the current nucleic acid based techniques are expensive, time-consuming, and inconvenient. Here, we developed a novel DNA-independent HBV genotyping tool based on a one-step fluorescent lateral flow immunoassay (LFIA). Epitope-targeting immunization and screening techniques were used to develop HBV genotype specific monoclonal antibodies (mAbs). These mAbs were used to develop a multitest LFIA with a matched scanning luminoscope for HBV genotyping (named the GT-LFIA). The performance of this novel assay was carefully evaluated in well-characterized clinical cohorts. The GT-LFIA, which can specifically differentiate HBV genotypes A, B, C, and D in a pretreatment-free single test, was successfully developed using four genotype specific mAbs. The detection limits of the GT-LFIA for HBV genotypes A, B, C, and D were 2.5-10.0 IU HBV surface antigen/mL, respectively. Among the sera from 456 CHB patients, 439 (96.3%; 95% confidence interval (CI), 94.1-97.8%) were genotype-differentiable by the GT-LFIA and 437 (99.5%; 95% CI, 98.4-99.9%) were consistent with viral genome sequencing. In the 21 patients receiving nucleos(t)ide analogue therapy, for end-of-treatment specimens that were HBV DNA undetectable and were not applicable for DNA-dependent genotyping, the GT-LFIA presented genotyping results that were consistent with those obtained in pretreatment specimens by viral genome sequencing and the GT-LFIA. In conclusion, the novel GT-LFIA is a convenient, fast, and reliable tool for differential HBV genotyping, especially in patients with low or undetectable HBV DNA levels.

  9. Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis.

    PubMed

    Houghton, Raymond L; Reed, Dana E; Hubbard, Mark A; Dillon, Michael J; Chen, Hongjing; Currie, Bart J; Mayo, Mark; Sarovich, Derek S; Theobald, Vanessa; Limmathurotsakul, Direk; Wongsuvan, Gumphol; Chantratita, Narisara; Peacock, Sharon J; Hoffmaster, Alex R; Duval, Brea; Brett, Paul J; Burtnick, Mary N; Aucoin, David P

    2014-03-01

    Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.

  10. Field-Usable Lateral Flow Immunoassay for the Rapid Detection of White Spot Syndrome Virus (WSSV)

    PubMed Central

    Kulabhusan, Prabir Kumar; Rajwade, Jyutika M.; Sugumar, Vimal; Taju, Gani; Sahul Hameed, A. S.

    2017-01-01

    Background White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses. Methods/Principal Findings Here, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional “gold standard test”, viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen’s kappa coefficient of 0.983 suggested "very good agreement” between the developed LFIA and the conventional one-step PCR. Conclusion The LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry. PMID:28046005

  11. Field-Usable Lateral Flow Immunoassay for the Rapid Detection of White Spot Syndrome Virus (WSSV).

    PubMed

    Kulabhusan, Prabir Kumar; Rajwade, Jyutika M; Sugumar, Vimal; Taju, Gani; Sahul Hameed, A S; Paknikar, Kishore M

    2017-01-01

    White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses. Here, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional "gold standard test", viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen's kappa coefficient of 0.983 suggested "very good agreement" between the developed LFIA and the conventional one-step PCR. The LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry.

  12. Sensitive immunochemical approaches for quantitative (FPIA) and qualitative (lateral flow tests) determination of gentamicin in milk.

    PubMed

    Beloglazova, N V; Shmelin, P S; Eremin, S A

    2016-01-01

    Three kinds of immunoassays for the determination of gentamicin in milk samples were developed and validated. First, a fast and easily-performed fluorescence polarization immunoassay was used for characterization of the employed polyclonal antibody. The calculated Kaff were (1.9±0.4)×10(9)М(-1) and (6.0±0.2)×10(6)М(-1) for the high- and low-affinity fractions respectively. The assay was characterized with a good sensitivity, the limit of detection being 5μgkg(-1). Two different kinds of detection labels, i.e. colloidal gold (CG) and quantum dots (QDs), were evaluated for use in lateral-flow format with respect to rapid visual on-site testing. The cut-off levels for both qualitative formats were selected based on the maximum level for gentamicin in milk established by the European Commission, 100μgkg(-1), resulting in a 10μgkg(-1) cut-off considering sample dilution. The intra-laboratory validation was performed with sterilized milk samples artificially spiked with gentamicin at concentrations less than, equal to, and greater than the cut-off level. It was shown that milk products could be analyzed without any sample preparation, except for dilution with the buffer solution. The rates of false-positive and false-negative results were below 5% for both labels. The different developed immunoassays were tested towards gentamicin determination in artificially-spiked and naturally contaminated milk samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Rapid Detection of Listeria by Bacteriophage Amplification and SERS-Lateral Flow Immunochromatography

    PubMed Central

    Stambach, Nicholas R.; Carr, Stephanie A.; Cox, Christopher R.; Voorhees, Kent J.

    2015-01-01

    A rapid Listeria detection method was developed utilizing A511 bacteriophage amplification combined with surface-enhanced Raman spectroscopy (SERS) and lateral flow immunochromatography (LFI). Anti-A511 antibodies were covalently linked to SERS nanoparticles and printed onto nitrocellulose membranes. Antibody-conjugated SERS nanoparticles were used as quantifiable reporters. In the presence of A511, phage-SERS nanoparticle complexes were arrested and concentrated as a visible test line, which was interrogated quantitatively by Raman spectroscopy. An increase in SERS intensity correlated to an increase in captured phage-reporter complexes. SERS limit of detection was 6 × 106 pfu·mL−1, offering detection below that obtainable by the naked eye (LOD 6 × 107 pfu·mL−1). Phage amplification experiments were carried out at a multiplicity of infection (MOI) of 0.1 with 4 different starting phage concentrations monitored over time using SERS-LFI and validated by spot titer assay. Detection of L. monocytogenes concentrations of 1 × 107 colony forming units (cfu)·mL−1, 5 × 106 cfu·mL−1, 5 × 105 cfu·mL−1 and 5 × 104 cfu·mL−1 was achieved in 2, 2, 6, and 8 h, respectively. Similar experiments were conducted at a constant starting phage concentration (5 × 105 pfu·mL−1) with MOIs of 1, 2.5, and 5 and were detected in 2, 4, and 5 h, respectively. PMID:26694448

  14. Blood coagulation screening using a paper-based microfluidic lateral flow device.

    PubMed

    Li, H; Han, D; Pauletti, G M; Steckl, A J

    2014-10-21

    A simple approach to the evaluation of blood coagulation using a microfluidic paper-based lateral flow assay (LFA) device for point-of-care (POC) and self-monitoring screening is reported. The device utilizes whole blood, without the need for prior separation of plasma from red blood cells (RBC). Experiments were performed using animal (rabbit) blood treated with trisodium citrate to prevent coagulation. CaCl2 solutions of varying concentrations are added to citrated blood, producing Ca(2+) ions to re-establish the coagulation cascade and mimic different blood coagulation abilities in vitro. Blood samples are dispensed into a paper-based LFA device consisting of sample pad, analytical membrane and wicking pad. The porous nature of the cellulose membrane separates the aqueous plasma component from the large blood cells. Since the viscosity of blood changes with its coagulation ability, the distance RBCs travel in the membrane in a given time can be related to the blood clotting time. The distance of the RBC front is found to decrease linearly with increasing CaCl2 concentration, with a travel rate decreasing from 3.25 mm min(-1) for no added CaCl2 to 2.2 mm min(-1) for 500 mM solution. Compared to conventional plasma clotting analyzers, the LFA device is much simpler and it provides a significantly larger linear range of measurement. Using the red colour of RBCs as a visible marker, this approach can be utilized to produce a simple and clear indicator of whether the blood condition is within the appropriate range for the patient's condition.

  15. Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis

    PubMed Central

    Houghton, Raymond L.; Reed, Dana E.; Hubbard, Mark A.; Dillon, Michael J.; Chen, Hongjing; Currie, Bart J.; Mayo, Mark; Sarovich, Derek S.; Theobald, Vanessa; Limmathurotsakul, Direk; Wongsuvan, Gumphol; Chantratita, Narisara; Peacock, Sharon J.; Hoffmaster, Alex R.; Duval, Brea; Brett, Paul J.; Burtnick, Mary N.; AuCoin, David P.

    2014-01-01

    Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the “gold standard” for the diagnosis of melioidosis; results can take 3–7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation. PMID:24651568

  16. Continuous enrichment of circulating tumor cells using a microfluidic lateral flow filtration chip.

    PubMed

    Lee, Sung-Woo; Hyun, Kyung-A; Kim, Seung-Il; Kang, Ji-Yoon; Jung, Hyo-Il

    2015-01-16

    The isolation and characterization of circulating tumor cells (CTC) is of great importance in cancer diagnosis and prognosis. Highly sensitive detection of CTCs can be very difficult because they are extremely rare (i.e., 1-5 CTCs per 10(9) erythrocytes) in blood. Recently, various devices have been developed that exploit biochemical (affinity-based) and physical (size or density) methods. Antibody-based isolation has its own limitations, as the expression level of the epitopes for an antibody varies due to the heterogeneity of cancer cells. Harsh conditions associated with physical methods can cause the deformation and damage of CTCs during the isolation process. Here, we propose a microfluidic lateral flow filtration (μ-LaFF) chip in which lateral flow was combined with vertical flow into the filter to capture the CTCs gently. The CTCs experienced weak shear flow owing to the lateral flow and traveled alongside the filter channel until finally being captured. The vertical flow in the filter held the captured cells tightly and served as an exit for uncaptured hematological cells (white and red blood cells). From our μ-LaFF chip we obtained a high capture efficiency (95%) and purity (99%), minimizing any damage to the CTCs. Our μ-LaFF technology is expected to be useful in the diagnosis and prognosis of various cancers.

  17. Continuous-flow automation of the Lactobacillus casei serum folate assay.

    PubMed Central

    Tennant, G B

    1977-01-01

    A method is described for the continuous-flow automation of the serum folate assay using Lactobacillus casei. The total incubation period is approximately four hours. The growth response of the organism to folate is estimated by measuring the rate of reduction of 2,3,5-triphenyl tetrazolium chloride (TTC). A simple continuous culture apparatus is used to grow the inoculum. Supplementation of the assay medium is necessary to obtain parallel results. A statistical assessment shows a favourable comparison with the whole-serum tube assay using a chloramphenicol resistant strain of L. casei. The method is less sensitive to inhibitory substances than the tube assay. PMID:415069

  18. Evaluation of a lateral flow immunoassay for field identification of Solenopsis invicta (Hymenoptera: Formicidae) in Australia

    USDA-ARS?s Scientific Manuscript database

    In an effort to improve surveillance capacity for the exotic red imported fire ant, Solenopsis invicta, a lateral flow immunoassay (LFA) was recently evaluated by Biosecurity Queensland staff in Australia. The purpose of the research was to assess the ability of the fire ant LFA to discriminate S. i...

  19. Ultrasensitive detection of microbial cells using magnetic focus enhanced lateral flow sensors.

    PubMed

    Ren, Wen; Cho, Il-Hoon; Zhou, Zhongwu; Irudayaraj, Joseph

    2016-04-07

    We report on an improved lateral flow immunoassay (LFIA) sensor with a magnetic focus for ultrasensitive naked-eye detection of pathogenic microorganisms at a near single cell limit without any pre-enrichment steps, by allowing the magnetic probes to focus the labelled pathogens to the target zone of the LF strip.

  20. Lateral flow immunoassay for the rapid detection of citrus tristeza virus

    USDA-ARS?s Scientific Manuscript database

    A lateral flow methodology was developed using gold nanoparticles for rapid detection of Citrus tristeza virus (CTV). The test strip was based on a sandwich immunoassay and could be accomplished within 10 minutes. A sample was considered negative for CTV when only the control line appeared; whereas,...

  1. A silicon dioxide modified magnetic nanoparticles-labeled lateral flow strips for HBs antigen.

    PubMed

    Zhang, Xueqing; Jiang, Lin; Zhang, Chunlei; Li, Ding; Wang, Can; Gao, Feng; Cui, Daxiang

    2011-12-01

    Herein we reported a new type of silicon dioxide wrapped magnetic nanoparticles-labeled lateral flow strip for detection of HBs antigen in sera. The SiO2 wrapped Fe3O4 nanocomposites were prepared and characterized by HR-TEM, FTIR and magnetometer. As-prepared nanocomposites were used to label anti-HBV surface monoclonal antibody, the lateral flow strips were constructed, and 100 specimens of sera were collected and tested. Results showed that the prepared SiO2 wrapped Fe3O4 nanocomposites were shell/core structure, well dispersed, with the size of 25 nm in diameter, the thickness of the shell was about 3 nm, their magnetic saturation intensity was 44.3 meu g(-1). Clinical sera specimens test results showed that the prepared lateral flow strips were with the detection limitation of 5 pg/mL by naked eye observation, and 0.1 pg/mL by CCD reader or MAR Analyzer, specificity was 100%. In conclusion, one kind of silicon dioxide wrapped magnetic nanoparticles-labeled lateral flow strip for ultrasensitive detection of HBs antigen was successfully developed, its ease of use, sensitiveness and low-cost make it well-suited for population-based on-the-site hepatitis B screening.

  2. Lateral Migration and Rotational Motion of Elliptic Particles in Planar Poiseuille Flow

    NASA Technical Reports Server (NTRS)

    Qi, Dewei; Luo, Li-Shi; Aravamuthan, Raja; Strieder, William; Bushnell, Dennis M. (Technical Monitor)

    2002-01-01

    Simulations of elliptic particulate suspensions in the planar Poiseuille flow are performed by using the lattice Boltzmann equation. Effects of the multi-particle on the lateral migration and rotational motion of both neutrally and non-neutrally buoyant elliptic particles are investigated. Low and intermediate total particle volume fraction f(sub a) = 13%, 15%, and 40% are considered in this work.

  3. A lateral flow biosensor for detection of nucleic acids with high sensitivity and selectivity.

    PubMed

    Lie, Puchang; Liu, Jie; Fang, Zhiyuan; Dun, Boying; Zeng, Lingwen

    2012-01-07

    A lateral flow biosensor based on isothermal strand-displacement polymerase reaction and gold nanoparticles has been developed for the visual detection of nucleic acids with a detection limit of 0.01 fM. This journal is © The Royal Society of Chemistry 2012

  4. The performance characteristics of lateral flow devices with 2 strains of highly pathogenic avian influenza virus

    USDA-ARS?s Scientific Manuscript database

    Lateral flow devices (LFD) are commercially available and provide a fast, highly specific, on-site test for avian influenza. Because of the low analytic sensitivity of LFD tests at low virus concentrations, targeted sampling of sick and dead birds has been proposed in order to increase detection pr...

  5. Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay.

    PubMed

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-08-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87(N). Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

  6. Large-scale clinical validation of a lateral flow immunoassay for detection of cryptococcal antigen in serum and cerebrospinal fluid specimens.

    PubMed

    Suwantarat, Nuntra; Dalton, Justin B; Lee, Richard; Green, Rachel; Memon, Warda; Carroll, Karen C; Riedel, Stefan; Zhang, Sean X

    2015-05-01

    We compared a lateral flow immunoassay (LFA) to a currently used enzyme immunoassay for detection of cryptococcal antigen in 396 sera and 651 cerebrospinal fluid specimens. We found 97% concordance between the 2 assays. The LFA detected an additional 22 positives. Overall, the LFA had sensitivity of 100% and specificity of 99.6% for the diagnosis of cryptococcosis. The LFA is rapid, accurate, and easy to perform, and it is suitable for routine patient care testing.

  7. Flow systems exploiting in-line prior assays.

    PubMed

    Grassi, Viviane; Dias, Ana Cristi B; Zagatto, Elias A G

    2004-12-15

    An expert sequential injection system involving a prior assay is proposed for spectrophotometric determination of phosphate and eventually zinc in soil extracts. The result of phosphate determination is the basis for a concentration-oriented decision regarding to the need or not for zinc determination. Zinc was only determined if a threshold value (peak height corresponding to 5.0mgl(-1)P) was surpassed. The methods involved formation of molybdenum blue and the Rhodamine 6G/ammonium thiocyanate/Zn(2+) ternary complex. Variations in the threshold value were < 2% during 4h operating periods, false responses were not verified, and the analytical time was reduced in about 30%. Precise results (R.S.D. <3%P and < 1% Zn) in agreement with spectrophotometry and flame atomic absorption spectrometry were obtained. The innovation permits faster information processing, as well as a reduction in the number of measurements, number of analytical steps, laboratorial time, and consumption of sample and reagents, thus waste generation.

  8. VORSTAB: A computer program for calculating lateral-directional stability derivatives with vortex flow effect

    NASA Technical Reports Server (NTRS)

    Lan, C. Edward

    1985-01-01

    A computer program based on the Quasi-Vortex-Lattice Method of Lan is presented for calculating longitudinal and lateral-directional aerodynamic characteristics of nonplanar wing-body combination. The method is based on the assumption of inviscid subsonic flow. Both attached and vortex-separated flows are treated. For the vortex-separated flow, the calculation is based on the method of suction analogy. The effect of vortex breakdown is accounted for by an empirical method. A summary of the theoretical method, program capabilities, input format, output variables and program job control set-up are described. Three test cases are presented as guides for potential users of the code.

  9. An improved flow cytometry assay to monitor phagosome acidification.

    PubMed

    Colas, Chloé; Menezes, Shinelle; Gutiérrez-Martínez, Enric; Péan, Claire B; Dionne, Marc S; Guermonprez, Pierre

    2014-10-01

    Phago-lysosome formation is important for cell-autonomous immunity to intracellular pathogens, antigen presentation and metabolism. A hallmark feature of phago-lysosomal compartments is that they undergo progressive luminal acidification controlled by the activation of vacuolar V-ATPase. Acidification is required for many enzymatic processes taking place in phago-lysosomes, like proteolysis, and supports the microbicidal activity of macrophages. Here we present a new quantitative methodology to assess phagosome acidification by flow cytometry based on the use of bi-fluorescent particles. This method relies on the use of UV polystyrene beads labelled with the acid sensor pHrodo-succinimidyl ester (pHrodo(TM) SE red) and enables us to dissociate particle association with phagocytes from their engulfment in acidified compartments. This methodology is well suited to monitor the acidification of phagosomes formed in vivo after fluorescent bead administration.

  10. Effect of lateral mobility of fluorescent probes in lipid mixing assays of cell fusion.

    PubMed Central

    Huang, S K; Cheng, M; Hui, S W

    1990-01-01

    Monolayers of human erythrocytes, immobilized on a cover slip, were induced to fuse by polyethylene glycol (mol wt 8,000). The mobility of fluorescent probes, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadizol-4-yl)amino]dodecanoyl] phosphatidyl-choline (C12-NBD-PC), from labeled cells to unlabeled cells was monitored by video-enhanced fluorescence microscopy. A dequenching curve was obtained from the measurement of fluorescence intensities of pairs of fused cells over time. The dequenching curve and the curve obtained from macroscopic measurements of a cell monolayer (described in the preceding article) were compared and discussed. The slow probe transfer rate between a pair of fused cells was explained by a diffusion model based on membrane area conservation and the geometry of the fusion lumen. An equivalent lumen between two fused cells, thought to be the main rate limitation of probe mobility after fusion, was calculated to be approximately 130 nm in diameter. Lumens of 75 nm in diameter were observed by electron microscopy. Thus, the rate of macroscopic fluorescence dequenching depends not only upon the fusion efficiency, but also upon the number of simultaneous fusion partners, the geometry of their contact points, and the lateral mobility of the fluorescent probes through these points. The relative fusion efficiency can be derived only from the saturation dequenching values. Images FIGURE 3 FIGURE 5 FIGURE 7 PMID:2291938

  11. A bio-inspired real-time capable artificial lateral line system for freestream flow measurements.

    PubMed

    Abels, C; Qualtieri, A; De Vittorio, M; Megill, W M; Rizzi, F

    2016-06-03

    To enhance today's artificial flow sensing capabilities in aerial and underwater robotics, future robots could be equipped with a large number of miniaturized sensors distributed over the surface to provide high resolution measurement of the surrounding fluid flow. In this work we show a linear array of closely separated bio-inspired micro-electro-mechanical flow sensors whose sensing mechanism is based on a piezoresistive strain-gauge along a stress-driven cantilever beam, mimicking the biological superficial neuromasts found in the lateral line organ of fishes. Aiming to improve state-of-the-art flow sensing capability in autonomously flying and swimming robots, our artificial lateral line system was designed and developed to feature multi-parameter freestream flow measurements which provide information about (1) local flow velocities as measured by the signal amplitudes from the individual cantilevers as well as (2) propagation velocity, (3) linear forward/backward direction along the cantilever beam orientation and (4) periodicity of pulses or pulse trains determined by cross-correlating sensor signals. A real-time capable cross-correlation procedure was developed which makes it possible to extract freestream flow direction and velocity information from flow fluctuations. The computed flow velocities deviate from a commercial system by 0.09 m s(-1) at 0.5 m s(-1) and 0.15 m s(-1) at 1.0 m s(-1) flow velocity for a sampling rate of 240 Hz and a sensor distance of 38 mm. Although experiments were performed in air, the presented flow sensing system can be applied to underwater vehicles as well, once the sensors are embedded in a waterproof micro-electro-mechanical systems package.

  12. Evaluation of a new nanoparticle-based lateral-flow immunoassay for the exclusion of heparin-induced thrombocytopenia (HIT).

    PubMed

    Sachs, Ulrich J; von Hesberg, Jakob; Santoso, Sentot; Bein, Gregor; Bakchoul, Tamam

    2011-12-01

    Heparin-induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies (abs) that recognise platelet factor 4-heparin (PF4/hep) complexes. Several laboratory tests are available for the confirmation and/or refutation of HIT. A reliable and rapid single-sample test is still pending. It was the objective of this study to evaluate a new lateral-flow immunoassay based on nanoparticle technology. A cohort of 452 surgical and medical patients suspected of having HIT was evaluated. All samples were tested in two IgG-specific ELISAs, in a particle gel immunoassay (PaGIA) and in a newly developed lateral-flow immunoassay (LFI-HIT) as well as in a functional test (HIPA). Clinical pre-test probability was determined using 4T's score. Platelet-activating antibodies were present in 34/452 patients, all of whom had intermediate to high clinical probability. PF4/hep abs were detected in 79, 87, 86, and 63 sera using the four different immunoassays. The negative predictive values (NPV) were 100% for both ELISA tests and LFI-HIT but only 99.2% for PaGIA. There were less false positives (n=29) in the LFI-HIT compared to any other test. Additionally, significantly less time was required to perform LFI-HIT than to perform the other immunoassays. In conclusion, a newly developed lateral-flow assay, LFI-HIT, was capable of identifying all HIT patients in a cohort in a short period of time. Beside an NPV of 100%, the rate of false-positive signals is significantly lower with LFI-HIT than with other immunoassay(s). These performance characteristics suggest a high potency in reducing the risk and costs in patients suspected of having HIT.

  13. Flow induced by ependymal cilia dominates near-wall cerebrospinal fluid dynamics in the lateral ventricles.

    PubMed

    Siyahhan, Bercan; Knobloch, Verena; de Zélicourt, Diane; Asgari, Mahdi; Schmid Daners, Marianne; Poulikakos, Dimos; Kurtcuoglu, Vartan

    2014-05-06

    While there is growing experimental evidence that cerebrospinal fluid (CSF) flow induced by the beating of ependymal cilia is an important factor for neuronal guidance, the respective contribution of vascular pulsation-driven macroscale oscillatory CSF flow remains unclear. This work uses computational fluid dynamics to elucidate the interplay between macroscale and cilia-induced CSF flows and their relative impact on near-wall dynamics. Physiological macroscale CSF dynamics are simulated in the ventricular space using subject-specific anatomy, wall motion and choroid plexus pulsations derived from magnetic resonance imaging. Near-wall flow is quantified in two subdomains selected from the right lateral ventricle, for which dynamic boundary conditions are extracted from the macroscale simulations. When cilia are neglected, CSF pulsation leads to periodic flow reversals along the ventricular surface, resulting in close to zero time-averaged force on the ventricle wall. The cilia promote more aligned wall shear stresses that are on average two orders of magnitude larger compared with those produced by macroscopic pulsatile flow. These findings indicate that CSF flow-mediated neuronal guidance is likely to be dominated by the action of the ependymal cilia in the lateral ventricles, whereas CSF dynamics in the centre regions of the ventricles is driven predominantly by wall motion and choroid plexus pulsation.

  14. Flow induced by ependymal cilia dominates near-wall cerebrospinal fluid dynamics in the lateral ventricles

    PubMed Central

    Siyahhan, Bercan; Knobloch, Verena; de Zélicourt, Diane; Asgari, Mahdi; Schmid Daners, Marianne; Poulikakos, Dimos; Kurtcuoglu, Vartan

    2014-01-01

    While there is growing experimental evidence that cerebrospinal fluid (CSF) flow induced by the beating of ependymal cilia is an important factor for neuronal guidance, the respective contribution of vascular pulsation-driven macroscale oscillatory CSF flow remains unclear. This work uses computational fluid dynamics to elucidate the interplay between macroscale and cilia-induced CSF flows and their relative impact on near-wall dynamics. Physiological macroscale CSF dynamics are simulated in the ventricular space using subject-specific anatomy, wall motion and choroid plexus pulsations derived from magnetic resonance imaging. Near-wall flow is quantified in two subdomains selected from the right lateral ventricle, for which dynamic boundary conditions are extracted from the macroscale simulations. When cilia are neglected, CSF pulsation leads to periodic flow reversals along the ventricular surface, resulting in close to zero time-averaged force on the ventricle wall. The cilia promote more aligned wall shear stresses that are on average two orders of magnitude larger compared with those produced by macroscopic pulsatile flow. These findings indicate that CSF flow-mediated neuronal guidance is likely to be dominated by the action of the ependymal cilia in the lateral ventricles, whereas CSF dynamics in the centre regions of the ventricles is driven predominantly by wall motion and choroid plexus pulsation. PMID:24621815

  15. Magnetic Control of Lateral Migration of Ellipsoidal Microparticles in Microscale Flows

    NASA Astrophysics Data System (ADS)

    Zhou, Ran; Sobecki, Christopher A.; Zhang, Jie; Zhang, Yanzhi; Wang, Cheng

    2017-08-01

    Precise manipulations of nonspherical microparticles by shape have diverse applications in biology and biomedical engineering. Here, we study lateral migration of ellipsoidal paramagnetic microparticles in low-Reynolds-number flows under uniform magnetic fields. We show that magnetically induced torque alters the rotation dynamics of the particle and results in shape-dependent lateral migration. By adjusting the direction of the magnetic field, we demonstrate versatile control of the symmetric and asymmetric rotation of the particles, thereby controlling the direction of the particle's lateral migration. The particle rotations are experimentally measured, and their symmetry or asymmetry characteristics agree well with the prediction from a simple theory. The lateral migration mechanism is found to be valid for nonmagnetic particles suspended in a ferrofluid. Finally, we demonstrate shape-based sorting of microparticles by exploiting the proposed migration mechanism.

  16. Development of Multiple Cross Displacement Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Shigella spp.

    PubMed Central

    Wang, Yi; Wang, Yan; Xu, Jianguo; Ye, Changyun

    2016-01-01

    Shigella spp., the etiological agent of shigellosis or “bacillary dysentery,” are responsible for considerable morbidity and mortality in excess of a million deaths globally per year. Although PCR-based techniques (such as PCR-based dipstick biosensors) have been used for the molecular diagnosis of infectious disease, these assays were restricted due to the need for a sophisticated thermal cycling apparatus to denature target templates. To facilitate simple and rapid detection of target pathogens, we successfully devised an inexpensive, reliable and nearly instrument-free molecular technique, which incorporates multiple cross displacement amplification (MCDA) combined with a newly designed lateral flow biosensor (LFB) for visual, sensitive and specific detection of Shigella. The MCDA-LFB assay was conducted at 65°C for only 20 min during the amplification stage, and then products were directly analyzed on the biosensor, alleviating the use of special reagents, electrophoresis equipment and amplicon detection instruments. The entire process, including specimen processing (35 min), amplification (20) and detection (2–5 min), can be finished within 1 h. The MCDA-LFB assay demonstrated high specificity for Shigella detection. The analytical sensitivity of the assay was 10 fg of genomic templates per reaction in pure culture and 5.86 CFU per tube in human fecal samples, which was consistent with MCDA by colorimetric indicator, gel electrophoresis, real time turbidity and fluorescence detection. Hence, the simplicity, rapidity and nearly instrument-free platform of the MCDA-LFB assay make it practical for ‘on-site’ diagnosis, point-of-care testing and more. Moreover, the proof-of-concept approach can be reconfigured to detect a wide variety of target sequences by re-designing the specific MCDA primers. PMID:27917160

  17. Development of multiple cross displacement amplification label-based gold nanoparticles lateral flow biosensor for detection of Listeria monocytogenes

    PubMed Central

    Wang, Yi; Li, Hui; Wang, Yan; Li, Hua; Luo, Lijuan; Xu, Jianguo; Ye, Changyun

    2017-01-01

    Listeria monocytogenes, one of most problematic foodborne pathogens, is responsible for listeriosis in both humans and animals and mainly transmitted through the food chain. In this report, we propose a simple, rapid, and nearly instrument-free molecular technique using multiple cross displacement amplification (MCDA) label-based gold nanoparticles lateral flow biosensor (LFB) for specific, sensitive, and visual detection of L. monocytogenes. The MCDA-LFB method was carried out at a constant temperature (61°C) for only 20 min during the reaction stage, and then the amplification mixtures were directly detected by using LFB, eliminating the use of an electrophoresis instrument, special reagents, or amplicon analysis equipment. The whole procedure, from sample processing to result indicating, was finished within 1 h. The analytical specificity of MCDA-LFB method was successfully determined by distinguishing the target bacterium from other pathogens. The analytical sensitivity of the MCDA-LFB assay was 10 fg of genomic templates per reaction in pure culture, which was in complete accordance with MCDA by gel electrophoresis, real-time turbidity, and colorimetric indicator. The assay was also successfully applied to detecting L. monocytogenes in pork samples. Therefore, the rapidity, simplicity, and nearly equipment-free platform of the MCDA-LFB technique make it possible for food control, clinical diagnosis, and more. The proof-of-concept assay can be reconfigured to detect various target sequences by redesigning the specific MCDA primers. PMID:28138243

  18. Smartphone-Based Dual-Modality Imaging System for Quantitative Detection of Color or Fluorescent Lateral Flow Immunochromatographic Strips

    NASA Astrophysics Data System (ADS)

    Hou, Yafei; Wang, Kan; Xiao, Kun; Qin, Weijian; Lu, Wenting; Tao, Wei; Cui, Daxiang

    2017-04-01

    Nowadays, lateral flow immunochromatographic assays are increasingly popular as a diagnostic tool for point-of-care (POC) test based on their simplicity, specificity, and sensitivity. Hence, quantitative detection and pluralistic popular application are urgently needed in medical examination. In this study, a smartphone-based dual-modality imaging system was developed for quantitative detection of color or fluorescent lateral flow test strips, which can be operated anywhere at any time. In this system, the white and ultra-violet (UV) light of optical device was designed, which was tunable with different strips, and the Sobel operator algorithm was used in the software, which could enhance the identification ability to recognize the test area from the background boundary information. Moreover, this technology based on extraction of the components from RGB format (red, green, and blue) of color strips or only red format of the fluorescent strips can obviously improve the high-signal intensity and sensitivity. Fifty samples were used to evaluate the accuracy of this system, and the ideal detection limit was calculated separately from detection of human chorionic gonadotropin (HCG) and carcinoembryonic antigen (CEA). The results indicated that smartphone-controlled dual-modality imaging system could provide various POC diagnoses, which becomes a potential technology for developing the next-generation of portable system in the near future.

  19. In vivo lateral blood flow velocity measurement using speckle size estimation.

    PubMed

    Xu, Tiantian; Hozan, Mohsen; Bashford, Gregory R

    2014-05-01

    In previous studies, we proposed blood measurement using speckle size estimation, which estimates the lateral component of blood flow within a single image frame based on the observation that the speckle pattern corresponding to blood reflectors (typically red blood cells) stretches (i.e., is "smeared") if blood flow is in the same direction as the electronically controlled transducer line selection in a 2-D image. In this observational study, the clinical viability of ultrasound blood flow velocity measurement using speckle size estimation was investigated and compared with that of conventional spectral Doppler of carotid artery blood flow data collected from human patients in vivo. Ten patients (six male, four female) were recruited. Right carotid artery blood flow data were collected in an interleaved fashion (alternating Doppler and B-mode A-lines) with an Antares Ultrasound Imaging System and transferred to a PC via the Axius Ultrasound Research Interface. The scanning velocity was 77 cm/s, and a 4-s interval of flow data were collected from each subject to cover three to five complete cardiac cycles. Conventional spectral Doppler data were collected simultaneously to compare with estimates made by speckle size estimation. The results indicate that the peak systolic velocities measured with the two methods are comparable (within ±10%) if the scan velocity is greater than or equal to the flow velocity. When scan velocity is slower than peak systolic velocity, the speckle stretch method asymptotes to the scan velocity. Thus, the speckle stretch method is able to accurately measure pure lateral flow, which conventional Doppler cannot do. In addition, an initial comparison of the speckle size estimation and color Doppler methods with respect to computational complexity and data acquisition time indicated potential time savings in blood flow velocity estimation using speckle size estimation. Further studies are needed for calculation of the speckle stretch method

  20. Discharge flow of granular media from silos with a lateral orifice and injection of air

    NASA Astrophysics Data System (ADS)

    Aussillous, Pascale; Zhou, Yixian; Ruyer, Pierre; Lagrée, Pierre-Yves

    2017-06-01

    Few studies concern the prediction of the mass flow rate of a granular media discharged from a silo with a lateral orifice. However, this situation can have pratical interest considering a tank of granular material with a leak on its side. We studied experimentally the discharge of a vertical silo filled by spherical glass beads. We consider rectangular silos with a rectangular orifice. The impact of size, aspect ratio and position of the orifice and the effect of an additional air flow were studied. The measured parameters are the mass flow rate and the pressure along the silo, whereas the controlled parameters are the size of particles, and the flow rate of air. We identified two regimes of discharge according to the aspect ratio (of width to height) of the rectangular orifice. Increasing the air flow rate induces an increase of the granular media flow rate. Using a simple physical model to describe the grains and gas flow, we put in evidence the role played by the air pressure gradient at the outlet. Then we compared the experimental results with continuum Navier-Stokes simulations with the granular μ(I)-rheology. We showed that the continuum μ(I)-rheology describes well our discharge flow of granular media from silos, taking into account the effect of the position of the orifice as well as the coupling with the gas flow.

  1. Recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip for detection of Toxoplasma gondii in the environment.

    PubMed

    Wu, Y D; Xu, M J; Wang, Q Q; Zhou, C X; Wang, M; Zhu, X Q; Zhou, D H

    2017-08-30

    Toxoplasma gondii infects all warm-blooded vertebrates, resulting in a great threat to human health and significant economic loss to the livestock industry. Ingestion of infectious oocysts of T. gondii from the environment is the major source of transmission. Detection of T. gondii oocysts by existing methods is laborious, time-consuming and expensive. The objective of the present study was to develop a recombinase polymerase amplification (RPA) method combined with a lateral flow (LF) strip for detection of T. gondii oocysts in the soil and water. The DNA of T. gondii oocysts was amplified by a pair of specific primers based on the T. gondii B1 gene over 15min at a constant temperature ranging from 30°C to 45°C using RPA. The amplification product was visualized by the lateral flow (LF) strip within 5min using the specific probe added to the RPA reaction system. The sensitivity of the established assay was 10 times higher than that of nested PCR with a lower detection limit of 0.1 oocyst per reaction, and there was no cross-reactivity with other closely related protozoan species. Fifty environmental samples were further assessed for the detection validity of the LF-RPA assay (B1-LF-RPA) and compared with nested PCR based on the B1 gene sequence. The B1-LF-RPA and nested PCR both showed that 5 out of the 50 environmental samples were positive. The B1-LF-RPA method was also proven to be sufficiently tolerant of existing inhibitors in the environment. In addition, the advantages of simple operation, speediness and cost-effectiveness make B1-LF-RPA a promising molecular detection tool for T. gondii. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Chemical and isotopic characteristics of thermal fluids in the Long Valley caldera lateral flow system, California

    SciTech Connect

    Shevenell, L.; Goff, F.; Grigsby, C.O.; Janik, C.J.; Trujillo, P.E. Jr.; Counce, D.

    1987-01-01

    Chemical and isotopic data of thermal waters in Long Valley caldera have been used to identify both the origins and characteristics of the fluids and to evaluate mixing and boiling processes occurring within the lateral flow system of the caldera. Recharge to the Long Valley geothermal system occurs in the western part of the caldera with the water being heated at depth and flowing laterally eastward in the subsurface. The lateral flow system was recently intersected by the Shady Rest Continental Scientific Drilling Program (CSDP) corehole at 335 m (1100 ft) with fluids in this 202/sup 0/C zone being more concentrated than non-boiled fluids to the east. As the Na-K-HCO/sub 3/-Cl thermal fluids flow eastward, they are increasingly mixed with isotopically depleted, dilute groundwaters similar to cold waters east of Lake Crowley. Near surface boiling of Casa Diablo well fluids at 100/sup 0/C forms waters with the compositions of Colton and Casa Diablo hot springs. Waters to the east of the Casa Diablo area are mixtures of meteoric water and boiled thermal fluids with a composition close to that of Colton Hot Spring. There is no correlation between /sup 3/H and /sup 36/Cl in thermal fluids or between these components and conservative species, and it appears that cold fluids involved in mixing must be relatively old waters, low in both meteoric /sup 3/H and /sup 36/Cl.

  3. An experimental study of the lateral migration of a droplet in a creeping flow

    NASA Astrophysics Data System (ADS)

    Hiller, W.; Kowalewski, T. A.

    1986-01-01

    The distribution of droplets in a plane Hagen-Poiseuille flow of dilute suspensions has been measured by a special LDA technique. This method assumes a well defined relation between the velocity of the droplets and their lateral position in the channel. The measurements have shown that the droplet distribution is non-uniform and depends on the viscosity ratio between the droplets and the carrier liquid. The results have been compared with a theory by Chan and Leal describing the lateral migration of suspended droplets.

  4. Development and evaluation of flow through assay for detection of antibodies against porcine cysticercosis.

    PubMed

    Sreedevi, C; Hafeez, Md; Subramanyam, K V; Anand Kumar, P; Chengalva Rayulu, V

    2011-04-01

    A flow through assay (FTA) was developed on cellulose acetate membrane for the serodiagnosis of porcine cysticercosis using cyst fluid (CFA) and whole cyst antigens (WCA) of Taenia solium metacestode. The assay consisted of antigen of T. solium metacestode coated onto membrane, mounted on a flow-through test device to provide assay capture matrix. The optimum concentration of coating antigen was 250 ng. The protein A colloidal gold conjugate served as antigen-antibody detecting reagent. A total of 225 serum samples were tested using two antigens. Results were better with CFA (96.0% sensitivity; 96.0% specificity) compared to WCA (92.0% sensitivity; 96.0% specificity). The test was also compared with enzyme-linked immunosorbent assay. The ELISA showed 96 per cent sensitivity with both the antigens whereas; the specificity was 96 and 92 per cent with CFA and WCA respectively. The sensitivity and specificity of flow through assay agrees closely with those of the ELISA. The cross-reaction was observed in one out of eight hydatidosis positive pigs (12.5%) with CFA by both the assays. The highest diagnostic accuracy (96%) was obtained with CFA-FTA and CFA-ELISA. For its high sensitivity and sporadic cross-reactions, CFA-FTA appears to be suitable for practical use at field level without instrumentation.

  5. Occurrence and Relevance of Vertical and Lateral Preferential Flow Pathways across Land-uses and Landscapes

    NASA Astrophysics Data System (ADS)

    Weiler, M.

    2014-12-01

    There seems to be less and less doubt that preferential flow pathways in soils have a profound impact on hydrology by enhancing infiltration rates, reducing the filter function of soils or by enhancing fast subsurface flow in hillslopes. Soil hydrological or catchment models have been developed including the different kind of preferential flow pathways like earthworm like macropores (e.g. earthworm channels), pipes, roots, etc. and they have been successfully applied to make predictions at a range of spatial scales. One of the biggest issue using hydrological models including preferential flow routines is the parameterization. What are the landscape features influencing the occurrence and quantity of specific preferential flow features? Will certain macropres be more probable to occur under forest than under grassland soils? In this study, I will highlight several studies looking at the effect of land-use and landscape features on preferential flow properties and parameters. Several field experiments studied on the one side the properties among topographic locations or vegetation cover, but also at the hydrological functions and hence the relevance of preferential flow pathways. In the second part the soil hydrological model ROGER is introduced, which will further evaluate and predict the relevance of vertical and lateral preferential flow pathways at the plot, hillslope and catchment scale.

  6. Investigation of the effect of laterally ejected gas jets on flows

    NASA Astrophysics Data System (ADS)

    Patz, G.

    1986-02-01

    The effect of a gas jet, ejected through a hole in the wall, on the supersonic flow along that wall was simulated in a model measuring chamber in a shock tube. A head wave with an obliquely detaching shock was formed, accompanied by a pressure increase in front of the jet. This effect can be used for missile control. In the shock tube, the flow about a plane plate with a slit through which a gas jet was laterally ejected, was simulated. Flow visualization and wall pressure measurements in front of and behind the nozzle slit show the effect of the jet on the flow, and allow the analysis of the initial phase and the evaluation of the forces resulting from the wall pressure which add to the jet thrust.

  7. A flume experiment on the lateral distribution of driftwood according to piece characteristics and flow patterns

    NASA Astrophysics Data System (ADS)

    Ghaffarian, Hossein; Lopez, Diego; Piegay, Hervé; Riviere, Nicolas; Ruiz-Villanueva, Virginia

    2017-04-01

    The presence of driftwood is one of the influential components in river dynamics, especially in forested catchments and fluvial corridors. As they are transported by the flow, driftwoods can be trapped in critical sections of river (e.g. bridges, weirs or floodplain edges) and may increase the destructive effects of floods. Whereas many recent studies provided significant results on wood transport and jam formation, limited knowledge is available on the lateral distribution of wood in the river section during transport according to flow pattern. In this work we investigate the influence of flow and wood characteristics on the lateral distribution of wood pieces in a controlled laboratory experiment. The experiments are carried out in a straight rectangular (6 m long and 0.80 m wide) glass-walled flume, where different surface velocity profiles and flow conditions can be generated. Natural stems and rootstocks of different sizes (5 to 15 cm long and 0.5 to 1.5 cm in diameter) are dropped at the flume entrance and tracked with a camera as they are carried away by the flow. In addition to the flow characteristics, a special attention is given to the wood properties, in order to identify the influence of buoyancy (that can vary due to the immersed time as well as the type of wood) and geometry (e.g. stems, rootstocks or both) on the lateral distribution. An estimation of driftwood preferential paths and stream lines could provide useful insights into driftwood management and the prevention of the associated risks.

  8. Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira.

    PubMed

    Nurul Najian, A B; Engku Nur Syafirah, E A R; Ismail, Nabilah; Mohamed, Maizan; Yean, Chan Yean

    2016-01-15

    In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10(-1) genomic equivalent ml(-1). An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device.

  9. [Microleakage of root canal fillings with GuttaFlow and Resilon compared with lateral condensation].

    PubMed

    Kqiku, Lumnije; Miletic, Ivana; Gruber, Hans Jürgen; Anic, Ivica; Städtler, Peter

    2010-05-01

    Epiphany/Resilon and GuttaFlow are newly developed methods for obturation of the root canal system. Epiphany/Resilon is a thermoplastic, synthetic polymer-based root canal filling material which enables the bonding to the dentin root canal wall during root canal obturation. GuttaFlow is a cold flowable filling system for the obturation of root canals, combining sealer and gutta-percha in one product. The purpose of this study was to assess the leakage of the Epiphany/Resilon or GuttaFlow root canal filling compared with lateral condensation of gutta-percha. For this study were used 45 human extracted teeth, chemo mechanically prepared, divided into three groups and obturated with gutta-percha/AH Plus, Epiphany/Resilon and GuttaFlow. For dye penetration all teeth were centrifuged for three minutes at 30 g in 2% methylene blue and dissolved in 65% nitric acid for 3 days. The extracted methylene blue was determined with Photometer. Root Canal fillings with Epiphany/Resilon showed less dye penetration than lateral condensation of gutta-percha and GuttaFlow. Epiphany/Resilon is ideally suited as a root canal filling material.

  10. Image-based modelling of lateral magma flow: the Basement Sill, Antarctica

    NASA Astrophysics Data System (ADS)

    Petford, Nick; Mirhadizadeh, Seyed

    2017-05-01

    The McMurdo Dry Valleys magmatic system, Antarctica, provides a world-class example of pervasive lateral magma flow on a continental scale. The lowermost intrusion (Basement Sill) offers detailed sections through the now frozen particle microstructure of a congested magma slurry. We simulated the flow regime in two and three dimensions using numerical models built on a finite-element mesh derived from field data. The model captures the flow behaviour of the Basement Sill magma over a viscosity range of 1-104 Pa s where the higher end (greater than or equal to 102 Pa s) corresponds to a magmatic slurry with crystal fractions varying between 30 and 70%. A novel feature of the model is the discovery of transient, low viscosity (less than or equal to 50 Pa s) high Reynolds number eddies formed along undulating contacts at the floor and roof of the intrusion. Numerical tracing of particle orbits implies crystals trapped in eddies segregate according to their mass density. Recovered shear strain rates (10-3-10-5 s-1) at viscosities equating to high particle concentrations (around more than 40%) in the Sill interior point to shear-thinning as an explanation for some types of magmatic layering there. Model transport rates for the Sill magmas imply a maximum emplacement time of ca 105 years, consistent with geochemical evidence for long-range lateral flow. It is a theoretically possibility that fast-flowing magma on a continental scale will be susceptible to planetary-scale rotational forces.

  11. Mass flow rate of granular material in silos with lateral exit holes

    NASA Astrophysics Data System (ADS)

    Medina, Abraham; Serrano, Armando; Sanchez, Florencio

    2014-11-01

    In this work we have analyzed experimentally the mass flow rate, m', of the lateral outflow of cohesionless granular material through circular orifices of diameter D and rectangular and triangular slots of hydraulic diameter DH made in vertical walls of bins. Experiments were made in order to determine also the influence of the wall thickness of the bin, w. Geometrical and physical arguments, are given to get a general correlation for m' embracing both quantities, D (DH) and w. The angle of repose is also an important factor characterizing these flows.

  12. A highly sensitive europium nanoparticle-based lateral flow immunoassay for detection of chloramphenicol residue.

    PubMed

    Xia, Xiaohu; Xu, Ye; Ke, Rongqin; Zhang, Heng; Zou, Mingqiang; Yang, Wei; Li, Qingge

    2013-09-01

    A europium nanoparticle-based lateral flow immunoassay for highly sensitive detection of chloramphenicol residue was developed. The detection result could be either qualitatively resolved with naked eye or quantitatively analyzed with the assistance of a digital camera. In the qualitative mode, the limit of detection (LOD) was found to be 0.25 ng/mL. In the quantitative mode, the half-maximal inhibition concentration (IC50) was determined to be 0.45 ng/mL and the LOD can reach an ultralow level of 0.03 ng/mL, which is ~100 times lower than that of the conventional colloidal gold-based lateral flow immunoassay. Potential application of the established method was demonstrated by analyzing representative cow milk samples.

  13. Lateral flow immunoassay with the signal enhanced by gold nanoparticle aggregates based on polyamidoamine dendrimer.

    PubMed

    Shen, Guangyu; Xu, Hui; Gurung, Anant S; Yang, Yunhui; Liu, Guodong

    2013-01-01

    In order to amplify the signal in a gold nanoparticle-based lateral flow immunoassay, a simple and sensitive method utilizing gold nanoparticle aggregates as a colored reagent formed with a polyamidoamine dendrimer was developed. The results were compared with that achieved by employing the individual nanoparticles used in the conventional lateral flow immunoassay. Under the optimized experimental conditions, a detection limit of 0.1 ng mL⁻¹ for rabbit immunoglobulin G was achieved, which is almost 20-fold lower than that of the traditional method using individual gold nanoparticles. We believe that this simple, practical bioassay platform will be of interest for use in areas such as disease diagnostics, pathogen detection, and quality monitoring of food and water.

  14. Chemiluminescence lateral flow immunoassay based on Pt nanoparticle with peroxidase activity.

    PubMed

    Park, Jong-Min; Jung, Ha-Wook; Chang, Young Wook; Kim, Hyung-Seok; Kang, Min-Jung; Pyun, Jae-Chul

    2015-01-01

    A lateral flow immunoassay (LF-immunoassay) with an enhanced sensitivity and thermostability was developed by using Pt nanoparticles with a peroxidase activity. The Pt nanoparticles were synthesized by citrate reduction method, and the peroxidase activity of Pt nanoparticles was optimized by adjusting reaction conditions. The peroxidase activity was estimated by using Michaelis-Menten kinetics model with TMB as a chromogenic substrate. The kinetics parameters of KM and Vmax were calculated and compared with horseradish peroxidase (HRP). The thermal stability of the Pt nanoparticles was compared with horseradish peroxidase (HRP) according to the storage temperature and long-term storage period. The feasibility of lateral flow immunoassay with a chemiluminescent signal band was demonstrated by the detection of human chorionic gonadotropin (hCG) as a model analyte, and the sensitivity was determined to be improved by as much as 1000-fold compared to the conventional rapid test based on colored gold-colloids.

  15. In Planta Microsphere-Based Lateral Flow Leaf Biosensor in Maize.

    PubMed

    Wen, Jessica T; Castro, Carlos; Tsutsui, Hideaki

    2015-08-01

    Low-cost and quick detection of biotic stresses is critically important for protection of staple food crops such as maize in smallholder farms in developing countries, where access to improved seed varieties, fertilizers, and pesticides is limited due to financial and geographical reasons. Here, we report a new lateral flow detection technology directly integrated in a maize leaf, in which microspheres conjugated with analyte-specific capture antibodies are non-invasively injected. The antibody-conjugated microspheres capture and detect an analyte in a concentration-specific manner. In this study, we optimized microsphere size for effective infiltration and immobilization in the leaf, and further demonstrated detection of a fluorescent mock biomarker, fluorescein, in a live maize plant. This in planta lateral flow biosensor is the first of its kind and is expected to provide a low-cost and user-friendly detection method for biotic stresses in the field. © 2014 Society for Laboratory Automation and Screening.

  16. A new partitioning approach for nonlinear Muskingum flood routing models with lateral flow contribution

    NASA Astrophysics Data System (ADS)

    Ayvaz, M. Tamer; Gurarslan, Gurhan

    2017-10-01

    In this study, a new partitioning approach is proposed for nonlinear Muskingum flood routing models with lateral flow contribution. The proposed approach is used to partition the inflow hydrograph into different sub-regions so that each sub-region can have its own model parameters. The main advantage of the proposed approach is its independence from any kind of user intervention during generation of the sub-regions. This is a general and systematic solution approach and may be applied to all of the flood routing applications based on the Muskingum model. Applicability of the proposed approach is evaluated by solving four flood routing applications by considering lateral flow contribution. Identified results indicated that the proposed approach can be effectively used to improve the model identification performance more than 80% in the Muskingum flood routing models.

  17. Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum.

    PubMed

    Sun, Kui; Xing, Weiwei; Yu, Xinling; Fu, Wenliang; Wang, Yuanyuan; Zou, Minji; Luo, Zhihong; Xu, Donggang

    2016-08-31

    With the continuous decline in prevalence and intensity of Schistosoma japonicum infection in China, more accurate and sensitive methods suitable for field detection become much needed for schistosomiasis control. Here, a novel rapid and visual detection method based on the combination of recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) was developed to detect S. japonicum DNA in fecal samples. The LFD-RPA assay targeting SjR2 could detect 5 fg S. japonicum DNA, which was identical to qPCR and real-time RPA assay, and showed no cross-reaction with other parasites. The detection could be finished within 15-20 min at a wide temperature range (25-45 °C), and the results could be visualized by naked eye. The diagnostic validity of LFD-RPA assay was further assessed with 14 fecal samples of infected patients diagnosed by Kato-Katz method and 31 fecal samples of healthy persons, and compared with that of Enzyme-linked immunosorbent assay (ELSIA) and Indirect Hemagglutination Assay (IHA). The LFD-RPA assay showed 92.68 % sensitivity, 100 % specificity and excellent diagnostic agreement with the gold standard Kato-Katz test (k = 0.947, Z = 6.36, P < 0.001), whereas ELISA showed 85.71 % sensitivity, 93.55 % specificity, and substantial diagnostic agreement (k = 0.793, Z = 5.31, P < 0.001), and IHA showed 78.57 % sensitivity, 83.87 % specificity, and moderate diagnostic agreement (k = 0.600, Z = 4.05, P < 0.001), indicating that the LFD-RPA was much better than the traditional methods. The LFD-RPA assay established by us is a sensitive, specific, rapid and convenient method for the diagnosis of schistosomiasis, and shows a great potency in field application.

  18. Enhancing the lateral-flow immunoassay for detection of proteins using an aqueous two-phase micellar system.

    PubMed

    Mashayekhi, Foad; Le, Alexander M; Nafisi, Parsa M; Wu, Benjamin M; Kamei, Daniel T

    2012-10-01

    The lateral-flow (immuno)assay (LFA) has been widely investigated for the detection of molecular, macromolecular, and particle targets at the point-of-need due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements. However, for some analytes, such as certain proteins, the detection limit of LFA is inferior to lab-based assays, such as the enzyme-linked immunosorbent assay, and needs to be improved. One solution for improving the detection limit of LFA is to concentrate the target protein in a solution prior to the detection step. In this study, a novel approach was used in the context of an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 to concentrate a model protein, namely transferrin, prior to LFA. Proteins have been shown to partition, or distribute, fairly evenly between the two phases of an aqueous two-phase system, which in turn results in their limited concentration in one of the two phases. Therefore, larger colloidal gold particles decorated with antibodies for transferrin were used in the concentration step to bind to transferrin and aid its partitioning into the top, micelle-poor phase. By manipulating the volume ratio of the two coexisting micellar phases and combining the concentration step with LFA, the transferrin detection limit of LFA was improved by tenfold from 0.5 to 0.05 μg/mL in a predictive manner. In addition to enhancing the sensitivity of LFA, this universal concentration method could also be used to improve other detection assays.

  19. Numerical study on characteristics of supercavitating flow around the variable-lateral-force cavitator

    NASA Astrophysics Data System (ADS)

    Hu, Xiao; Gao, Ye; Shi, Xiao-tao

    2017-03-01

    A control scheme named the variable-lateral-force cavitator, which is focused on the control of lift force, drag force and lateral forces for underwater supercavity vehicles was proposed, and the supercavitating flow around the cavitator was investigated numerically using the mixture multiphase flow model. It is verified that the forces of pitching, yawing, drag and lift, as well as the supercavity size of the underwater vehicle can be effectively regulated through the movements of the control element of the variable-lateral-force cavitator in the radial and circumferential directions. In addition, if the control element on either side protrudes to a height of 5% of the diameter of the front cavitator, an amount of forces of pitching and yawing equivalent to 30% of the drag force will be produced, and the supercavity section appears concave inwards simultaneously. It is also found that both the drag force and lift force of the variable-lateral-force cavitator decline as the angle of attack increases.

  20. Influence of Lateral Flow on the Predisposition of Aspen Mortality during Drought

    NASA Astrophysics Data System (ADS)

    Tai, X.; Mackay, D. S.; Anderegg, W.; Sperry, J. S.

    2014-12-01

    Lateral subsurface flow can be critical to understanding the spatial soil moisture availability to plants, and when, where, and how drought are influencing individual plants. The concentration of intensive aspen damage in certain hillslopes with higher temperature and lower soil moisture suggests that soil augmentation/reduction from lateral redistribution could help explain the survivability of some aspen through its influence on soil water availability during drought. It remains unclear how lateral water redistribution helps to limit hydraulic impairment of aspen located in different topographic positions during a drought event. This study employed an integrated ecohydrology model, TREES, combining plant-water balance and canopy physiology, to examine the potential effects of lateral flow on hydraulic and metabolic performance of aspen, by exposing trees to a set of soil water conditions associated with different levels of water stress. Sap flux, soil moisture, meteorological and plant hydraulic data from aspen trees in Colorado that died (SAD) and those that lived were used to parameterize the model. Our goal was to quantify the extent to which lateral flow explained sudden aspen dieback. The results indicate that the predisposition of tree mortality is related to the level of soil water augmentation. A reduction of 30% soil water content could introduce 21.55% increase in the loss of hydraulic conductivity (PLC), 23.6% loss in canopy transpiration, 21.7% loss in GPP. It would also cause the frequency of greater than 50% PLC to increase from 42.1% of the time to 51% of the time, and the frequency of hitting the 88% PLC pressure to increase from 11% to 14% of the time. On the other hand, an augment of 30% soil water content could introduce 20.2% reduction in PLC, 16.4% gain in canopy transpiration, 16.5% gain in GPP. The frequency of greater than 50% PLC is reduced to 31% of the time and the frequency of hitting the 88% PLC pressure is reduced to 6% of the time

  1. Bead-Based Assays for Biodetection: From Flow-Cytometry to Microfluidics

    SciTech Connect

    Ozanich, Richard M.; Antolick, Kathryn C.; Bruckner-Lea, Cindy J.; Bunch, Kyle J.; Dockendorff, Brian P.; Grate, Jay W.; Nash, Michael A.; Tyler, Abby J.

    2009-05-04

    ABSTRACT The potential for the use of biological agents by terrorists is a real threat. Two approaches for detection of biological species will be described: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. The methods and automated fluidic systems used for trapping functionalized microbeads will be described. This approach allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive assays. The automated fluidic approach resulted in up to five-fold improvements in assay sensitivity/speed as compared to identical assays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based assays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (> 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100’s of picomolar range (10’s of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach. Video taping magnetic nanoparticle capture and release was used to improve understanding of the process and revealed interesting behavior.

  2. Point-of-care vertical flow allergen microarray assay: proof of concept.

    PubMed

    Chinnasamy, Thiruppathiraja; Segerink, Loes I; Nystrand, Mats; Gantelius, Jesper; Andersson Svahn, Helene

    2014-09-01

    Sophisticated equipment, lengthy protocols, and skilled operators are required to perform protein microarray-based affinity assays. Consequently, novel tools are needed to bring biomarkers and biomarker panels into clinical use in different settings. Here, we describe a novel paper-based vertical flow microarray (VFM) system with a multiplexing capacity of at least 1480 microspot binding sites, colorimetric readout, high sensitivity, and assay time of <10 min before imaging and data analysis. Affinity binders were deposited on nitrocellulose membranes by conventional microarray printing. Buffers and reagents were applied vertically by use of a flow controlled syringe pump. As a clinical model system, we analyzed 31 precharacterized human serum samples using the array system with 10 allergen components to detect specific IgE reactivities. We detected bound analytes using gold nanoparticle conjugates with assay time of ≤10 min. Microarray images were captured by a consumer-grade flatbed scanner. A sensitivity of 1 ng/mL was demonstrated with the VFM assay with colorimetric readout. The reproducibility (CV) of the system was <14%. The observed concordance with a clinical assay, ImmunoCAP, was R(2) = 0.89 (n = 31). In this proof-of-concept study, we demonstrated that the VFM assay, which combines features from protein microarrays and paper-based colorimetric systems, could offer an interesting alternative for future highly multiplexed affinity point-of-care testing. © 2014 American Association for Clinical Chemistry.

  3. Micro segmented-flow in biochemical and cell-based assays.

    PubMed

    Clausell-Tormos, Jenifer; Merten, Christoph A

    2012-01-01

    Micro-segmented flow (e.g. in microfluidic channels, capillaries or a length of tubing) has become a promising technique in modern biology. Compared to conventional formats such as microtiter plates, sample volumes can be reduced about 1000-fold, thus allowing a massive reduction of assay costs and the use of samples available in low quantities, only (e.g. primary cells). Furthermore, assays can be highly parallelized and performed at superb spatio-temporal resolution. Here, we review the state-of-the-art in micro-segmented flow as applied in biochemical, cell- and multicellular organisms-based assays. We discuss likely future applications such as single cell / single organism proteomics and transcriptomics and point out the specific advantages and limitations compared to emulsion-based (droplet-based) approaches.

  4. A rapid lateral flow immunoassay for the detection of tyrosine phosphatase-like protein IA-2 autoantibodies in human serum.

    PubMed

    Kikkas, Ingrid; Mallone, Roberto; Larger, Etienne; Volland, Hervé; Morel, Nathalie

    2014-01-01

    Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity.

  5. Rapid and visual detection of Mycobacterium tuberculosis complex using recombinase polymerase amplification combined with lateral flow strips.

    PubMed

    Ma, Qinglin; Liu, Houming; Ye, Feidi; Xiang, Guangxin; Shan, Wanshui; Xing, Wanli

    2017-08-26

    To definitively diagnose active pulmonary Tuberculosis (TB), Mycobacterium tuberculosis complex (MTBC) bacilli must be identified within clinical specimens from patients. In this study, we introduced a rapid and visual detection method of MTBC using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strips. The LF-RPA assay, read results with naked eyes, could detect as few as 5 genome copies of M. tuberculosis H37Rv (ATCC 27294) per reaction and had no cross-reactions with other control bacteria even using excessive amount of template DNA. The system could work well at a broad range of temperature 25-45 °C and reach detectable level even within 5 min. When testing a total of 137 clinical specimens, the sensitivity and specificity of the LF-RPA assay were 100% (95% CI: 95.94%-100%) and 97.92% (95% CI: 88.93%-99.95%), respectively, compared to culture identification method. Therefore, the LF-RPA system we have demonstrated is a rapid, simple, robust method for MTBC detection which, subject to the availability of a suitable sample extraction method, has the potentiality to diagnose TB at the point-of-care testing. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Tracking the amphibian pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans using a highly specific monoclonal antibody and lateral-flow technology.

    PubMed

    Dillon, Michael J; Bowkett, Andrew E; Bungard, Michael J; Beckman, Katie M; O'Brien, Michelle F; Bates, Kieran; Fisher, Matthew C; Stevens, Jamie R; Thornton, Christopher R

    2017-03-01

    The fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a lethal epizootic disease of amphibians. Rapid identification of the pathogen and biosecurity is essential to prevent its spread, but current laboratory-based tests are time-consuming and require specialist equipment. Here, we describe the generation of an IgM monoclonal antibody (mAb), 5C4, specific to Bd as well as the related salamander and newt pathogen Batrachochytrium salamandrivorans (Bsal). The mAb, which binds to a glycoprotein antigen present on the surface of zoospores, sporangia and zoosporangia, was used to develop a lateral-flow assay (LFA) for rapid (15 min) detection of the pathogens. The LFA detects known lineages of Bd and also Bsal, as well as the closely related fungus Homolaphlyctis polyrhiza, but does not detect a wide range of related and unrelated fungi and oomycetes likely to be present in amphibian habitats. When combined with a simple swabbing procedure, the LFA was 100% accurate in detecting the water-soluble 5C4 antigen present in skin, foot and pelvic samples from frogs, newts and salamanders naturally infected with Bd or Bsal. Our results demonstrate the potential of the portable LFA as a rapid qualitative assay for tracking these amphibian pathogens and as an adjunct test to nucleic acid-based detection methods.

  7. A Rapid Lateral Flow Immunoassay for the Detection of Tyrosine Phosphatase-Like Protein IA-2 Autoantibodies in Human Serum

    PubMed Central

    Kikkas, Ingrid; Mallone, Roberto; Larger, Etienne; Volland, Hervé; Morel, Nathalie

    2014-01-01

    Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity. PMID:25047039

  8. Design and application of a fish-shaped lateral line probe for flow measurement.

    PubMed

    Tuhtan, J A; Fuentes-Pérez, J F; Strokina, N; Toming, G; Musall, M; Noack, M; Kämäräinen, J K; Kruusmaa, M

    2016-04-01

    We introduce the lateral line probe (LLP) as a measurement device for natural flows. Hydraulic surveys in rivers and hydraulic structures are currently based on time-averaged velocity measurements using propellers or acoustic Doppler devices. The long-term goal is thus to develop a sensor system, which includes spatial gradients of the flow field along a fish-shaped sensor body. Interpreting the biological relevance of a collection of point velocity measurements is complicated by the fact that fish and other aquatic vertebrates experience the flow field through highly dynamic fluid-body interactions. To collect body-centric flow data, a bioinspired fish-shaped probe is equipped with a lateral line pressure sensing array, which can be applied both in the laboratory and in the field. Our objective is to introduce a new type of measurement device for body-centric data and compare its output to estimates of conventional point-based technologies. We first provide the calibration workflow for laboratory investigations. We then provide a review of two velocity estimation workflows, independent of calibration. Such workflows are required as existing field investigations consist of measurements in environments where calibration is not feasible. The mean difference for uncalibrated LLP velocity estimates from 0 to 50 cm/s under in a closed flow tunnel and open channel flume was within 4 cm/s when compared to conventional measurement techniques. Finally, spatial flow maps in a scale vertical slot fishway are compared for the LLP, direct measurements, and 3D numerical models where it was found that the LLP provided a slight overestimation of the current velocity in the jet and underestimated the velocity in the recirculation zone.

  9. Design and application of a fish-shaped lateral line probe for flow measurement

    NASA Astrophysics Data System (ADS)

    Tuhtan, J. A.; Fuentes-Pérez, J. F.; Strokina, N.; Toming, G.; Musall, M.; Noack, M.; Kämäräinen, J. K.; Kruusmaa, M.

    2016-04-01

    We introduce the lateral line probe (LLP) as a measurement device for natural flows. Hydraulic surveys in rivers and hydraulic structures are currently based on time-averaged velocity measurements using propellers or acoustic Doppler devices. The long-term goal is thus to develop a sensor system, which includes spatial gradients of the flow field along a fish-shaped sensor body. Interpreting the biological relevance of a collection of point velocity measurements is complicated by the fact that fish and other aquatic vertebrates experience the flow field through highly dynamic fluid-body interactions. To collect body-centric flow data, a bioinspired fish-shaped probe is equipped with a lateral line pressure sensing array, which can be applied both in the laboratory and in the field. Our objective is to introduce a new type of measurement device for body-centric data and compare its output to estimates of conventional point-based technologies. We first provide the calibration workflow for laboratory investigations. We then provide a review of two velocity estimation workflows, independent of calibration. Such workflows are required as existing field investigations consist of measurements in environments where calibration is not feasible. The mean difference for uncalibrated LLP velocity estimates from 0 to 50 cm/s under in a closed flow tunnel and open channel flume was within 4 cm/s when compared to conventional measurement techniques. Finally, spatial flow maps in a scale vertical slot fishway are compared for the LLP, direct measurements, and 3D numerical models where it was found that the LLP provided a slight overestimation of the current velocity in the jet and underestimated the velocity in the recirculation zone.

  10. Estimation of lateral water flow and bromide transport in a subsurface seepage irrigation system.

    PubMed

    Ouyang, Y

    2009-01-01

    Subsurface seepage irrigation is a common method used by growers in the Tri-County Agricultural Area (TCAA), Florida, USA, owing to its cost-effectiveness and low maintenance requirements. This study investigated the lateral flow of the perched water and the lateral transport of bromide (Br-) in this irrigation system in the TCAA and estimated the potential discharge of Br- into the drainage canals at the edges of the field, using the Visual MODFLOW/ MT3DMS models in conjunction with field experiments. Simulations showed that the perched water flowed from the northeast to the southwest of the field. Migration of the Br- plume from the source areas toward the canals was very slow and varied depending on the selection of the outer Br- concentration contour levels. However, the lateral transport of Br- from the perched water into the canals occurred after about 61 days. The simulations further revealed that the rate of perched water Br- discharge into the canals averaged 8.6 g day(-1) during a 30-day discharge period (from 61 to 91 days). This rate is very important for estimating Br- discharge into the canals and could also provide useful information for evaluating dissolved nutrient discharge into canals from the subsurface seepage irrigation system.

  11. Laterality of Stance during Optic Flow Stimulation in Male and Female Young Adults

    PubMed Central

    Persiani, Michela; Piras, Alessandro; Squatrito, Salvatore; Raffi, Milena

    2015-01-01

    During self-motion, the spatial and temporal properties of the optic flow input directly influence the body sway. Men and women have anatomical and biomechanical differences that influence the postural control during visual stimulation. Given that recent findings suggest a peculiar role of each leg in the postural control of the two genders, we investigated whether the body sway during optic flow perturbances is lateralized and whether anteroposterior and mediolateral components of specific center of pressure (COP) parameters of the right and left legs differ, reexamining a previous experiment (Raffi et al. (2014)) performed with two, side-by-side, force plates. Experiments were performed on 24 right-handed and right-footed young subjects. We analyzed five measures related to the COP of each foot and global data: anteroposterior and mediolateral range of oscillation, anteroposterior and mediolateral COP velocity, and sway area. Results showed that men consistently had larger COP parameters than women. The values of the COP parameters were correlated between the two feet only in the mediolateral axis of women. These findings suggest that optic flow stimulation causes asymmetry in postural balance and different lateralization of postural controls in men and women. PMID:26539509

  12. Image-based modelling of lateral magma flow: the Basement Sill, Antarctica

    PubMed Central

    Mirhadizadeh, Seyed

    2017-01-01

    The McMurdo Dry Valleys magmatic system, Antarctica, provides a world-class example of pervasive lateral magma flow on a continental scale. The lowermost intrusion (Basement Sill) offers detailed sections through the now frozen particle microstructure of a congested magma slurry. We simulated the flow regime in two and three dimensions using numerical models built on a finite-element mesh derived from field data. The model captures the flow behaviour of the Basement Sill magma over a viscosity range of 1–104 Pa s where the higher end (greater than or equal to 102 Pa s) corresponds to a magmatic slurry with crystal fractions varying between 30 and 70%. A novel feature of the model is the discovery of transient, low viscosity (less than or equal to 50 Pa s) high Reynolds number eddies formed along undulating contacts at the floor and roof of the intrusion. Numerical tracing of particle orbits implies crystals trapped in eddies segregate according to their mass density. Recovered shear strain rates (10−3–10−5 s−1) at viscosities equating to high particle concentrations (around more than 40%) in the Sill interior point to shear-thinning as an explanation for some types of magmatic layering there. Model transport rates for the Sill magmas imply a maximum emplacement time of ca 105 years, consistent with geochemical evidence for long-range lateral flow. It is a theoretically possibility that fast-flowing magma on a continental scale will be susceptible to planetary-scale rotational forces. PMID:28573002

  13. Honeybees' Speed Depends on Dorsal as Well as Lateral, Ventral and Frontal Optic Flows

    PubMed Central

    Portelli, Geoffrey; Ruffier, Franck; Roubieu, Frédéric L.; Franceschini, Nicolas

    2011-01-01

    Flying insects use the optic flow to navigate safely in unfamiliar environments, especially by adjusting their speed and their clearance from surrounding objects. It has not yet been established, however, which specific parts of the optical flow field insects use to control their speed. With a view to answering this question, freely flying honeybees were trained to fly along a specially designed tunnel including two successive tapering parts: the first part was tapered in the vertical plane and the second one, in the horizontal plane. The honeybees were found to adjust their speed on the basis of the optic flow they perceived not only in the lateral and ventral parts of their visual field, but also in the dorsal part. More specifically, the honeybees' speed varied monotonically, depending on the minimum cross-section of the tunnel, regardless of whether the narrowing occurred in the horizontal or vertical plane. The honeybees' speed decreased or increased whenever the minimum cross-section decreased or increased. In other words, the larger sum of the two opposite optic flows in the horizontal and vertical planes was kept practically constant thanks to the speed control performed by the honeybees upon encountering a narrowing of the tunnel. The previously described ALIS (“AutopiLot using an Insect-based vision System”) model nicely matches the present behavioral findings. The ALIS model is based on a feedback control scheme that explains how honeybees may keep their speed proportional to the minimum local cross-section of a tunnel, based solely on optic flow processing, without any need for speedometers or rangefinders. The present behavioral findings suggest how flying insects may succeed in adjusting their speed in their complex foraging environments, while at the same time adjusting their distance not only from lateral and ventral objects but also from those located in their dorsal visual field. PMID:21589861

  14. Honeybees' speed depends on dorsal as well as lateral, ventral and frontal optic flows.

    PubMed

    Portelli, Geoffrey; Ruffier, Franck; Roubieu, Frédéric L; Franceschini, Nicolas

    2011-05-12

    Flying insects use the optic flow to navigate safely in unfamiliar environments, especially by adjusting their speed and their clearance from surrounding objects. It has not yet been established, however, which specific parts of the optical flow field insects use to control their speed. With a view to answering this question, freely flying honeybees were trained to fly along a specially designed tunnel including two successive tapering parts: the first part was tapered in the vertical plane and the second one, in the horizontal plane. The honeybees were found to adjust their speed on the basis of the optic flow they perceived not only in the lateral and ventral parts of their visual field, but also in the dorsal part. More specifically, the honeybees' speed varied monotonically, depending on the minimum cross-section of the tunnel, regardless of whether the narrowing occurred in the horizontal or vertical plane. The honeybees' speed decreased or increased whenever the minimum cross-section decreased or increased. In other words, the larger sum of the two opposite optic flows in the horizontal and vertical planes was kept practically constant thanks to the speed control performed by the honeybees upon encountering a narrowing of the tunnel. The previously described ALIS ("AutopiLot using an Insect-based vision System") model nicely matches the present behavioral findings. The ALIS model is based on a feedback control scheme that explains how honeybees may keep their speed proportional to the minimum local cross-section of a tunnel, based solely on optic flow processing, without any need for speedometers or rangefinders. The present behavioral findings suggest how flying insects may succeed in adjusting their speed in their complex foraging environments, while at the same time adjusting their distance not only from lateral and ventral objects but also from those located in their dorsal visual field.

  15. Image-based modelling of lateral magma flow: the Basement Sill, Antarctica.

    PubMed

    Petford, Nick; Mirhadizadeh, Seyed

    2017-05-01

    The McMurdo Dry Valleys magmatic system, Antarctica, provides a world-class example of pervasive lateral magma flow on a continental scale. The lowermost intrusion (Basement Sill) offers detailed sections through the now frozen particle microstructure of a congested magma slurry. We simulated the flow regime in two and three dimensions using numerical models built on a finite-element mesh derived from field data. The model captures the flow behaviour of the Basement Sill magma over a viscosity range of 1-10(4) Pa s where the higher end (greater than or equal to 10(2) Pa s) corresponds to a magmatic slurry with crystal fractions varying between 30 and 70%. A novel feature of the model is the discovery of transient, low viscosity (less than or equal to 50 Pa s) high Reynolds number eddies formed along undulating contacts at the floor and roof of the intrusion. Numerical tracing of particle orbits implies crystals trapped in eddies segregate according to their mass density. Recovered shear strain rates (10(-3)-10(-5) s(-1)) at viscosities equating to high particle concentrations (around more than 40%) in the Sill interior point to shear-thinning as an explanation for some types of magmatic layering there. Model transport rates for the Sill magmas imply a maximum emplacement time of ca 10(5) years, consistent with geochemical evidence for long-range lateral flow. It is a theoretically possibility that fast-flowing magma on a continental scale will be susceptible to planetary-scale rotational forces.

  16. Rapid detection of methicillin-resistant Staphylococcus aureus in pork using a nucleic acid-based lateral flow immunoassay.

    PubMed

    Zhang, Hongwei; Ma, Luyao; Ma, Lina; Hua, Marti Z; Wang, Shuo; Lu, Xiaonan

    2017-02-21

    Methicillin-resistant Staphylococcus aureus (MRSA) is considered as one of the leading causes of food poisonings worldwide. Due to the high prevalence and extensive challenges in clinical treatment, a rapid and accurate detection method is required to differentiate MRSA from other S. aureus isolated from foods. Since the methicillin resistance of S. aureus is due to the acquisition of the mecA gene from staphylococcal chromosome cassette, the presence of the mecA gene is interpreted as a marker for the identification of MRSA. In this study, a low-cost lateral flow immunoassay (LFI) strip was used to detect the mecA amplicons subsequent to polymerase chain reaction (PCR). The specificity of this PCR-LFI assay was tested between MRSA and methicillin-susceptive S. aureus. Both the test line and control line were shown up on the LFI strip for MRSA, whereas only the control line developed for methicillin-susceptive S. aureus. The detection limit of PCR-LFI assay was 20fg for genomic DNA (100 times more sensitive than gel electrophoresis) and 2×10(0)CFU per 100g of pork products after enrichment at 37°C for 48h. The total detection time of using LFI was 3min, which was faster than the conventional electrophoresis (~45min). With the performance of PCR-LFI, 7 out of 42 S. aureus isolates were identified to be MRSA from imported pork products, which was consistent to the standardized minimum inhibitory concentration assay. This mecA-based PCR-LFI strip can be used for rapid and accurate detection of MRSA isolated from commercial pork products. Copyright © 2016. Published by Elsevier B.V.

  17. Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick

    PubMed Central

    2014-01-01

    Background Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions. Results A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids. Conclusions Our results indicate that the methodology here reported constitutes a step forward in the development

  18. Evaluation of the Sepsis Flow Chip assay for the diagnosis of blood infections

    PubMed Central

    Galiana, Antonio; Coy, Javier; Gimeno, Adelina; Guzman, Noemi Marco; Rosales, Francisco; Merino, Esperanza; Royo, Gloria; Rodríguez, Juan Carlos

    2017-01-01

    Background Blood infections are serious complex conditions that generally require rapid diagnosis and treatment. The big challenge is to reduce the time necessary to make a diagnosis with current clinical microbiological methods so as to improve the treatment given to patients. Methods In this study, we assess for the first time the Sepsis Flow Chip assay, which is a novel diagnostic assay for simultaneous rapid-detection of the vast majority of bloodstream pathogens, including Gram-positive and Gram-negative bacteria and fungi, in the same assay, and for the detection of most common antibiotic resistance genes. The SFC assay is based on multiplex PCR and low density DNA arrays. Results Positive blood cultures from 202 consecutive bacteremia patients were analyzed by SFC assay and the results were compared with the results obtained by the gold standard methodology used in clinical microbiology diagnostic laboratories (EUCAST guidelines). SFC assay overall sensitivity and specificity for bacterial identification were 93.3% and 100% respectively and sensitivity and specificity for the identification of antibiotic genetic resistance determinants were 93.6% and 100% respectively. Conclusions This is the first evaluation of SFC assay in clinical samples. This new method appears to be very promising by combining the high number of distinct pathogens and genetic resistance determinants identified in a single assay. Further investigations should be done to evaluate the usefulness of this assay in combination with clinical multidisciplinary groups (stewardship), in order for the results to be applied appropriately to the management of patients`infectious processes. PMID:28542614

  19. Point-of-care coagulation monitoring: first clinical experience using a paper-based lateral flow diagnostic device.

    PubMed

    Hegener, Michael A; Li, Hua; Han, Daewoo; Steckl, Andrew J; Pauletti, Giovanni M

    2017-09-01

    Vitamin K antagonists such as warfarin are the most widely used class of oral anticoagulants. Due to a narrow therapeutic window, patients on warfarin require regular monitoring. Self-testing using point-of-care (POC) diagnostic devices is available, but cost makes this monitoring method beyond reach for many. The main objective of this research was to assess the clinical utility of a low-cost, paper-based lateral flow POC diagnostic device developed for anticoagulation monitoring without the need for a separate electronic reader. Custom-fabricated lateral flow assay (LFA) test strips comprised of a glass fiber sample pad, a nitrocellulose analytical membrane, a cellulose wicking pad, and a plastic backing card were assembled in a plastic cassette. Healthy volunteers and patients on warfarin therapy were recruited for this prospective study. For each participant, a whole blood sample was collected via fingerstick to determine: (1) international normalized ratio (INR) using the CoaguChek® XS coagulometer, (2) hematocrit by centrifugation, and (3) red blood cell (RBC) travel distance on the experimental LFA device after 240 s using digital image analysis. RBC travel distance measured on the LFA device using blood samples obtained from warfarin patients positively correlated with increasing INR value and the LFA device had the capability to statistically distinguish between healthy volunteer INR values and those for patients groups with INR ≥ 2.6. From these data, it is predicted that this low-cost, paper-based LFA device can have clinical utility for identifying anticoagulated patients taking vitamin K antagonists who are outside of the desired therapeutic efficacy window.

  20. Development of a paper-based lateral flow immunoassay for simultaneous detection of lipopolysaccharides of Salmonella serovars.

    PubMed

    Schenk, Florian; Weber, Patricia; Vogler, Julian; Hecht, Lars; Dietzel, Andreas; Gauglitz, Günter

    2017-10-02

    Lateral flow type detection is becoming interesting not only in regions with a poor medical infrastructure but also for practitioners in day-to-day clinical work or for veterinary control in case of possible epidemics. In this work, we describe the first steps of development of a multi-channel strip with potential internal calibration of multiparametric and colorimetric lateral flow assays for the simultaneous detection of the lipopolysaccharides (LPS) of Salmonella typhimurium (S. typhimurium) and Salmonella enteritidis (S. enteritidis). We structured four channels in the nitrocellulose membrane with a Yb:KGW solid-state femtosecond laser ("cold" ablation process) to form distinct tracks of porous material and used gold nanoparticles for the labeling of the antibodies. In addition, calibration curves of the spot intensities of both serovars are presented, and it was shown that no cross reactivity between the different capture antibodies and LPS occurred. Finally, we detected LPS of both Salmonella serovars simultaneously. The color changes (spot intensities of the reaction zones) were evaluated using the open-source image-processing program ImageJ. Graphical abstract Multiparametric testing, strip A was tested with LPS S. enteritidis ( c=0.01 g/L) and LPS S.typhimurium ( c=0.0001 g/L), strip B with LPS S. enteritidis ( c=0.001 g/L) and LPS S. typhimurium ( c=0.001g/L) and strip C with LPS S. enteritidis (c=0.0001 g/L) and LPS S. typhimurium ( c=0.01 g/L), and read-out.

  1. Lateral and subsurface flows impact arctic coastal plain lake water budgets

    USGS Publications Warehouse

    Koch, Joshua C.

    2016-01-01

    Arctic thaw lakes are an important source of water for aquatic ecosystems, wildlife, and humans. Many recent studies have observed changes in Arctic surface waters related to climate warming and permafrost thaw; however, explaining the trends and predicting future responses to warming is difficult without a stronger fundamental understanding of Arctic lake water budgets. By measuring and simulating surface and subsurface hydrologic fluxes, this work quantified the water budgets of three lakes with varying levels of seasonal drainage, and tested the hypothesis that lateral and subsurface flows are a major component of the post-snowmelt water budgets. A water budget focused only on post-snowmelt surface water fluxes (stream discharge, precipitation, and evaporation) could not close the budget for two of three lakes, even when uncertainty in input parameters was rigorously considered using a Monte Carlo approach. The water budgets indicated large, positive residuals, consistent with up to 70% of mid-summer inflows entering lakes from lateral fluxes. Lateral inflows and outflows were simulated based on three processes; supra-permafrost subsurface inflows from basin-edge polygonal ground, and exchange between seasonally drained lakes and their drained margins through runoff and evapotranspiration. Measurements and simulations indicate that rapid subsurface flow through highly conductive flowpaths in the polygonal ground can explain the majority of the inflow. Drained lakes were hydrologically connected to marshy areas on the lake margins, receiving water from runoff following precipitation and losing up to 38% of lake efflux to drained margin evapotranspiration. Lateral fluxes can be a major part of Arctic thaw lake water budgets and a major control on summertime lake water levels. Incorporating these dynamics into models will improve our ability to predict lake volume changes, solute fluxes, and habitat availability in the changing Arctic.

  2. Lateral Flow across Multi-parallel Columns and Their Implications on Large-Scale Evapotranspiration Modeling

    NASA Astrophysics Data System (ADS)

    Sun, D.; Zhu, J.

    2011-12-01

    Evapotranspiration (ET, i.e., evaporation and plant transpiration) is an important component in hydrological cycle, especially for semi-arid and arid environments. The representation of soil hydrologic processes and parameters at scales different from the scale at which observations and measurements are made is a major challenge. Large scale evapotranspiration is often quantified through simulation of multiple columns of independent one-dimensional local scale vertical flow. The soil column used in each simulation is considered homogeneous for the purpose of modeling over short depths. A main limitation is that this purely one-dimensional modeling approach does not consider interaction between columns. Lateral flows might be significant for long and narrow tubes and heterogeneous hydraulic properties and plant characteristics. This study is to quantify the significance of lateral flow and examine whether using this one-dimensional modeling approach may introduce unacceptable errors for large scale evapotranspiration simulations using a three-dimensional modeling appraoch. Instead of using convenient parallel column models of independent hydrologic processes, this study simulates three-dimensional transpiration and evaporation in multiple columns which allow lateral interactions. Specifically, we examined the impact of plant rooting density, depth, pattern and other characteristics on the accuracy of this commonly used one-dimensional approximation of hydrological processes. In addition, the influence of spatial variability of hydraulic properties on the validity of the one-dimensional approach and the difference of wetting and drying processes are discussed. The results provide applicable guidance for applications of one-dimensional approach to simulate large scale evapotranspiration in a heterogeneous landscape.

  3. Photoluminescent lateral-flow immunoassay revealed by graphene oxide: highly sensitive paper-based pathogen detection.

    PubMed

    Morales-Narváez, Eden; Naghdi, Tina; Zor, Erhan; Merkoçi, Arben

    2015-08-18

    A paper-based lateral flow immunoassay for pathogen detection that avoids the use of secondary antibodies and is revealed by the photoluminescence quenching ability of graphene oxide is reported. Escherichia coli has been selected as a model pathogen. The proposed device is able to display a highly specific and sensitive performance with a limit of detection of 10 CFU mL(-1) in standard buffer and 100 CFU mL(-1) in bottled water and milk. This low-cost disposable and easy-to-use device will prove valuable for portable and automated diagnostics applications.

  4. A glass fiber sheet-based electroosmotic lateral flow immunoassay for point-of-care testing.

    PubMed

    Oyama, Yuriko; Osaki, Toshihisa; Kamiya, Koki; Kawano, Ryuji; Honjoh, Tsutomu; Shibata, Haruki; Ide, Toru; Takeuchi, Shoji

    2012-12-21

    We have developed a quantitative immunoassay chip targeting point-of-care testing. To implement a lateral flow immunoassay, a glass fiber sheet was chosen as the material for the microfluidic channel in which the negative charge on the fiber surfaces efficiently generates the electroosmotic flow (EOF). The EOF, in turn, allows controllable bound/free separation of antigen/antibody interactions on the chip and enables precise determination of the antigen concentration. In addition, the defined size of the porous matrix was suitable for the filtration of undesired large particles. We confirmed the linear relationship between the concentration of analyte and the resulting fluorescence intensity from the immunoassay of two model analytes, C-reactive protein (CRP) and insulin, demonstrating that analyte concentration was quantitatively determined within the developed chip in 20 min. The limits of detection were 8.5 ng mL(-1) and 17 ng mL(-1) for CRP and insulin, respectively.

  5. Imaging lateral groundwater flow in the shallow subsurface using stochastic temperature fields

    NASA Astrophysics Data System (ADS)

    Fairley, Jerry P.; Nicholson, Kirsten N.

    2006-04-01

    Although temperature has often been used as an indication of vertical groundwater movement, its usefulness for identifying horizontal fluid flow has been limited by the difficulty of obtaining sufficient data to draw defensible conclusions. Here we use stochastic simulation to develop a high-resolution image of fluid temperatures in the shallow subsurface at Borax Lake, Oregon. The temperature field inferred from the geostatistical simulations clearly shows geothermal fluids discharging from a group of fault-controlled hydrothermal springs, moving laterally through the subsurface, and mixing with shallow subsurface flow originating from nearby Borax Lake. This interpretation of the data is supported by independent geochemical and isotopic evidence, which show a simple mixing trend between Borax Lake water and discharge from the thermal springs. It is generally agreed that stochastic simulation can be a useful tool for extracting information from complex and/or noisy data and, although not appropriate in all situations, geostatistical analysis may provide good definition of flow paths in the shallow subsurface. Although stochastic imaging techniques are well known in problems involving transport of species, e.g. delineation of contaminant plumes from soil gas survey data, we are unaware of previous applications to the transport of thermal energy for the purpose of inferring shallow groundwater flow.

  6. Touch at a distance sensing: lateral-line inspired MEMS flow sensors.

    PubMed

    Prakash Kottapalli, Ajay Giri; Asadnia, Mohsen; Miao, Jianmin; Triantafyllou, Michael

    2014-11-07

    Evolution bestowed the blind cavefish with a resourcefully designed lateral-line of sensors that play an essential role in many important tasks including object detection and avoidance, energy-efficient maneuvering, rheotaxis etc. Biologists identified the two types of vital sensors on the fish bodies called the superficial neuromasts and the canal neuromasts that are responsible for flow sensing and pressure-gradient sensing, respectively. In this work, we present the design, fabrication and experimental characterization of biomimetic polymer artificial superficial neuromast micro-sensor arrays. These biomimetic micro-sensors demonstrated a high sensitivity of 0.9 mV/(m s(-1)) and 0.022 V/(m s(-1)) and threshold velocity detection limits of 0.1 m s(-1) and 0.015 m s(-1) in determining air and water flows respectively. Experimental results demonstrate that the biological canal inspired polymer encapsulation on the array of artificial superficial neuromast sensors is capable of filtering steady-state flows that could otherwise significantly mask the relevant oscillatory flow signals of high importance.

  7. Flow-induced differential lateral migration of deformable particles by inner/outer viscosity ratio

    NASA Astrophysics Data System (ADS)

    Chen, Yeng-Long; Wang, Shih-Hao; Yeh, Wei-Ting

    2016-11-01

    We investigate the practicality of flow-driven separation of deformable particles (DP) such as cells, droplets, and capsules in microfluidic flow. We use lattice Boltzmann-immersed boundary method to model the hydrodynamic coupling between DP and the fluid. We find that whether a DP migrates towards the wall or to the center at steady state depends strongly on particle Reynolds number Re, capillary numbers Ca, and viscosity ratio λ. The lateral steady state position d* and velocity is determined by the competition between the inertia- and deformation-driven forces. In the deformation-dominated regime (Ca >> Re), DP migrates towards the channel centerline and flow faster for sufficiently small λ. In the inertia-dominated regime (Ca<flow slower for small λ. For sufficiently large λ, DP migrates towards the wall as the inertia-driven lift effects increase and the particle velocity decreases. In the intermediate regime (Ca Re), we find that d* has non-monotonic dependence on λ, leading to complicated dependence of particle velocity. We find that the non-monotonic trend is a consequence of inertia-deformation coupling, and only occurs if the inertia- and deformation-driven lift effects are comparable. This result could provide be further utilized for separating soft particles with different internal fluid property. MOST Taiwan, NCTS.

  8. Measurement of Separase Proteolytic Activity in Single Living Cells by a Fluorogenic Flow Cytometry Assay

    PubMed Central

    Haaß, Wiltrud; Kleiner, Helga; Müller, Martin C.; Hofmann, Wolf-Karsten; Fabarius, Alice; Seifarth, Wolfgang

    2015-01-01

    ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic

  9. Rapid parallel flow cytometry assays of active GTPases using effector beads.

    PubMed

    Buranda, Tione; BasuRay, Soumik; Swanson, Scarlett; Agola, Jacob; Bondu, Virginie; Wandinger-Ness, Angela

    2013-11-15

    We describe a rapid assay for measuring the cellular activity of small guanine triphosphatases (GTPases) in response to a specific stimulus. Effector-functionalized beads are used to quantify in parallel multiple GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus.

  10. A six-color flow cytometry assay for immunophenotyping classical Hodgkin lymphoma in lymph nodes.

    PubMed

    Fromm, Jonathan R; Wood, Brent L

    2014-03-01

    We have recently demonstrated that classical Hodgkin lymphoma (CHL) can be immunophenotyped by flow cytometry (FC), thus obviating the need for immunohistochemistry in many cases. The previously described nine-color assay, however, cannot be used by laboratories that do not have access to a nine- or ten-color flow cytometer. Therefore, a six-color FC tube was designed employing the following combination: CD64-FITC/CD30-PE/CD40-PeCy5.5/CD20-PECy7/CD95-APC/CD3-APC-H7. To validate this assay, we analyzed 408 tissue specimens (including 55 CHL cases, 26 of which had been previously cryopreserved). Specimen inclusion criteria included the identification of an abnormal population by FC or (if no abnormal population was identified) greater than 50,000 viable events and specimen age less than 4 days. All FC studies were examined blinded to any clinical, laboratory, or histologic information. The diagnostic sensitivity and specificity of the six-color FC assay was 85.4% and 99.7%, respectively. Taken together, these results suggest that the six-color FC assay has acceptable sensitivity and specificity for clinical use, allowing more FC laboratories to immunophenotype CHL by this method.

  11. [Fluoroimmunoassay and Magnetic Lateral Flow Immunoassay for the Detection of Ractopamine].

    PubMed

    Wang, Song-bai; Zhang, Yan; Wei, Yan-li; An, Wen-ting; Wang, Yu; Shuang, Shao-min

    2015-11-01

    A fluoroimmunoassay based on quantum dots (QDs) and a lateral flow immunoassay system based on the magnetic beads (MB) were constructed to detect ractopamine (RAG) in urine samples. The monoclonal antibody (Ab1) against RAC was conjugated with QDs or MB as detector reagent, respectively. They apply a competitive format using an immobilized RAC conjugate and free RAC present in samples. That is to say, the concentration of RAC in the sample was negative related to the fluorescense intensity of QDs or the color density of MB. Results showed that the limit of detection (LOD) of fluorescence immunoassay method is 1 ng · mL⁻¹ and analysis time is 4 h, while the visual LOD was 10 ng · mL⁻¹ and analysis time was 15 min in magnetic lateral flow immunoassay system (MFLIS). Taken into consideration of the advantages and disadvantages of the two methods, it was suitable for the trace detection of RAC using fluoroimmunoassay while it was appropriate for point-of-care tesing of RAC by MFLIS.

  12. FRET on lateral flow test strip to enhance sensitivity for detecting cancer biomarker.

    PubMed

    Wang, Jidong; Cao, Fengjing; He, Songliang; Xia, Yong; Liu, Xinyu; Jiang, Wenxiao; Yu, Yangyang; Zhang, Huisheng; Chen, Wenwen

    2018-01-01

    Fluorescence resonance energy transfer (FRET) between fluorescein isothiocyanate (FITC) and gold nanoparticles (Au NPs) is introduced in the lateral flow strip to detect cancer biomarker CEA with the color and fluorescence dual-readout. Anti-CEA monoclonal antibody coated Au NPs were on the conjugate pad and FITC labelled antibody (FITC-Ab) for CEA was coated on the test line. All the reagents were general in the lateral flow strip or commercially available and no new materials or technique were involved, which make our proposal a more universal method and easier to operate. With the addition of CEA on the sample pad, anti-CEA monoclonal antibody coated Au NPs-CEA-FITC-Ab complex formed on the test line, leading to a megascopic red line and simultaneous quenched fluorescence of FITC via FRET. The visual limit of detection (LOD) through distinguishing red color change was 10ng/mL and the LOD by differentiating fluorescence intensity was 0.1ng/mL, which was two orders of magnitude lower than that without considering fluorescence in the strip. And the linear range changed from 10-80ng/mL to 5-80ng/mL with the analysis of fluorescence change. Meanwhile, the feasibility of our method applied in real clinical samples was also confirmed. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. High content evaluation of shear dependent platelet function in a microfluidic flow assay.

    PubMed

    Hansen, Ryan R; Wufsus, Adam R; Barton, Steven T; Onasoga, Abimbola A; Johnson-Paben, Rebecca M; Neeves, Keith B

    2013-02-01

    The high blood volume requirements and low throughput of conventional flow assays for measuring platelet function are unsuitable for drug screening and clinical applications. In this study, we describe a microfluidic flow assay that uses 50 μL of whole blood to measure platelet function on ~300 micropatterned spots of collagen over a range of physiologic shear rates (50-920 s(-1)). Patterning of collagen thin films (CTF) was achieved using a novel hydrated microcontact stamping method. CTF spots of 20, 50, and 100 μm were defined on glass substrates and consisted of a dense mat of nanoscale collagen fibers (3.74 ± 0.75 nm). We found that a spot size of greater than 20 μm was necessary to support platelet adhesion under flow, suggesting a threshold injury size is necessary for stable platelet adhesion. Integrating 50 μm CTF microspots into a multishear microfluidic device yielded a high content assay from which we extracted platelet accumulation metrics (lag time, growth rate, total accumulation) on the spots using Hoffman modulation contrast microscopy. This method has potential broad application in identifying platelet function defects and screening, monitoring, and dosing antiplatelet agents.

  14. High content evaluation of shear dependent platelet function in a microfluidic flow assay

    PubMed Central

    Hansen, Ryan R.; Wufsus, Adam R.; Barton, Steven T.; Onasoga, Abimbola A.; Johnson-Paben, Rebecca M.; Neeves, Keith B.

    2012-01-01

    The high blood volume requirements and low throughput of conventional flow assays for measuring platelet function are unsuitable for drug screening and clinical applications. In this study, we describe a microfluidic flow assay that uses 50 μL of whole blood to measure platelet function on ~300 micropatterned spots of collagen over a range of physiologic shear rates (50–920 s−1). Patterning of collagen thin films (CTF) was achieved using a novel hydrated microcontact stamping method. CTF spots of 20, 50, and 100 μm were defined on glass substrates and consisted of a dense mat of nanoscale collagen fibers (3.74 ± 0.75 nm). We found that a spot size of greater than 20 μm was necessary to support platelet adhesion under flow, suggesting a threshold injury is necessary for stable platelet adhesion. Integrating 50 μm CTF microspots into a multishear microfluidic device yielded a high content assay from which we extracted platelet accumulation metrics (lag time, growth rate, total accumulation) on the spots using Hoffman modulation contrast microscopy. This method has potential broad application in identifying platelet function defects and screening, monitoring and dosing antiplatelet agents. PMID:23001359

  15. Ultrarapid detection of pathogenic bacteria using a 3D immunomagnetic flow assay.

    PubMed

    Lee, Wonjae; Kwon, Donghoon; Chung, Boram; Jung, Gyoo Yeol; Au, Anthony; Folch, Albert; Jeon, Sangmin

    2014-07-01

    We developed a novel 3D immunomagnetic flow assay for the rapid detection of pathogenic bacteria in a large-volume food sample. Antibody-functionalized magnetic nanoparticle clusters (AbMNCs) were magnetically immobilized on the surfaces of a 3D-printed cylindrical microchannel. The injection of a Salmonella-spiked sample solution into the microchannel produced instant binding between the AbMNCs and the Salmonella bacteria due to their efficient collisions. Nearly perfect capture of the AbMNCs and AbMNCs-Salmonella complexes was achieved under a high flow rate by stacking permanent magnets with spacers inside the cylindrical separator to maximize the magnetic force. The concentration of the bacteria in solution was determined using ATP luminescence measurements. The detection limit was better than 10 cfu/mL, and the overall assay time, including the binding, rinsing, and detection steps for a 10 mL sample took less than 3 min. To our knowledge, the 3D immunomagnetic flow assay described here provides the fastest high-sensitivity, high-capacity method for the detection of pathogenic bacteria.

  16. Quantitative data analysis methods for bead-based DNA hybridization assays using generic flow cytometry platforms.

    PubMed

    Corrie, S R; Lawrie, G A; Battersby, B J; Ford, K; Rühmann, A; Koehler, K; Sabath, D E; Trau, M

    2008-05-01

    Bead-based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two-sided t-tests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5-labeled synthetic cytokeratin 19 (K19) RNA of c.a. 1 x 10(4) to 500 x 10(4) molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead-based cytometry assays to ensure reproducibility in inter- and intra-platform comparisons of data between laboratories. (c) 2008 International Society for Advancement of Cytometry.

  17. Development of improved enzyme-based and lateral flow immunoassays for rapid and accurate serodiagnosis of canine brucellosis.

    PubMed

    Cortina, María E; Novak, Analía; Melli, Luciano J; Elena, Sebastián; Corbera, Natalia; Romero, Juan E; Nicola, Ana M; Ugalde, Juan E; Comerci, Diego J; Ciocchini, Andrés E

    2017-09-01

    Brucellosis is a widespread zoonotic disease caused by Brucella spp. Brucella canis is the etiological agent of canine brucellosis, a disease that can lead to sterility in bitches and dogs causing important economic losses in breeding kennels. Early and accurate diagnosis of canine brucellosis is central to control the disease and lower the risk of transmission to humans. Here, we develop and validate enzyme and lateral flow immunoassays for improved serodiagnosis of canine brucellosis using as antigen the B. canis rough lipopolysaccharide (rLPS). The method used to obtain the rLPS allowed us to produce more homogeneous batches of the antigen that facilitated the standardization of the assays. To validate the assays, 284 serum samples obtained from naturally infected dogs and healthy animals were analyzed. For the B. canis-iELISA and B. canis-LFIA the diagnostic sensitivity was of 98.6%, and the specificity 99.5% and 100%, respectively. We propose the implementation of the B. canis-LFIA as a screening test in combination with the highly accurate laboratory g-iELISA. The B. canis-LFIA is a rapid, accurate and easy to use test, characteristics that make it ideal for the serological surveillance of canine brucellosis in the field or veterinary laboratories. Finally, a blind study including 1040 serum samples obtained from urban dogs showed a prevalence higher than 5% highlighting the need of new diagnostic tools for a more effective control of the disease in dogs and therefore to reduce the risk of transmission of this zoonotic pathogen to humans. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. The influence of tree species on soil moisture dynamics and lateral water flow during intense rainfall

    NASA Astrophysics Data System (ADS)

    Jost, G.; Schume, H.; Hager, H.

    2009-12-01

    This study investigates how different tree species influence soil hydrological properties that are relevant for the rainfall - runoff response of a given soil type. We hypothesize that for the same soil type, tree species with different rooting systems and different water consumption lead to different soil moisture dynamics and lateral flow processes during rainfall and hence to different runoff responses. To test this hypothesis, we compare soil moisture patterns and interflow at different soil depths in a Norway spruce (Picea abies (L.) Karst) forest and in a European beech (Fagus sylvatica L) forest during sprinkling experiments on two 6 m x 10 m hillslopes with the same soil type. Spruce with a shallow rooting system and beech with a deep rooting system are two of the most important tree species in Central Europe. At each hillslope, volumetric water content was measured in 6 minute intervals with 48 TDR wave-guides during and after sprinkling with intensities of 100 mm/h and 60 mm/h (for 1 hour). TDRs were installed in 12 soil pits, whereby a single soil pit consisted of four 20 cm buriable TDR installed in 10 cm, 30 cm, 50 cm and 70 cm soil depth. Surface and shallow interflow at 10 cm soil depth and interflow at soil depths of 30 cm and 60 cm was automatically recorded. In addition, stand scale soil water recharge patterns were derived from manual TDR measurements at 196 locations with two different soil depths in each forest. Despite the high rainfall intensities, surface flow under beech and spruce was negligible. Soil moisture patterns of lateral cross sections during and after the sprinkling reveal the different dynamics of the two forest types: The deeper rooting system of beech seems to direct more water towards deeper soil horizons, from where the watertable raises into the top soil with high lateral conductivity. Towards the end of the sprinkling, the deep soil horizons are around saturation, whereas the topsoil remains substantially below saturation

  19. Lateral flow in the middle crust - Analogue experiments from the Svecofennian orogen

    NASA Astrophysics Data System (ADS)

    Nikkilä, Kaisa; Koyi, Hemin; Korja, Annakaisa; Eklund, Olav

    2013-04-01

    The exposed Svecofennian crust (50-65 km) has been suggested to have thickened in continental accretion between Archean and Paleoproterozoic terranes, probably at a high convergence rate. It is likely that this thickened orogen experienced lateral spreading during its final stages. This post-orogenic event has reshaped the collisional framework and modified its bulk appearance. In this study, we have used scaled analogue centrifuge modeling to simulate extensional lateral flow at the Archean- Paleoproterozoic boundary zone during final stages of the Svecofennian orogeny. The analogue models simulate both the evolution of a mechanical boundary between two rheologically different tectonic blocks, and the role of pre-existing weaknesses at moderate angles (representing the old stacking structures). In models the upper layer is brittle, the middle layer is ductile, and the lower layer is more viscous. The layers represent upper, middle and lower crust, respectively. The Proterozoic layers have lower viscosity values than the Archean layers at similar depths. The materials are based on the plastilina modelling putty, which is mixed with acid oil, silicone, sweetener and/or barium sulphate to get the appropriate composition for each layer. Both the Archean and the Paleoproterozoic blocks have a low-viscous middle crust. The three layered models are extended unilaterally. The model results show that during extension the rheologically different layers deform and spread at different rates during the tectonic collapse. This results in 1) vertical rotation of the Archean and Proterozoic boundary; 2) the pre-existed faults become listric and discontinuous; and 3) the upward flow of the low viscosity middle layer to fill the newly-formed gaps between the upper layer blocks. The experiments show geometrically similar crustal-scale structures to those observed in the deep seismic reflection profiles (FIRE). Thus it is possible that lateral flow has taken place in the core of the

  20. Detection of influenza virus using a lateral flow immunoassay for amplified DNA by a microfluidic RT-PCR chip.

    PubMed

    Nagatani, Naoki; Yamanaka, Keiichiro; Ushijima, Hiromi; Koketsu, Ritsuko; Sasaki, Tadahiro; Ikuta, Kazuyoshi; Saito, Masato; Miyahara, Toshiro; Tamiya, Eiichi

    2012-08-07

    Influenza virus RNA was amplified by a continuous-flow polydimethylsiloxane microfluidic RT-PCR chip within 15-20 min. The amplified influenza virus RNA was observed with the naked eye, as the red color at the test line, using a lateral flow immunoassay within 1 min.

  1. Magnetic Lateral Flow Strip for the Detection of Cocaine in Urine by Naked Eyes and Smart Phone Camera.

    PubMed

    Wu, Jing; Dong, Mingling; Zhang, Cheng; Wang, Yu; Xie, Mengxia; Chen, Yiping

    2017-06-05

    Magnetic lateral flow strip (MLFS) based on magnetic bead (MB) and smart phone camera has been developed for quantitative detection of cocaine (CC) in urine samples. CC and CC-bovine serum albumin (CC-BSA) could competitively react with MB-antibody (MB-Ab) of CC on the surface of test line of MLFS. The color of MB-Ab conjugate on the test line relates to the concentration of target in the competition immunoassay format, which can be used as a visual signal. Furthermore, the color density of the MB-Ab conjugate can be transferred into digital signal (gray value) by a smart phone, which can be used as a quantitative signal. The linear detection range for CC is 5-500 ng/mL and the relative standard deviations are under 10%. The visual limit of detection was 5 ng/mL and the whole analysis time was within 10 min. The MLFS has been successfully employed for the detection of CC in urine samples without sample pre-treatment and the result is also agreed to that of enzyme-linked immunosorbent assay (ELISA). With the popularization of smart phone cameras, the MLFS has large potential in the detection of drug residues in virtue of its stability, speediness, and low-cost.

  2. Highly sensitive and selective lateral flow immunoassay based on magnetic nanoparticles for quantitative detection of carcinoembryonic antigen.

    PubMed

    Liu, Fangming; Zhang, Honglian; Wu, Zhenhua; Dong, Haidao; Zhou, Lin; Yang, Dawei; Ge, Yuqing; Jia, Chunping; Liu, Huiying; Jin, Qinghui; Zhao, Jianlong; Zhang, Qiqing; Mao, Hongju

    2016-12-01

    Carcinoembryonic antigen (CEA) is an important biomarker in cancer diagnosis. Here, we present an efficient, selective lateral-flow immunoassay (LFIA) based on magnetic nanoparticles (MNPs) for in situ sensitive and accurate point-of-care detection of CEA. Signal amplification mechanism involved linking of detection MNPs with signal MNPs through biotin-modified single-stranded DNA (ssDNA) and streptavidin. To verify the effectiveness of this modified LFIA system, the sensitivity and specificity were evaluated. Sensitivity evaluation showed a broad detection range of 0.25-1000ng/ml for CEA protein by the modified LFIA, and the limit of detection (LOD) of the modified LFIA was 0.25ng/ml, thus producing significant increase in detection threshold compared with the traditional LFIA. The modified LFIA could selectively recognize CEA in presence of several interfering proteins. In addition, this newly developed assay was applied for quantitative detection of CEA in human serum specimens collected from 10 randomly selected patients. The modified LFIA system detected minimum 0.27ng/ml of CEA concentration in serum samples. The results were consistent with the clinical data obtained using commercial electrochemiluminescence immunoassay (ECLIA) (p<0.01). In conclusion, the MNPs based LFIA system not only demonstrated enhanced signal to noise ratio, it also detected CEA with higher sensitivity and selectivity, and thus has great potential to be commercially applied as a sensitive tumor marker filtration system.

  3. Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on "Road Closure".

    PubMed

    Wang, Dian-Bing; Tian, Bo; Zhang, Zhi-Ping; Wang, Xu-Ying; Fleming, Joy; Bi, Li-Jun; Yang, Rui-Fu; Zhang, Xian-En

    2015-05-15

    Detection of Bacillus anthracis in the field, whether as a natural infection or as a biothreat remains challenging. Here we have developed a new lateral-flow immunochromatographic assay (LFIA) for B. anthracis spore detection based on the fact that conjugates of B. anthracis spores and super-paramagnetic particles labeled with antibodies will block the pores of chromatographic strips and form retention lines on the strips, instead of the conventionally reported test lines and control lines in classic LFIA. As a result, this new LFIA can simultaneously realize optical, magnetic and naked-eye detection by analyzing signals from the retention lines. As few as 500-700 pure B. anthracis spores can be recognized with CV values less than 8.31% within 5 min of chromatography and a total time of 20 min. For powdery sample tests, this LFIA can endure interference from 25% (w/v) milk, 10% (w/v) baking soda and 10% (w/v) starch without any sample pre-treatment, and has a corresponding detection limit of 6×10(4) spores/g milk powder, 2×10(5) spores/g starch and 5×10(5) spores/g baking soda. Compared with existing methods, this new approach is very competitive in terms of sensitivity, specificity, cost and ease of operation. This proof-of-concept study can also be extended for detection of many other large-sized analytes.

  4. A novel double antibody sandwich-lateral flow immunoassay for the rapid and simple detection of hepatitis C virus.

    PubMed

    Xiang, Tingxiu; Jiang, Zheng; Zheng, Jian; Lo, Chaoyu; Tsou, Harry; Ren, Guosheng; Zhang, Jun; Huang, Ailong; Lai, Guoqi

    2012-11-01

    The objective of this study was to screen for antigens of the hepatitis C virus (HCV) to establish a new double antibody sandwich-lateral flow immunoassay (DAS-LFIA) method for testing the presence of anti-HCV antibodies in human serum or plasma. A series of different recombinant HCV proteins in Escherichia coli cells were constructed, expressed, purified and the new DAS-LFIA strip was developed. The sensitivity and specificity of new the DAS-LFIA strip were evaluated by detecting 23 HCV-positive sera, a set of quality control references for anti-HCV detection that contain known amounts of anti-HCV antibodies, and 8 HCV-negative sera. A total of 300 clinical serum samples was examined by both the new DAS-LFIA strip and enzyme-linked immunosorbent assay (ELISA). Data were analyzed using SPSS 11.5 software. The sensitivity and specificity of the new DAS-LFIA strip were 100%. The lowest test line of the HCV DAS-LFIA strips was 2 NCU/ml. Additionally, the concordance between the new DAS-LFIA strip and ELISA methods was 94.33%. In conclusion, our new testing method is rapid, simple, sensitive and specifically detects the presence of anti-HCV antibodies in human serum or plasma. Therefore, it may be used for monitoring HCV.

  5. Development and Validation of a Lateral Flow Immunoassay Test Kit for Dual Detection of Casein and β-Lactoglobulin Residues.

    PubMed

    Masiri, Jongkit; Barrios-Lopez, Brianda; Benoit, Lora; Tamayo, Joshua; Day, Jeffrey; Nadala, Cesar; Sung, Shao-Lei; Samadpour, Mansour

    2016-03-01

    Allergies to cow's milk are very common and can present as life-threatening anaphylaxis. Consequently, food labeling legislation mandates that foods containing milk residues, including casein and/or β-lactoglobulin, provide an indication of such on the product label. Because contamination with either component independent of the other can occur during food manufacturing, effective allergen management measures for containment of milk residues necessitates the use of dual screening methods. To assist the food industry in improving food safety practices, we have developed a rapid lateral flow immunoassay test kit that reliably reports both residues down to 0.01 μg per swab and 0.1 ppm of protein for foods. The assay utilizes both sandwich and competitive format test lines and is specific for bovine milk residues. Selectivity testing using a panel of matrices with potentially interfering substances, including commonly used sanitizing agents, indicated reduction in the limit of detection by one-to fourfold. With food, residues were easily detected in all cow's milk-based foods tested, but goat and sheep milk residues were not detected. Specificity analysis revealed no cross-reactivity with common commodities, with the exception of kidney beans when present at high concentrations (> 1%). The development of a highly sensitive and rapid test method capable of detecting trace amounts of casein and/or β-lactoglobulin should aid food manufacturers and regulatory agencies in monitoring for milk allergens in environmental and food samples.

  6. Developmental validation of RSID-saliva: a lateral flow immunochromatographic strip test for the forensic detection of saliva.

    PubMed

    Old, Jennifer B; Schweers, Brett A; Boonlayangoor, Pravat W; Reich, Karl A

    2009-07-01

    Current methods for forensic identification of saliva generally assay for the enzymatic activity of alpha-amylase, an enzyme long associated with human saliva. Here, we describe the Rapid Stain IDentification (RSID-Saliva), a lateral flow immunochromatographic strip test that uses two antisalivary amylase monoclonal antibodies to detect the presence of salivary amylase, rather than the activity of the enzyme. We demonstrate that RSID-Saliva is accurate, reproducible, and highly sensitive for human saliva; RSID-Saliva detects less than 1 microL of saliva. The sensitivity of RSID-Saliva allows investigators to sample a fraction of a questioned stain while retaining the majority for DNA-STR analysis. We demonstrate that RSID-Saliva identifies saliva from a variety of materials (e.g., cans, bottles, envelopes, and cigarette-butts) and it does not cross-react with blood, semen, urine, or vaginal fluid. RSID-Saliva is a useful forensic test for determining which evidentiary items contain saliva and thus may yield a DNA profile.

  7. Rapid and simple detection of Japanese encephalitis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick.

    PubMed

    Deng, Jieru; Pei, Jingjing; Gou, Hongchao; Ye, Zuodong; Liu, Cuicui; Chen, Jinding

    2015-03-01

    Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in geographical areas, such as Asia and Western Pacific, where it is a threat to human and animal health. To control this disease, it is necessary to develop a rapid, simple, accurate method for diagnosis. In this study, a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) coupled with a lateral flow dipstick (LFD) has been developed to detect JEV (JEV RT-LAMP-LFD). The entire assay can be completed within 70 min, and in this study, no false positive results were observed when other pathogens were tested, indicating that the assay is a highly specific method for the detection of JEV. Additionally, the sensitivity of the RT-LAMP-LFD assay for SA14-14-2 strain was 50 pg of RNA, which was similar to that of RT-PCR and RT-LAMP combined with gel electrophoresis, and was 10-fold more sensitive than RT-LAMP combined with calcein. The limit of detection for this assay was 5 pg of RNA. In addition, no false positive results were obtained with 14 serum samples. Our results indicate that this RT-LAMP-LFD assay will be of great value for JEV infection testing due to its rapid and highly specific and sensitive properties. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Heat Transfer to Anode of Arc as Function of Transverse Magnetic Field and Lateral Gas Flow Velocity

    NASA Astrophysics Data System (ADS)

    Zama, Yoshiyuki; Shiino, Toru; Ishii, Yoko; Maeda, Yoshifumi; Yamamoto, Shinji; Iwao, Toru

    2016-10-01

    Gas tungsten arc welding has useful joining technology because of high-energy and high-current characteristics. It can be flexible from the transverse magnetic field and lateral gas flow velocity. In this case, the weld defect occurs. In this research, the heat transfer to the anode of the arc as a function of the transverse magnetic field and lateral gas flow velocity is elucidated. That magnetic flux density and lateral gas velocity were varied from 0 to 3 mT and 0 to 50?m?s -1, respectively. The axial plasma gas argon flow rates were 3?slm. A transverse magnetic field is applied to the arc using Helmholtz coil. The anode is used by a water-cooled copper plate, and the heat transfer is measured by temperature of cooled water. As a result, the arc is deflected by the Lorentz force and lateral gas convection. Thus, the heat transfer to the anode of the arc decreases with increasing the transverse magnetic field and lateral gas flow velocity. In addition, the heat transfer to the anode changes with different attachments modes. The lateral gas flow causes a convective heat loss from the arc to the chamber walls.

  9. Influence of the lateral wall velocity on three-dimensional disturbance development in plane Poiseuille-Couette flow

    NASA Astrophysics Data System (ADS)

    Savenkov, I. V.

    2017-05-01

    The linear stage of three-dimensional disturbance development in the Poiseuille-Couette flow in the case when both walls can move in the lateral direction is investigated by applying the asymptotic triple-deck theory. It is shown that the lateral wall velocity has no effect on the streamwise velocity of a wave packet. The packet does not bifurcate, but drifts in the lateral direction at the speed equal to the arithmetic mean of the walls' speeds. Characteristic "ripples" in the lateral direction are observed at the stage of packet formation.

  10. Serological detection of Helicobacter pylori by a flow microsphere immunofluorescence assay.

    PubMed Central

    Best, L M; Veldhuyzen van Zanten, S J; Bezanson, G S; Haldane, D J; Malatjalian, D A

    1992-01-01

    A flow cytometric immunofluorescence assay (FMIA) for the detection of immunoglobulin G antibodies to Helicobacter pylori was developed. A multicomponent antigen was prepared and used to coat carboxylated polystyrene microspheres for reaction with patient sera followed by fluorescein isothiocyanate-labelled goat anti-human immunoglobulin G. The reacted microspheres were collected with a flow cytometer, and fluorescence was quantitated relative to the cutoff value provided by pooled sera from patients in whom H. pylori could not be demonstrated by culture or histology. Serum samples from 28 H. pylori-positive patients and 27 H. pylori-negative patients were tested by FMIA. Additionally, an in-house enzyme-linked immunosorbent assay (ELISA) employing the same antigen preparation and a commercially available ELISA were used to assay the patient population. Both the FMIA and in-house ELISA were 100% sensitive and 89% specific with positive and negative predictive values of 90 and 100% and no equivocal results. The commercial ELISA was 96% sensitive and 89% specific with positive and negative predictive values of 90 and 96% and five equivocal results. FMIA provides a rapid, inexpensive, and easily performed means for serodiagnosis of H. pylori. Images PMID:1400995

  11. Influence of vertical and lateral heat transfer on permafrost thaw, peatland landscape transition, and groundwater flow

    USGS Publications Warehouse

    Kurylyk, Barret; Masaki, Masaki; Quinton, William L.; McKenzie, Jeffrey M.; Voss, Clifford I.

    2016-01-01

    Recent climate change has reduced the spatial extent and thickness of permafrost in many discontinuous permafrost regions. Rapid permafrost thaw is producing distinct landscape changes in the Taiga Plains of the Northwest Territories, Canada. As permafrost bodies underlying forested peat plateaus shrink, the landscape slowly transitions into unforested wetlands. The expansion of wetlands has enhanced the hydrologic connectivity of many watersheds via new surface and near-surface flow paths, and increased streamflow has been observed. Furthermore, the decrease in forested peat plateaus results in a net loss of boreal forest and associated ecosystems. This study investigates fundamental processes that contribute to permafrost thaw by comparing observed and simulated thaw development and landscape transition of a peat plateau-wetland complex in the Northwest Territories, Canada from 1970 to 2012. Measured climate data are first used to drive surface energy balance simulations for the wetland and peat plateau. Near-surface soil temperatures simulated in the surface energy balance model are then applied as the upper boundary condition to a three-dimensional model of subsurface water flow and coupled energy transport with freeze-thaw. Simulation results demonstrate that lateral heat transfer, which is not considered in many permafrost models, can influence permafrost thaw rates. Furthermore, the simulations indicate that landscape evolution arising from permafrost thaw acts as a positive feedback mechanism that increases the energy absorbed at the land surface and produces additional permafrost thaw. The modeling results also demonstrate that flow rates in local groundwater flow systems may be enhanced by the degradation of isolated permafrost bodies.

  12. Influence of vertical and lateral heat transfer on permafrost thaw, peatland landscape transition, and groundwater flow

    NASA Astrophysics Data System (ADS)

    Kurylyk, Barret L.; Hayashi, Masaki; Quinton, William L.; McKenzie, Jeffrey M.; Voss, Clifford I.

    2016-02-01

    Recent climate change has reduced the spatial extent and thickness of permafrost in many discontinuous permafrost regions. Rapid permafrost thaw is producing distinct landscape changes in the Taiga Plains of the Northwest Territories, Canada. As permafrost bodies underlying forested peat plateaus shrink, the landscape slowly transitions into unforested wetlands. The expansion of wetlands has enhanced the hydrologic connectivity of many watersheds via new surface and near-surface flow paths, and increased streamflow has been observed. Furthermore, the decrease in forested peat plateaus results in a net loss of boreal forest and associated ecosystems. This study investigates fundamental processes that contribute to permafrost thaw by comparing observed and simulated thaw development and landscape transition of a peat plateau-wetland complex in the Northwest Territories, Canada from 1970 to 2012. Measured climate data are first used to drive surface energy balance simulations for the wetland and peat plateau. Near-surface soil temperatures simulated in the surface energy balance model are then applied as the upper boundary condition to a three-dimensional model of subsurface water flow and coupled energy transport with freeze-thaw. Simulation results demonstrate that lateral heat transfer, which is not considered in many permafrost models, can influence permafrost thaw rates. Furthermore, the simulations indicate that landscape evolution arising from permafrost thaw acts as a positive feedback mechanism that increases the energy absorbed at the land surface and produces additional permafrost thaw. The modeling results also demonstrate that flow rates in local groundwater flow systems may be enhanced by the degradation of isolated permafrost bodies.

  13. Broken flow symmetry explains the dynamics of small particles in deterministic lateral displacement arrays.

    PubMed

    Kim, Sung-Cheol; Wunsch, Benjamin H; Hu, Huan; Smith, Joshua T; Austin, Robert H; Stolovitzky, Gustavo

    2017-06-27

    Deterministic lateral displacement (DLD) is a technique for size fractionation of particles in continuous flow that has shown great potential for biological applications. Several theoretical models have been proposed, but experimental evidence has demonstrated that a rich class of intermediate migration behavior exists, which is not predicted. We present a unified theoretical framework to infer the path of particles in the whole array on the basis of trajectories in a unit cell. This framework explains many of the unexpected particle trajectories reported and can be used to design arrays for even nanoscale particle fractionation. We performed experiments that verify these predictions and used our model to develop a condenser array that achieves full particle separation with a single fluidic input.

  14. LATERAL HEAT FLOW INFRARED THERMOGRAPHY FOR THICKNESS INDEPENDENT DETERMINATION OF THERMAL DIFFUSIVITY IN CFRP

    SciTech Connect

    Tralshawala, Nilesh; Howard, Don; Knight, Bryon; Plotnikov, Yuri; Ringermacher, Harry

    2008-02-28

    In conventional infrared thermography, determination of thermal diffusivity requires thickness information. Recently GE has been experimenting with the use of lateral heat flow to determine thermal diffusivity without thickness information. This work builds on previous work at NASA Langley and Wayne State University but we incorporate thermal time of flight (tof) analysis rather than curve fitting to obtain quantitative information. We have developed appropriate theoretical models and a tof based data analysis framework to experimentally determine all components of thermal diffusivity from the time-temperature measurements. Initial validation was carried out using finite difference simulations. Experimental validation was done using anisotropic carbon fiber reinforced polymer (CFRP) composites. We found that in the CFRP samples used, the in-plane component of diffusivity is about eight times larger than the through-thickness component.

  15. Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection.

    PubMed

    Chen, Mu-Xin; Chen, Jia-Xu; Chen, Shao-Hong; Huang, Da-Na; Ai, Lin; Zhang, Ren-Li

    2016-06-01

    Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.

  16. Broken flow symmetry explains the dynamics of small particles in deterministic lateral displacement arrays

    PubMed Central

    Kim, Sung-Cheol; Wunsch, Benjamin H.; Hu, Huan; Smith, Joshua T.; Stolovitzky, Gustavo

    2017-01-01

    Deterministic lateral displacement (DLD) is a technique for size fractionation of particles in continuous flow that has shown great potential for biological applications. Several theoretical models have been proposed, but experimental evidence has demonstrated that a rich class of intermediate migration behavior exists, which is not predicted. We present a unified theoretical framework to infer the path of particles in the whole array on the basis of trajectories in a unit cell. This framework explains many of the unexpected particle trajectories reported and can be used to design arrays for even nanoscale particle fractionation. We performed experiments that verify these predictions and used our model to develop a condenser array that achieves full particle separation with a single fluidic input. PMID:28607075

  17. Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

    PubMed Central

    Chen, Mu-Xin; Chen, Jia-Xu; Chen, Shao-Hong; Huang, Da-Na; Ai, Lin; Zhang, Ren-Li

    2016-01-01

    Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis. PMID:27417097

  18. Visual detection of nucleic acids based on lateral flow biosensor and hybridization chain reaction amplification.

    PubMed

    Ying, Na; Ju, Chuanjing; Li, Zhongyi; Liu, Wensen; Wan, Jiayu

    2017-03-01

    In this study, a new lateral flow nucleic acid biosensor (LFNAB) using hybridization chain reaction (HCR) for signal amplification was developed for visual detection of nucleic acids with high sensitivity and low cost. A "sandwich-type" detection strategy was employed in our design. The sandwich system of capture probe (CP)/target DNA/reporter probe (RP)-HCR complexes was fabricated as the sensing platform. As the initiator strand, reporter probe propagated a chain reaction of hybridization events between the two hairpin probes modified with biotin, and determined whether long nicked DNA polymers were formed. The biotin-labeled double-strand DNA polymers then introduced numerous Streptavidin (SA)-labeled gold nanoparticles (AuNPs) on the lateral flow device. The CP/target DNA/RP-HCR complexes were captured on the test zone by the specific reaction between anti-Fam monoclonal antibody (anti-Fam mAb) on the test zone and Fam of the complexes. The accumulation of AuNPs on the test zone of the biosensor enabled the visual detection of specific sequences. The detection limit of specific DNA was as low as 1.76pM, which was about 2 orders lower than that of the LFNAB without HCR amplification. And the detection limit of Salmonella was 3×10(3)cfumL(-1). In conclusion, this visual detection system, HCR-LFNAB, is suitable for non-specialist personnel and point-of-care (POC) diagnosis in low-resource settings.

  19. A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin

    PubMed Central

    Maass, Katie F.; Kulkarni, Chethana; Quadir, Mohiuddin A.; Hammond, Paula T.; Betts, Alison M.; Wittrup, K. Dane

    2015-01-01

    Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell’s ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 105 – 1010 doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell’s ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4 – 12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug’s single-cell potency and can be used for any fluorescent or fluorescently-labeled drug, including nanoparticles or antibody-drug conjugates. PMID:26344409

  20. A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin.

    PubMed

    Maass, Katie F; Kulkarni, Chethana; Quadir, Mohiuddin A; Hammond, Paula T; Betts, Alison M; Wittrup, Karl Dane

    2015-12-01

    Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell's ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 10(5) -10(10) doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell's ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4-12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug's single-cell potency and can be used for any fluorescent or fluorescently labeled drug, including nanoparticles or antibody-drug conjugates.

  1. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays

    PubMed Central

    Urbani, Francesca; Proietti, Enrico

    2013-01-01

    The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM-) based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT) combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma. PMID:24195078

  2. High Throughput Flow Cytometry Bead-based Multiplex Assay for Identification of Rho GTPase Inhibitors

    PubMed Central

    Surviladze, Zurab; Young, Susan M; Sklar, Larry A

    2015-01-01

    Summary Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly. Herein we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases. PMID:22144280

  3. Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout

    PubMed Central

    Löf, Liza; Ebai, Tonge; Dubois, Louise; Wik, Lotta; Ronquist, K. Göran; Nolander, Olivia; Lundin, Emma; Söderberg, Ola; Landegren, Ulf; Kamali-Moghaddam, Masood

    2016-01-01

    Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells. However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs – in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification. PMID:27681459

  4. Functional assay of antiplatelet drugs based on margination of platelets in flowing blood

    PubMed Central

    Eichinger, Colin D.; Fogelson, Aaron L.; Hlady, Vladimir

    2016-01-01

    A novel functional assay of antiplatelet drug efficacy was designed by utilizing the phenomena of platelet margination in flowing blood and transient platelet contacts with surface-immobilized platelet agonists. Flow margination enhances transient contacts of platelets with the walls of flow chambers covered with surface-immobilized proteins. Depending on the type and the surface density of the immobilized agonists, such transient interactions could “prime” the marginated platelet subpopulation for enhanced activation and adhesion downstream. By creating an upstream surface patch with an immobilized platelet agonist, platelet flow margination was used to test how effective antiplatelet drugs are in suppressing downstream platelet activation and adhesion. The platelet adhesion downstream was measured by a so-called “capture” patch region close to the distal end of the flow chamber. Platelet adhesion downstream was found to be dose-dependent on the upstream surface coverage of the “priming” patch, with immobilized fibrinogen acting as a platelet agonist. Several antiplatelet agents (acetylsalicylic acid, eptifibatide, and tirofiban) were evaluated for their efficacy in attenuating downstream adhesion after upstream platelet priming. The activation of the platelet population was found to be dependent on both the extent of the upstream agonist stimulus and the antiplatelet drug concentration. Such a relationship provides an opportunity to measure the efficacy of specific antiplatelet agents against the type and concentration of upstream platelet agonists. PMID:27030476

  5. Rapid and sensitive detection of the food allergen glycinin in powdered milk using a lateral flow colloidal gold immunoassay strip test.

    PubMed

    Wang, Yao; Deng, Ruiguang; Zhang, Gaiping; Li, Qingmei; Yang, Jifei; Sun, Yaning; Li, Zhixi; Hu, Xiaofei

    2015-03-04

    A rapid immunochromatographic lateral flow test strip in a sandwich format was developed with the colloidal gold-labeled mouse antiglycinin monoclonal antibody (mAb) and rabbit antiglycinin polyclonal antibody (pAb) to specifically identify glycinin, a soybean allergen. The test strip is composed of a sample pad, a conjugate reagent pad, an absorbent pad, and a test membrane containing a control line and a test line. This test strip has high sensitivity, and results can be obtained within 10 min without sophisticated procedures. The limit of detection (LOD) of the test strip was calculated to be 0.69 mg/kg using an optical density scanner that measures relative optical density. The assay showed high specificity for glycinin, with no cross-reactions with other soybean proteins or other food allergens. The recoveries of the lateral flow test strip in detecting glycinin in powdered milk samples ranged between 80.5 and 89.9% with relative standard deviations of less than 5.29% (intra-assay) and 6.72% (interassay). Therefore, the test strip is useful as a quantitative, semiquantitative, or qualitative detection method for glycinin in powdered milk. In addition, the test strip can be used to detect glycinin in other processed foods and may be a valuable tool in identifying effective approaches for reducing the impact of glycinin.

  6. Inhibitory modulation of medial prefrontal cortical activation on lateral orbitofrontal cortex-amygdala information flow.

    PubMed

    Chang, Chun-Hui; Ho, Ta-Wen

    2017-09-01

    The basolateral complex of the amygdala (BLA) receives input from the lateral orbitofrontal cortex (lOFC) for cue-outcome contingencies and the medial prefrontal cortex (mPFC) for emotion control. Here we examined how the mPFC modulates lOFC-BLA information flow. We found that the majority of BLA neurons responsive to lOFC stimulation were also responsive to mPFC stimulation. Activation of the mPFC exerted an inhibitory modulation of the lOFC-BLA pathway, which was reversed with intra-amygdala blockade of GABAergic receptors. mPFC tetanus potentiated the lOFC-BLA pathway, but did not alter its inhibitory modulatory gating. These results show that the mPFC potently inhibits lOFC drive of the BLA in a GABA-dependent manner, which is informative in understanding the normal and potential pathophysiological state of emotion and contingency associations in regulating behaviour. Several neocortical projections converge onto the basolateral complex of the amygdala (BLA), including the lateral orbitofrontal cortex (lOFC) and the medial prefrontal cortex (mPFC). Lateral orbitofrontal input to the BLA is important for cue-outcome contingencies, while medial prefrontal input is essential for emotion control. In this study, we examined how the mPFC, specifically the infralimbic division of the mPFC, modulates lOFC-BLA information flow, using combined in vivo extracellular single-unit recordings and pharmacological manipulations in anaesthetized rats. We found that the majority (over 95%) of BLA neurons that responded to lOFC stimulation also responded to mPFC stimulation. Compared to basal condition, pharmacological (N-methyl-d-aspartate) or electrical activation of the mPFC exerted an inhibitory modulation of the lOFC-BLA pathway, which was reversed with intra-amygdala blockade of GABAergic receptors with combined GABAA and GABAB antagonists (bicuculline and saclofen). Moreover, mPFC tetanus potentiated the lOFC-BLA pathway, but mPFC tetanus or low-frequency stimulation did

  7. The role of the lateral line and vision on body kinematics and hydrodynamic preference of rainbow trout in turbulent flow.

    PubMed

    Liao, James C

    2006-10-01

    The ability to detect water flow using the hair cells of the lateral line system is a unique feature found in anamniotic aquatic vertebrates. Fishes use their lateral line to locate prey, escape from predators and form cohesive schooling patterns. Despite the prevalence of complex flows in nature, almost nothing is known about the function of the lateral line and its relationship to other sensory modalities for freely swimming fishes in turbulent flows. Past studies indicate that under certain conditions the lateral line is not needed to swim steadily in uniform flow. This paper examines how the lateral line and vision affect body kinematics and hydrodynamic habitat selection of rainbow trout (Oncorhynchus mykiss) exposed to vortices generated behind a cylinder. Trout Kármán gaiting (i.e. exploiting vortices to hold station in a vortex street) with a pharmacologically blocked lateral line display altered kinematics; body wavelength and wave speed increase compared to control animals. When visual cues are withheld by performing experiments in the dark, almost all Kármán gait kinematics measured for fish with and without a functional lateral line are the same. The lateral line, rather than vision, plays a larger role in affecting body kinematics when trout hold station in a vortex street. Trout show a preference to Kármán gait in the light but not in the dark, which may be attributed to physiological state rather than hydrodynamic or sensorimotor reasons. In the dark, trout both with and without a functional lateral line hold station near the downstream suction region of the cylinder wake (i.e. entraining) and avoid the vortex street. Vision therefore plays a larger role in the preference to associate with a turbulent vortex street. Trout in the light with a blocked lateral line show individual variation in their preference to Kármán gait or entrain. In the dark, entraining trout with an intact lateral line will alternate between right and left sides of the

  8. Effects of Soluble Surfactant on Lateral Migration of a Bubble in a Shear Flow

    NASA Astrophysics Data System (ADS)

    Muradoglu, Metin; Tryggvason, Gretar

    2014-11-01

    Motivated by the recent experimental study of Takagi et al. (2008), direct numerical simulations are performed to examine effects of soluble surfactant on the lateral migration of a deformable bubble in a pressure-driven channel flow. The interfacial and bulk surfactant concentration evolution equations are solved fully coupled with the incompressible Navier-Stokes equations. A non-linear equation of state is used to relate interfacial surface tension to surfactant concentration at the interface. A multiscale method is developed to handle the mass exchange between the interface and bulk fluid at high Peclet numbers, using a boundary-layer approximation next to the bubble and a relatively coarse grid for the rest of the flow. It is found that the surfactant induced Marangoni stresses can dominate over the shear-induced lift force and thus alter the behavior of the bubble completely, i.e., the contaminated bubble drifts away from the channel wall and stabilizes at the center of the channel in contrast with the corresponding clean bubble that drifts toward the wall and stabilizes near the wall. The Scientific and Technical Research Council of Turkey (TUBITAK), Grant 112M181 and Turkish Academy of Sciences (TUBA).

  9. Acoustic programming in step-split-flow lateral-transport thin fractionation.

    PubMed

    Ratier, Claire; Hoyos, Mauricio

    2010-02-15

    We propose a new separation scheme for micrometer-sized particles combining acoustic forces and gravitational field in split-flow lateral-transport thin (SPLITT)-like fractionation channels. Acoustic forces are generated by ultrasonic standing waves set up in the channel thickness. We report on the separation of latex particles of two different sizes in a preliminary experiment using this proposed hydrodynamic acoustic sorter, HAS. Total binary separation of 5 and 10 microm diameter particles has been achieved. Numerical simulations of trajectories of particles flowing through a step-SPLITT under the conditions which combine acoustic standing waves and gravity show a very good agreement with the experiment. Calculations in order to compare separations obtained by the acoustic programming s-SPLITT fractionation and the conventional SPLITT fractionation show that the improvement in separation time is around 1 order of magnitude and could still be improved; this is the major finding of this work. This separation technique can be extended to biomimetic particles and blood cells.

  10. Detection of staphylococcal enterotoxin B in milk and milk products using immunodiagnostic lateral flow devices.

    PubMed

    Boyle, Thomas; Njoroge, Joyce M; Jones, Robert L; Principato, Maryann

    2010-01-01

    Staphylococcal enterotoxin B (SEB) is an extracellular pyrotoxin produced by Staphylococcus aureus, a known etiologic agent of food poisoning in humans. Lateral flow immunochromatographic devices (LFDs) designed for the environmental detection of SEB were adapted for use in this study to detect SEB in milk containing 2% fat, chocolate-flavored milk, and milk-derived products such as yogurt, infant formula, and ice cream. The advantage of using LFDs in these particular food products was its ease and speed of use with no additional extraction methods needed. No false positives were observed with any of the products used in this study. Dilution of the samples overcame the Hook effect and permitted capillary flow into the membrane. Thus, semisolid products such as ice cream and some yogurts, and products containing thickeners needed to be diluted using a phosphate-buffered saline-based buffer, pH 7.2. SEB was easily detected at concentrations of 5 microg/mL and 500 ng/mL when the LFDs were used. SEB was also reliably detected at concentrations below 5 and 0.25 ng/mL, which may induce serious disease.

  11. Distributed flow estimation and closed-loop control of an underwater vehicle with a multi-modal artificial lateral line.

    PubMed

    DeVries, Levi; Lagor, Francis D; Lei, Hong; Tan, Xiaobo; Paley, Derek A

    2015-03-25

    Bio-inspired sensing modalities enhance the ability of autonomous vehicles to characterize and respond to their environment. This paper concerns the lateral line of cartilaginous and bony fish, which is sensitive to fluid motion and allows fish to sense oncoming flow and the presence of walls or obstacles. The lateral line consists of two types of sensing modalities: canal neuromasts measure approximate pressure gradients, whereas superficial neuromasts measure local flow velocities. By employing an artificial lateral line, the performance of underwater sensing and navigation strategies is improved in dark, cluttered, or murky environments where traditional sensing modalities may be hindered. This paper presents estimation and control strategies enabling an airfoil-shaped unmanned underwater vehicle to assimilate measurements from a bio-inspired, multi-modal artificial lateral line and estimate flow properties for feedback control. We utilize potential flow theory to model the fluid flow past a foil in a uniform flow and in the presence of an upstream obstacle. We derive theoretically justified nonlinear estimation strategies to estimate the free stream flowspeed, angle of attack, and the relative position of an upstream obstacle. The feedback control strategy uses the estimated flow properties to execute bio-inspired behaviors including rheotaxis (the tendency of fish to orient upstream) and station-holding (the tendency of fish to position behind an upstream obstacle). A robotic prototype outfitted with a multi-modal artificial lateral line composed of ionic polymer metal composite and embedded pressure sensors experimentally demonstrates the distributed flow sensing and closed-loop control strategies.

  12. Single cell kinase signaling assay using pinched flow coupled droplet microfluidics.

    PubMed

    Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S; Tan Shao Weng, Daniel; Thakor, Nitish V; Teck Lim, Chwee; Chen, Chia-Hung

    2014-05-01

    Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib.

  13. Lateral flow immunoassay with upconverting nanoparticle-based detection for indirect measurement of interferon response by the level of MxA.

    PubMed

    Juntunen, Etvi; Salminen, Teppo; Talha, Sheikh M; Martiskainen, Iida; Soukka, Tero; Pettersson, Kim; Waris, Matti

    2017-04-01

    Myxovirus resistance protein A (MxA) is a biomarker of interferon-induced gene expression state involved in many viral infections and some autoimmune disorders. It has a variety of potential utilities in clinical diagnostics, including distinguishing between bacterial and viral infections. Currently, MxA-assays are used for monitoring of IFN-β therapy in multiple sclerosis (MS) patients. As a proof-of-concept for rapid quantitative measurement of interferon response, a lateral flow immunoassay (LFIA) with upconverting nanoparticle (UCNP) reporters was developed and evaluated with clinical whole blood samples to assess the potential for a rapid and user-friendly quantitative assay for MxA, since the currently available rapid test for MxA (FebriDX) produces only qualitative result. The high detection sensitivity enabled by the UCNP reporter technology allowed the sample pre-treatment with dilution of whole blood into lysis buffer at a detectable analyte concentration. The assay can be done within 2 hr and the results correlate with the reference MxA-ELISA, which requires an overnight incubation. With 36 samples, R(2) for linear regression was 0.86. The assay detected 96% of the IFN-β responders with 89% specificity using a cut-off level of 100 μg/L for an elevated MxA-concentration. J. Med. Virol. 89:598-605, 2017. © 2016 Wiley Periodicals, Inc.

  14. Bead-based assays for biodetection: from flow-cytometry to microfluidics

    NASA Astrophysics Data System (ADS)

    Ozanich, Richard M., Jr.; Antolick, Kathryn; Bruckner-Lea, Cynthia J.; Bunch, Kyle J.; Dockendorff, Brian P.; Grate, Jay W.; Nash, Michael A.; Tyler, Abby; Warner, Cynthia L.; Warner, Marvin G.

    2009-05-01

    The potential for the use of biological agents by terrorists is a real threat. Two approaches for antibody-based detection of biological species are described in this paper: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. These approaches both involve the use of automated fluidic systems for trapping antibody-functionalized microbeads, which allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive immunoassays. The automated fluidic approach resulted in up to five-fold improvements in immunoassay sensitivity/speed as compared to identical immunoassays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based immunoassays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (>= 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100's of picomolar range (10's of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach.

  15. Early events in macrophage killing of Aspergillus fumigatus conidia: new flow cytometric viability assay.

    PubMed

    Marr, K A; Koudadoust, M; Black, M; Balajee, S A

    2001-11-01

    Detailed investigations of macrophage phagocytosis and killing of Aspergillus fumigatus conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active A. fumigatus conidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.

  16. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    NASA Astrophysics Data System (ADS)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  17. MicroRNA detection using lateral flow nucleic acid strips with gold nanoparticles.

    PubMed

    Hou, Shao-Yi; Hsiao, Yi-Ling; Lin, Ming-Shu; Yen, Chun-Che; Chang, Chi-Sheng

    2012-09-15

    In this study, the tested microRNA and the detection probe perfectly match with the capture probe instead of the traditional sandwich methods in which the tested oligonucleotide matches with the detection and capture probes. To avoid non-specific signals, mung-bean nuclease, a single-strand-specific nuclease, catalyzes the degradation of the capture probe if there is no tested miRNA in the samples. The gold nanoparticles conjugate the thiol-DNA as the detection probe and the biotin-single strand DNA serves as the capture probe. The avidin-biotin-Au-sample complex is captured by the anti-avidin antibody immobilized on a flow strip. The detection and quantification of the gold nanoparticle signal indicate the existence and quantity of the target miRNA. One fmol and five amol of the synthetic microRNA were detected without and with the silver enhancement, respectively. This highly sensitive and specific assay takes about 70 min after the RNA purification and preparation. It is simple, convenient, fast, and suitable for point-of-care. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Development of a Lateral Flow Immunoassay for the Rapid Diagnosis of Invasive Candidiasis.

    PubMed

    He, Zheng-Xin; Shi, Lan-Chun; Ran, Xiang-Yang; Li, Wei; Wang, Xian-Ling; Wang, Fu-Kun

    2016-01-01

    Early and accurate diagnosis of invasive candidiasis (IC) is very important. In this study, a lateral flow immunoassay (LFIA) was developed to detect antibody against Candida albicans enolase (Eno). Colloidal gold particle labeled mouse anti human IgG (1.0 mg/L) was used as the detector reagent. Recombinant enolase (rEno, 1.0 mg/L) and goat anti IgG (1.0 mg/L) were immobilized in test and control lines, respectively, of a nitrocellulose membrane, acting as the capture reagents. The LFIA was used to detect anti Eno in 38 sera from clinically proven IC patients, as well as in 50 healthy control subjects. Compared with an indirect ELISA designed as a reference test, the specificity and sensitivity of the LFIA were 98.2 and 84.8%, respectively. Excellent agreement between the results obtained by ELISA and the LFIA (κ = 0.851) was observed in this study. In addition, the agreement between the blood culture results and LFIA test is strong (κ = 0.658). The data presented in the study indicate that the LFIA test is a suitable tool for the serological surveillance of IC in the field or in poorly equipped laboratories.

  19. Rapid lateral-flow immunoassay for the quantum dot-based detection of puerarin.

    PubMed

    Qu, Huihua; Zhang, Yue; Qu, Baoping; Kong, Hui; Qin, Gaofeng; Liu, Shuchen; Cheng, Jinjun; Wang, Qingguo; Zhao, Yan

    2016-07-15

    In this study, a rapid (within 10min) quantitative lateral-flow immunoassay using a quantum dots (QDs)-antibody probe was developed for the analysis of puerarin (PUE) in water and biological samples. The competitive immunoassay was based on anti-PUE monoclonal antibody conjugated with QDs (detection reagent). Secondary antibody was immobilized on one end of a nitrocellulose membrane (control line) and PUE-bovine serum albumin conjugate was immobilized on the other end (test line). In the quantitative experiment, the detection results were scanned using a membrane strip reader and a detection curve (regression equation: y=-0.11ln(x)+0.979, R(2)=0.9816) representing the averages of the scanned data was obtained. This curve was linear from 1 to 10μg/mL. The IC50 value was 75.58ng/mL and the qualitative detection limit of PUE was 5.8ng/mL. The recovery of PUE added to phosphate-buffered saline and biological samples was in the range of 97.38-116.56%. To our knowledge, this is the first report of the quantitative detection of a natural product by QDs-based immunochromatography, which represents a powerful tool for rapidly screening PUE in plant materials and other biological samples.

  20. Development of a Rainbow Lateral Flow Immunoassay for the Simultaneous Detection of Four Mycotoxins.

    PubMed

    Foubert, Astrid; Beloglazova, Natalia V; Gordienko, Anna; Tessier, Mickael D; Drijvers, Emile; Hens, Zeger; De Saeger, Sarah

    2016-12-12

    A multiplex lateral flow immunoassay (LFIA) for the determination of the mycotoxins deoxynivalenol, zearalenone, and T2/HT2-toxin in barley was developed with luminescent quantum dots (QDs) as label. The synthesized QDs were hydrophilized by two strategies, that is, coating with an amphiphilic polymer or silica. The water-soluble QDs were compared with regard to their bioconjugation with monoclonal antibody (mAb) and were tested on a LFIA. Silica-coated QDs that contained epoxy groups were most promising. Therefore, green, orange, and red epoxy-functionalized silica-coated QDs were conjugated with anti-ZEN, anti-DON, and anti-T2 mAb, respectively. The LFIA was developed in accordance with the European Commission legal limits with cutoff limits of 1000, 80, and 80 μg/kg for deoxynivalenol, zearalenone, and T2/HT2-toxin, respectively. The LFIA gave a fast result (15 min) with a low false-negative rate (<5%), and the results were easy to interpret without any sophisticated equipment.

  1. Increased sensitivity of lateral flow immunoassay for ochratoxin A through silver enhancement.

    PubMed

    Anfossi, L; Di Nardo, F; Giovannoli, C; Passini, C; Baggiani, C

    2013-12-01

    Silver nucleation on gold has been exploited for signal amplification and has found application in several qualitative and quantitative bio-sensing techniques, thanks to the simplicity of the method and the high sensitivity achieved. Very recently, this technique has been tentatively applied to improve the performance of gold-based immunoassays. In this work, the exploitation of the signal amplification due to silver deposition on gold nanoparticles has been first applied to a competitive lateral flow immunoassay (LFIA). The signal enhancement due to silver allowed us to strongly reduce the amount of the competitor and of specific antibodies employed to build an LF device for measuring ochratoxin A (OTA), thus permitting the attainment of a highly sensitive assessment of OTA contamination, with a sensitivity gain of more than 10-fold compared to the gold-based LFIA that used the same immunoreagents and to all previously reported LFIA for measuring OTA. In addition, a less sensitive "quantitative" LFIA could be established, by suitably tuning competitor and antibody amounts, which was characterized by reproducible and accurate OTA determinations (RSD% 6-12%, recovery% 82-117%). The quantitative system allowed a reliable OTA quantification in wines and grape musts at the microgram per liter level requested by the European legislation, as demonstrated by a highly results obtained through the quantitative silver-enhanced LFIA and a reference HPLC-FLD on 30 samples.

  2. A novel method to detect Listeria monocytogenes via superparamagnetic lateral flow immunoassay.

    PubMed

    Shi, Lei; Wu, Feng; Wen, Yiming; Zhao, Fang; Xiang, Junjian; Ma, Lan

    2015-01-01

    A novel strip test system combining immunomagnetic separation with lateral flow immunoassay (LFIA) was established for the accurate detection of Listeria monocytogenes. In this system, a pair of matched monoclonal antibodies was used to construct a sandwich immunoassay, in which superparamagnetic particles were coupled with one of the antibodies as a labeled antibody to capture the target bacteria, while the other antibody was immobilized on the detection zone. After a 20-min reaction, the strips were analyzed by a novel instrument which could detect the magnetic signal of the immunocomplex in a magnetic field. Sensitivity evaluation showed that the limit of detection (LOD) of the superparamagnetic LFIA system for L. monocytogenes was 10(4) CFU/mL, which was at least one log lower than conventional LFIA. No cross-reaction was observed when Salmonella, Escherichia coli O157:H7, or three types of harmless Listeria strains were tested. Further evaluation with actual food samples indicated that the superparamagnetic LFIA system showed 100 % concordance with real-time PCR. Therefore, this novel superparamagnetic LFIA system could be used as a rapid, sensitive, and specific method for the detection of L. monocytogenes.

  3. Signal-Amplified Lateral Flow Test Strip for Visual Detection of Cu2+

    PubMed Central

    Xue, Juanjuan; Dong, Jinbo; Cai, Jia; Hua, Xiude; Wang, Minghua; Zhang, Cunzheng; Liu, Fengquan

    2017-01-01

    A signal-amplified lateral flow test strip (SA-LFTS) for the detection of Cu2+ in aqueous solution was constructed based on Cu+-catalyzed click chemistry and hybridization of single-stranded DNA (ssDNA). Alkyne and azide modified ssDNA acted as specific elements for Cu2+ recognition, and a chemical ligation product formed through Cu+-catalyzed alkyne–azide cycloaddition. Hybridization of ssDNA-labeled gold nanoparticles resulted in high sensitivity, and the output signal could be observed directly by the naked eye. Using the developed SA-LFTS under optimal conditions, Cu2+ could be detected rapidly with limit of detections of 5 nM and 4.2 nM by visual observation and quantitative analysis, respectively. The sensitivity (i.e. the visual limit of detection) of the SA-LFTS was 80-times higher than that of traditional LFTS. The SA-LFTS was applied to the determination of Cu2+ in municipal water and river water samples with the results showing good recovery and accuracy. The developed test strip is promising for point-of-care applications and detection of Cu2+ in the field. PMID:28072878

  4. Effects of different extraction buffers on peanut protein detectability and lateral flow device (LFD) performance.

    PubMed

    Rudolf, J; Ansari, P; Kern, C; Ludwig, T; Baumgartner, S

    2012-01-01

    The accidental uptake of peanuts can cause severe health reactions in allergic individuals. Reliable determination of traces of peanuts in food products is required to support correct labelling and therefore minimise consumers' risk. The immunoanalytical detectability of potentially allergenic peanut proteins is dependent on previous heat treatment, the extraction capacity of the applied buffer and the specificity of the antibody. In this study a lateral flow device (LFD) for the detection of peanut protein was developed and the capacity of 30 different buffers to extract proteins from mildly and strongly roasted peanut samples as well as their influence on the test strip performance were investigated. Most of the tested buffers showed good extraction capacity for putative Ara h 1 from mildly roasted peanuts. Protein extraction from dark-roasted samples required denaturing additives, which were proven to be incompatible with LFD performance. High-pH buffers increased the protein yield but inhibited signal generation on the test strip. Overall, the best results were achieved using neutral phosphate buffers but equal detectability of differently altered proteins due to food processing cannot be assured yet for immunoanalytical methods.

  5. Development of a Lateral Flow Immunoassay for the Rapid Diagnosis of Invasive Candidiasis

    PubMed Central

    He, Zheng-Xin; Shi, Lan-Chun; Ran, Xiang-Yang; Li, Wei; Wang, Xian-Ling; Wang, Fu-Kun

    2016-01-01

    Early and accurate diagnosis of invasive candidiasis (IC) is very important. In this study, a lateral flow immunoassay (LFIA) was developed to detect antibody against Candida albicans enolase (Eno). Colloidal gold particle labeled mouse anti human IgG (1.0 mg/L) was used as the detector reagent. Recombinant enolase (rEno, 1.0 mg/L) and goat anti IgG (1.0 mg/L) were immobilized in test and control lines, respectively, of a nitrocellulose membrane, acting as the capture reagents. The LFIA was used to detect anti Eno in 38 sera from clinically proven IC patients, as well as in 50 healthy control subjects. Compared with an indirect ELISA designed as a reference test, the specificity and sensitivity of the LFIA were 98.2 and 84.8%, respectively. Excellent agreement between the results obtained by ELISA and the LFIA (κ = 0.851) was observed in this study. In addition, the agreement between the blood culture results and LFIA test is strong (κ = 0.658). The data presented in the study indicate that the LFIA test is a suitable tool for the serological surveillance of IC in the field or in poorly equipped laboratories. PMID:27679622

  6. A lateral flow biosensor for the detection of human pluripotent stem cells.

    PubMed

    Wu, Wei; Yu, Luxin; Fang, Zhiyuan; Lie, Puchang; Zeng, Lingwen

    2013-05-15

    A lateral flow biosensor based on immunoassay has been developed for the detection of human stem cells for the first time. Antibody specific for a human stem cell surface antigen, SSEA-4, is coated onto gold nanoparticles, whereas antibody against another human pluripotent stem cell surface antigen, SSEA-3, is immobilized on the test zone of the NC membrane. Target cells bind to the antibody coated on the gold nanoparticles to form nanoparticles-stem cell complexes, and the complexes are then captured by another antibody immobilized on the test zone to form a red line for visual detection. This biosensor has been successfully applied to human embryonic stem cells and induced pluripotent stem cells. It is capable of detecting a minimum of 10,000 human embryonic stem cells by the naked eye and 7000 cells with a portable strip reader within 20 min. This approach has also shown excellent specificity to distinguish other types of cells. The biosensor shows great promise for specific and handy detection of human pluripotent stem cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Dynamic topography, gravity and the role of lateral viscosity variations from inversion of global mantle flow

    NASA Astrophysics Data System (ADS)

    Yang, Ting; Gurnis, Michael

    2016-11-01

    Lateral viscosity variations (LVVs) in the mantle influence geodynamic processes and their surface expressions. With the observed long-wavelength geoid, free-air anomaly, gravity gradient in three directions and discrete, high-accuracy residual topography, we invert for depth- and temperature-dependent and tectonically regionalized mantle viscosity with a mantle flow model. The inversions suggest that long-wavelength gravitational and topographic signals are mainly controlled by the radial viscosity profile; the pre-Cambrian lithosphere viscosity is slightly (˜ one order of magnitude) higher than that of oceanic and Phanerozoic lithosphere; plate margins are substantially weaker than plate interiors; and viscosity has only a weak apparent, dependence on temperature, suggesting either a balancing between factors or a smoothing of actual higher amplitude, but short wavelength, LVVs. The predicted large-scale lithospheric stress regime (compression or extension) is consistent with the world stress map (thrust or normal faulting). Both recent compiled high-accuracy residual topography and the predicted dynamic topography yield ˜1 km amplitude long-wavelength dynamic topography, inconsistent with recent studies suggesting amplitudes of ˜100 to ˜500 m. Such studies use a constant, positive admittance (transfer function between topography and gravity), in contrast to the evidence which shows that the earth has a spatially and wavelength-dependent admittance, with large, negative admittances between ˜4000 and ˜104 km wavelengths.

  8. Development of a Smartphone-based reading system for lateral flow immunoassay.

    PubMed

    Lee, Sangdae; Kim, Giyoung; Moon, Jihea

    2014-11-01

    This study was conducted to develop and evaluate the performance of the Smartphone-based reading system for the lateral flow immunoassay (LFIA). Smartphone-based reading system consists of a Samsung Galaxy S2 Smartphone, Smartphone application, and a LFIA reader. LFIA reader is composed of the close-up lens with a focal length up to 30 mm, white LED light, lithium polymer battery, and main body. The Smartphone application for image acquisition and data analysis was developed on the Android platform. The standard curve was obtained by plotting the measured P(T)/P(c) or A(T)/A(c) ratio versus Salmonella standard concentration. The mean, standard deviation (SD), recovery, and relative standard deviation (RSD) were also calculated using additional experimental results. These data were compared with that obtained from the benchtop LFIA reader. The LOD in both systems was observed with 10(6) CFU/mL. The results show high accuracy and good reproducibility with a RSD less than 10% in the range of 10(6) to 10(9) CFU/mL. Due to the simple structure, good sensitivity, and high accuracy of the Smartphone-based reading system, this system can be substituted for the benchtop LFIA reader for point-of-care medical diagnostics.

  9. Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample.

    PubMed

    Saidin, Syazwan; Yunus, Muhammad Hafiznur; Othman, Nurulhasanah; Lim, Yvonne Ai-Lian; Mohamed, Zeehaida; Zakaria, Nik Zairi; Noordin, Rahmah

    2017-05-01

    Entamoeba histolytica infection remains a public health concern in developing countries. Early diagnosis of amoebiasis can avoid disease complications, thus this study was aimed at developing a test that can rapidly detect the parasite antigens in stool samples. Rabbits were individually immunized with recombinant pyruvate phosphate dikinase (rPPDK) and E. histolytica excretory-secretory antigens to produce polyclonal antibodies. A rapid dipstick test was produced using anti-rPPDK PAb lined on the dipstick as capture reagent and anti-EhESA PAb conjugated to colloidal gold as the detector reagent. Using E. histolytica-spiked in stool sample of a healthy individual, the detection limit of the dipstick test was found to be 1000 cells ml(-1). Meanwhile when rPPDK was spiked in the stool sample, the minimum concentration detected by the dipstick test was 0.1 μg ml(-1). The performances of the dipstick, commercial Techlab E. histolytica II enzyme-linked immunosorbent assays (ELISA) and real-time PCR were compared using 70 stool samples from patients infected with Entamoeba species (n = 45) and other intestinal pathogens (n = 25). When compared to real-time PCR, the diagnostic sensitivity of the dipstick for detection of E. histolytica was 65.4% (n = 17/26); while the diagnostic specificity when tested with stool samples containing other intestinal pathogens was 92% (23/25). In contrast, Techlab E. histolytica II ELISA detected 19.2% (5/26) of the E. histolytica-positive samples as compared to real-time PCR. The lateral flow dipstick test produced in this study enabled rapid detection of E. histolytica, thus it showed good potential to be further developed into a diagnostic tool for intestinal amoebiasis.

  10. Detection of single-nucleotide polymorphisms in Plasmodium falciparum by PCR primer extension and lateral flow immunoassay.

    PubMed

    Moers, A P H A; Hallett, R L; Burrow, R; Schallig, H D F H; Sutherland, C J; van Amerongen, A

    2015-01-01

    The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries.

  11. Factors influencing the detection limit of the lateral-flow sandwich immunoassay: a case study with potato virus X.

    PubMed

    Safenkova, Irina; Zherdev, Anatoly; Dzantiev, Boris

    2012-06-01

    Key factors influencing the analyte detection limit of the sandwich immunochromatographic assay (ICA), namely, the size of gold nanoparticles, the antibody concentration, the conjugation pH, and characteristics of membranes, are discussed. The impacts of these factors were quantitatively characterized and compared for the first time using the same antigen (potato virus X). The antibody-colloidal gold conjugates synthesized at pH 9.0-9.5 (the pH was examined in the range from 7.5 to 10.0) and at an antibody concentration of 15 μg/mL (the concentration was tested from 10 to 100 μg/mL) demonstrated maximum binding with the analyte. The relationship between the size of gold nanoparticles and the ICA detection limit was determined. The detection limit decreases from 80 to 3 ng/mL (for antibodies with K (D) = 1.0 × 10(-9) M, data were obtained using a BIAcore X instrument) for a series of particles with a diameter from 6.4 to 33.4 nm (electron microscopy and dynamic light scattering data). In the case of larger particles (52 nm in diameter), the detection limit increases and reaches 9 ng/mL. A 10 mM phosphate buffer, pH 8, and a 50 mM phosphate buffer, pH 7, were the conditions of choice for the deposition of reactants. Taking into account these facts, we developed a lateral-flow test system for the rapid (10 min) detection of potato virus X in plant leaves. The ICA provided a visual detection limit of 3 ng/mL. In the case of the instrumental processing, potato virus X can be determined in the concentration range from 3 to 300 ng/mL with a detection limit 2 ng/mL.

  12. Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor.

    PubMed

    Ang, Geik Yong; Yu, Choo Yee; Yean, Chan Yean

    2012-01-01

    In the field of diagnostics, molecular amplification targeting unique genetic signature sequences has been widely used for rapid identification of infectious agents, which significantly aids physicians in determining the choice of treatment as well as providing important epidemiological data for surveillance and disease control assessment. We report the development of a rapid nucleic acid lateral flow biosensor (NALFB) in a dry-reagent strip format for the sequence-specific detection of single-stranded polymerase chain reaction (PCR) amplicons at ambient temperature (22-25°C). The NALFB was developed in combination with a linear-after-the-exponential PCR assay and the applicability of this biosensor was demonstrated through detection of the cholera toxin gene from diarrheal-causing toxigenic Vibrio cholerae. Amplification using the advanced asymmetric PCR boosts the production of fluorescein-labeled single-stranded amplicons, allowing capture probes immobilized on the NALFB to hybridize specifically with complementary targets in situ on the strip. Subsequent visual formation of red lines is achieved through the binding of conjugated gold nanoparticles to the fluorescein label of the captured amplicons. The visual detection limit observed with synthetic target DNA was 0.3 ng and 1 pg with pure genomic DNA. Evaluation of the NALFB with 164 strains of V. cholerae and non-V. cholerae bacteria recorded 100% for both sensitivity and specificity. The whole procedure of the low-cost NALFB, which is performed at ambient temperature, eliminates the need for preheated buffers or additional equipment, greatly simplifying the protocol for sequence-specific PCR amplicon analysis.

  13. Detection of Single-Nucleotide Polymorphisms in Plasmodium falciparum by PCR Primer Extension and Lateral Flow Immunoassay

    PubMed Central

    Moers, A. P. H. A.; Hallett, R. L.; Burrow, R.; Schallig, H. D. F. H.; Sutherland, C. J.

    2014-01-01

    The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries. PMID:25367901

  14. Lateral Preferential Flow in Soil Pipes on Hillslopes in the Catskill Mountains, New York, USA

    NASA Astrophysics Data System (ADS)

    Harpold, A. A.; Steenhuis, T. S.; Dahlke, H. E.

    2006-12-01

    Lateral preferential flow has been shown to be a significant factor controlling the timing and volume of hillslope runoff. In addition, preferential flow, including pipeflow, can reduce the contact time of contaminants with the soil matrix and thus profoundly alter runoff chemistry. This study examines the importance of soil pipes on hydrologic response and runoff chemistry from a hillslope in the Catskill Mountains of New York State. The pipes examined are unique in location, depth, and flow characteristics from previously published studies in North America. The implications of pipeflow on hydrologic process understanding and land management in both agricultural and pristine watersheds in the Catskills are numerous. Therefore, chemical tracers and hydrometric techniques are used to determine the hydrologic response characteristics, contributing area, and nutrient transport capacity of the pipes and non-invasive geophysical methods are used to investigate the morphology of the pipes and their importance in landscape formation. This study was conducted on a hillslope in the Town Brook watershed in the Catskill Mountains. Soil pipes were initially identified by visual and auditory reconnaissance. Soil pipe locations and frequency were further defined using ground penetrating radar (GPR). Additional pipe characteristics were estimated using simple tracer studies (using dye and salt) and by measuring the size of particles ejected by the pipe. After identification, the hillslope was instrumented with equipment capable of measuring the hydrologic response of the pipe, including a weir and tipping buckets measuring pipe outflow, a network of piezometers and tensiometers, and automated rain gauge. Water quality measurements were collected using automated samplers and event-based grab samples at several locations: upslope surface water, soil moisture (using a cluster of lysimeters), rainfall, pipe outflow, and stream water at the outlet of the subcatchment. Mixing models

  15. Redox-magnetohydrodynamics, flat flow profile-guided enzyme assay detection: toward multiple, parallel analyses.

    PubMed

    Sahore, Vishal; Fritsch, Ingrid

    2014-10-07

    A proof-of-concept superparamagnetic microbead-enzyme complex was integrated with microfluidics pumped by redox-magneto-hydrodynamics (MHD) to take advantage of the magnet (0.56 T) beneath the chip and the uniform flat flow profile, as a first step toward developing multiple, parallel chemical analyses on a chip without the need for independent channels. The superparamagnetic beads were derivatized with alkaline phosphatase (a common enzyme label for biochemical assays) and magnetically immobilized at three different locations on the chip with one directly on the path to the detector and the other two locations adjacent to, but off the path, by a distance >5 times the detector diameter. Electroactive p-aminophenol, enzymatically generated at the bead-enzyme complex from its electroinactive precursor p-aminophenyl phosphate in a solution containing a redox species [Ru(NH3)6](3+/2+) for pumping and Tris buffer, was transported by redox-MHD and detected with square wave voltammetry at a 312 μm diameter gold microdisk stationed 2 mm downstream from the bead-complex on the flow path. Oppositely biased pumping electrodes, consisting of 2.5 cm long gold bands and separated by 5.6 mm, flanked the active flow region containing the bead-enzyme complex and detection site. The signal from adjacent paths was only 20% of that for the direct path and ≤8% when pumping electrodes were inactive.

  16. FRET and Flow Cytometry Assays to Measure Proteopathic Seeding Activity in Biological Samples.

    PubMed

    Furman, Jennifer L; Diamond, Marc I

    2017-01-01

    Transcellular propagation of protein aggregates-or seeds-is increasingly implicated as a mechanism for disease progression in many neurodegenerative disorders, including Alzheimer's disease and the related tauopathies. While neuropathology generally originates in one discrete brain region, pathology progresses as disease severity advances, often along discrete neural networks. The stereotypical spread of tau pathology suggests that cell-to-cell transfer of toxic protein aggregates could underlie disease progression, and recent studies implicate seeding as a proximal marker of disease, as compared to standard histological and biochemical analyses. Commonly used metrics for protein aggregation detection, however, lack sensitivity, are not quantitative, and/or undergo subjective classification. Here, we describe a FRET and flow cytometry cell-based assay that allows for rapid and quantitative detection of protein aggregates from human and rodent biological specimens.

  17. Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system.

    PubMed

    Hartwell, Supaporn Kradtap; Somprayoon, Duangporn; Kongtawelert, Prachya; Ongchai, Siriwan; Arppornchayanon, Olarn; Ganranoo, Lucksagoon; Lapanantnoppakhun, Somchai; Grudpan, Kate

    2007-09-26

    Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.

  18. A novel quantitative kinase assay using bacterial surface display and flow cytometry.

    PubMed

    Henriques, Sónia Troeira; Thorstholm, Louise; Huang, Yen-Hua; Getz, Jennifer A; Daugherty, Patrick S; Craik, David J

    2013-01-01

    The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli) to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development.

  19. Modeling performance of a two-dimensional capsule in a microchannel flow: long-term lateral migration.

    PubMed

    Li, Hua; Ma, Gang

    2010-08-01

    The long-term lateral migration of a two-dimensional elastic capsule in a microchannel is studied numerically in this paper. The numerical method combines a finite volume technique for solving the fluid problem with a front tracking technique for capturing and tracking the capsule membrane. The capsule is modeled as a liquid medium enclosed by a thin membrane which has linear elastic properties. The capsule, whose initial shape is circle and which starts from a near-center position or a near-wall position, experiences tilting and membrane tank-treading, and migrates laterally when moving along the surrounding flow. The lateral migration demonstrates the existence of lift effect of surrounding flow on moving capsule. Before capsule approaches to the microchannel centerline closely, lower membrane dilation modulus and lower viscosity ratio tend to result in faster lateral migration. The initial position also influences the performance behavior of capsule, despite the lateral migration of capsule is a quasisteady process. Small difference in capsule behavior when capsule is not near to the microchannel centerline might lead to significant difference in capsule behavior when capsule approaches closely to the centerline. When capsules are near to microchannel wall, the effect of the wall on capsule behavior might dominate, leading to relatively faster lateral migration. When capsules are not far from microchannel centerline, the effect of the nonlinearity of Poiseuille flow might dominate, resulting in relatively slower lateral movement. When capsules are located closely to the centerline, they behave differently, where the reason still remains poorly understood and it will be one of our future studies. The comparison between the capsule behavior from the present simulation and that by the migration law proposed by Coupier [Phys. Fluids 20, 111702 (2008)] shows that the behavioral agreement for near-wall capsule is better than that for near-center capsule, and the best

  20. Impact of lateral flow on the transition from connected to disconnected stream-aquifer systems

    NASA Astrophysics Data System (ADS)

    Xian, Yang; Jin, Menggui; Liu, Yanfeng; Si, Aonan

    2017-05-01

    Understanding the mechanisms by which stream water infiltrates through streambeds to recharge groundwater systems is essential to sustainable management of scarce water resources in arid and semi-arid areas. An inverted water table (IWT) can develop under a stream in response to the desaturation between the stream and underlying aquifer as the system changes from a connected to disconnected status. However, previous studies have suggested that the IWT can only occur at the bottom of a low permeability streambed in which only the vertical flow between the stream and groundwater during disconnection was assumed. In the present study, numerical simulations revealed that the lateral flow induced by capillarity or heterogeneity also plays an essential role on interactions between streams and aquifers. Three pathways were identified for the transition from connection to disconnection in homogenous systems; notably, the lowest point of an IWT can develop not only at the bottom of the streambed but also within the streambed or the aquifer in response to the initial desaturation at, above, or below the interface between the streambed and aquifer (IBSA), respectively. A sensitivity analysis indicated that in wide streams, the lowest point of an IWT only occurs at the bottom of the streambed; however, for a stream half width of 1 m above a 6 m thick sandy loam streambed, the lowest point occurs in the streambed as stream depth is less than 0.5 m. This critical stream depth increases with streambed thickness and decreases with stream width. Thus, in narrow streams the lowest point can also develop in a thick streambed under a shallow stream. In narrow streams, the lowest point also forms in the aquifer if the ratio of the hydraulic conductivity of the streambed to that of the aquifer is greater than the ratio of the streambed thickness to the sum of the stream depth and the streambed thickness; correspondingly, the streambed is thin but relatively permeable and the stream is

  1. CdSe/ZnS Quantum Dot-Labeled Lateral Flow Strips for Rapid and Quantitative Detection of Gastric Cancer Carbohydrate Antigen 72-4.

    PubMed

    Yan, Xinyu; Wang, Kan; Lu, Wenting; Qin, Weijian; Cui, Daxiang; He, Jinghua

    2016-12-01

    Carbohydrate antigen 72-4 (CA72-4) is an important biomarker associated closely with diagnosis and prognosis of early gastric cancer. How to realize quick, sensitive, specific, and quantitative detection of CA72-4 in clinical specimens has become a great requirement. Herein, we reported a CdSe/ZnS quantum dot-labeled lateral flow test strip combined with a charge-coupled device (CCD)-based reader was developed for rapid, sensitive, and quantitative detection of CA72-4. Two mouse monoclonal antibodies (mAbs) against CA72-4 were employed. One of them was coated as a test line, while another mAb was labeled with quantum dots and coated onto conjugate pad. The goat anti-mouse IgG was immobilized as a control line. After sample was added, a sandwich structure was formed with CA72-4 and these two mAbs. The fluorescent signal from quantum dots (QD)-labeled mAb in sandwich structure was related to the amount of detected CA72-4. A CCD-based reader was used to realize quantitative detection of CA72-4. Results showed that developed QD-labeled lateral flow strips to detect CA72-4 biomarker with the sensitivity of 2 IU/mL and 10 min detection time. One hundred sera samples from clinical patients with gastric cancer and healthy people were used to confirm specificity of this strip method; results showed that established strip method own 100 % reproducibility and 100 % specificity compared with Roche electrochemiluminescence assay results. In conclusion, CdSe/ZnS quantum dot-labeled lateral flow strips for detection of CA72-4 could realize rapid, sensitive, and specific detection of clinical samples and could own great potential in clinical translation in near future.

  2. CdSe/ZnS Quantum Dot-Labeled Lateral Flow Strips for Rapid and Quantitative Detection of Gastric Cancer Carbohydrate Antigen 72-4

    NASA Astrophysics Data System (ADS)

    Yan, Xinyu; Wang, Kan; Lu, Wenting; Qin, Weijian; Cui, Daxiang; He, Jinghua

    2016-03-01

    Carbohydrate antigen 72-4 (CA72-4) is an important biomarker associated closely with diagnosis and prognosis of early gastric cancer. How to realize quick, sensitive, specific, and quantitative detection of CA72-4 in clinical specimens has become a great requirement. Herein, we reported a CdSe/ZnS quantum dot-labeled lateral flow test strip combined with a charge-coupled device (CCD)-based reader was developed for rapid, sensitive, and quantitative detection of CA72-4. Two mouse monoclonal antibodies (mAbs) against CA72-4 were employed. One of them was coated as a test line, while another mAb was labeled with quantum dots and coated onto conjugate pad. The goat anti-mouse IgG was immobilized as a control line. After sample was added, a sandwich structure was formed with CA72-4 and these two mAbs. The fluorescent signal from quantum dots (QD)-labeled mAb in sandwich structure was related to the amount of detected CA72-4. A CCD-based reader was used to realize quantitative detection of CA72-4. Results showed that developed QD-labeled lateral flow strips to detect CA72-4 biomarker with the sensitivity of 2 IU/mL and 10 min detection time. One hundred sera samples from clinical patients with gastric cancer and healthy people were used to confirm specificity of this strip method; results showed that established strip method own 100 % reproducibility and 100 % specificity compared with Roche electrochemiluminescence assay results. In conclusion, CdSe/ZnS quantum dot-labeled lateral flow strips for detection of CA72-4 could realize rapid, sensitive, and specific detection of clinical samples and could own great potential in clinical translation in near future.

  3. Improving lateral-flow immunoassay (LFIA) diagnostics via biomarker enrichment for mHealth.

    PubMed

    Lai, James J; Stayton, Patrick S

    2015-01-01

    Optical detection technologies based on mobile devices can be utilized to enable many mHealth applications, including a reader for lateral-flow immunoassay (LFIA). However, an intrinsic challenge associated with LFIA for clinical diagnostics is the limitation in sensitivity. Therefore, rapid and simple specimen processing strategies can directly enable more sensitive LFIA by purifying and concentrating biomarkers. Here, a binary reagent system is presented for concentrating analytes from a larger volume specimen to improve the malaria LFIA's limit of detection (LOD). The biomarker enrichment process utilizes temperature-responsive gold-streptavidin conjugates, biotinylated antibodies, and temperature-responsive magnetic nanoparticles. The temperature-responsive gold colloids were synthesized by modifying the citrate-stabilized gold colloids with a diblock copolymer, containing a thermally responsive poly(N-isopropylacrylamide) (pNIPAAm) segment and a gold-binding block composed of NIPAAm-co-N,N-dimethylaminoethylacrylamide. The gold-streptavidin conjugates were synthesized by conjugating temperature-responsive gold colloids with streptavidin via covalent linkages using carbodiimide chemistry chemistry. The gold conjugates formed half-sandwiches, gold labeled biomarker, by complexing with biotinylated antibodies that were bound to Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria antigen. When a thermal stimulus was applied in conjunction with a magnetic field, the half-sandwiches and temperature-responsive magnetic nanoparticles that were both decorated with pNIPAAm formed large aggregates that were efficiently magnetically separated from human plasma. The binary reagent system was applied to a large volume (500 μL) specimen for concentrating biomarker 50-fold into a small volume and applied directly to an off-the-shelf malaria LFIA to improve the signal-to-noise ratio.

  4. Rapid Molecular Detection of Multidrug-Resistant Tuberculosis by PCR-Nucleic Acid Lateral Flow Immunoassay.

    PubMed

    Kamphee, Hatairat; Chaiprasert, Angkana; Prammananan, Therdsak; Wiriyachaiporn, Natpapas; Kanchanatavee, Airin; Dharakul, Tararaj

    2015-01-01

    Several existing molecular tests for multidrug-resistant tuberculosis (MDR-TB) are limited by complexity and cost, hindering their widespread application. The objective of this proof of concept study was to develop a simple Nucleic Acid Lateral Flow (NALF) immunoassay as a potential diagnostic alternative, to complement conventional PCR, for the rapid molecular detection of MDR-TB. The NALF device was designed using antibodies for the indirect detection of labeled PCR amplification products. Multiplex PCR was optimized to permit the simultaneous detection of the drug resistant determining mutations in the 81-bp hot spot region of the rpoB gene (rifampicin resistance), while semi-nested PCR was optimized for the S315T mutation detection in the katG gene (isoniazid resistance). The amplification process additionally targeted a conserved region of the genes as Mycobacterium tuberculosis (Mtb) DNA control. The optimized conditions were validated with the H37Rv wild-type (WT) Mtb isolate and Mtb isolates with known mutations (MT) within the rpoB and katG genes. Results indicate the correct identification of WT (drug susceptible) and MT (drug resistant) Mtb isolates, with the least limit of detection (LOD) being 104 genomic copies per PCR reaction. NALF is a simple, rapid and low-cost device suitable for low resource settings where conventional PCR is already employed on a regular basis. Moreover, the use of antibody-based NALF to target primer-labels, without the requirement for DNA hybridization, renders the device generic, which could easily be adapted for the molecular diagnosis of other infectious and non-infectious diseases requiring nucleic acid detection.

  5. Membrane-based lateral flow immunochromatographic strip with nanoparticles as reporters for detection: A review.

    PubMed

    Huang, Xiaolin; Aguilar, Zoraida P; Xu, Hengyi; Lai, Weihua; Xiong, Yonghua

    2016-01-15

    Membrane-based lateral flow immunochromatographic strip (LFICS) is widely used in various fields because of its simplicity, rapidity (detection within 10min), and low cost. However, early designs of membrane-based LFICS for preliminary screening only provide qualitative ("yes/no" signal) or semi-quantitative results without quantitative information. These designs often suffer from low-signal intensity and poor sensitivity and are only capable of single analyte detection, not simultaneous multiple detections. The performance of existing techniques used for detection using LFICS has been considerably improved by incorporating different kinds of nanoparticles (NPs) as reporters. NPs can serve as alternative labels and improve analytical sensitivity or limit of detection of LFICS because of their unique properties, such as optical absorption, fluorescence spectra, and magnetic properties. The controlled manipulation of NPs allows simultaneous or multiple detections by using membrane-based LFICS. In this review, we discuss how colored (e.g., colloidal gold, carbon, and colloidal selenium NPs), luminescent (e.g., quantum dots, up-converting phosphor NPs, and dye-doped NPs), and magnetic NPs are integrated into membrane-based LFICS for the detection of target analytes. Gold NPs are also featured because of their wide applications. Different types and unique properties of NPs are briefly explained. This review focuses on examples of NP-based LFICS to illustrate novel concepts in various devices with potential applications as screening tools. This review also highlights the superiority of NP-based approaches over existing conventional strategies for clinical analysis, food safety, and environmental monitoring. This paper is concluded by a short section on future research trends regarding NP-based LFICS. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Multiplex Lateral Flow Immunoassays Based on Amorphous Carbon Nanoparticles for Detecting Three Fusarium Mycotoxins in Maize.

    PubMed

    Zhang, Xiya; Yu, Xuezhi; Wen, Kai; Li, Chenglong; Mujtaba Mari, Ghulam; Jiang, Haiyang; Shi, Weimin; Shen, Jianzhong; Wang, Zhanhui

    2017-09-13

    The detecting labels used for lateral flow immunoassays (LFAs) have been traditionally gold nanoparticles (GNPs) and, more recently, luminescent nanoparticles, such as quantum dots (QDs). However, these labels have low sensitivity and are costly, in particular, for trace detection of mycotoxins in cereals. Here, we provided a simple preparation procedure for amorphous carbon nanoparticles (ACNPs) and described multiplex LFAs employing ACNPs as labels (ACNP-LFAs) for detecting three Fusarium mycotoxins. The analytical performance of ACNPs in LFA was compared to GNPs and QDs using the same immunoreagents, except for the labels, allowing for their analytical characteristics to be objectively compared. The visual limit of detection for ACNP-LFAs in buffer was 8-fold better than GNPs and 2-fold better than QDs. Under optimized conditions, the quantitative limit of detection of ACNP-LFAs in maize was as low as 20 μg/kg for deoxynivalenol, 13 μg/kg for T-2 toxin, and 1 μg/kg for zearalenone. These measurements were much lower than the action level of these mycotoxins in maize. The accuracy and precision of the ACNP-LFAs were evaluated by analysis of spiked and incurred maize samples with recoveries of 84.6-109% and coefficients of variation below 13%. The results of ACNP-LFAs using naturally incurred maize samples showed good agreement with results from high-performance liquid chromatography-tandem mass spectrometry, indicating that ACNPs were more sensitive labels than and a promising alternative to GNPs used in LFAs for detecting mycotoxins in cereals.

  7. Immunochromatographic lateral flow test for detection of antibodies to Equine infectious anemia virus.

    PubMed

    Alvarez, I; Gutierrez, G; Barrandeguy, M; Trono, K

    2010-08-01

    The purpose of this study was to develop and evaluate a simple immunochromatographic lateral flow (ICLF) test for specific detection of Equine infectious anemia virus (EIAV) antibodies in equine sera. Viral recombinant p26 capsid protein (rp26) was used as the capture protein in the test line and as the detector reagent conjugated to colloidal gold. The performance of rp26-ICLF was evaluated, and the results obtained were compared with a commercially available agar gel immunodiffusion (AGID) test used as a standard of comparison according to international guidelines. The values obtained for comparative diagnostic sensitivity (98.3%), diagnostic specificity (87.4%) and concordance (92.4%) were similar to those reported for other ICLF tests for animal infectious diseases. Very good repeatability and reproducibility, as well as a total agreement with blind previous results from three proficiency test panels, were obtained, thus indicating that rp26-ICLF is a precise test. The end point of the twofold serial dilution of serum samples was the same as, and even better than, the AGID test, thus demonstrating the same analytical sensitivity as that of the reference method for EIA diagnosis. No cross-reactivity was observed when serum samples from horses with other infectious diseases were analyzed. rp26-ICLF proved to be a precise and rapid test suitable for field screening in veterinary practice, since minimal equipment and operator expertise are required. However, further research should be carried out to increase the level of sensitivity. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  8. Integrated OLED as excitation light source in fluorescent lateral flow immunoassays.

    PubMed

    Venkatraman, Vishak; Steckl, Andrew J

    2015-12-15

    The integration of organic light emitting diodes (OLEDs) as excitation light sources for quantum dot-based fluorescent lateral flow immunoassay systems (LFIA) was investigated. This approach has the potential to deliver a sensitive visible detection scheme for low-cost, disposable lab-on-chip point-of-care (POC) diagnosis system. Thin film phosphorescent green OLEDs fabricated on plastic substrates were integrated on-chip to excite the test line of a quantum dot-based LFIA (QD-LFIA). OLEDs were fabricated by sequential deposition of organic thin films (total of ~100 nm) onto ITO-coated PET substrates. CdSe/ZnS QDs emitting at 655 nm and Au nanoparticles (NP - 10 nm size) conjugated antibodies were used for the fluorescence QD-LFIA and conventional reflection-mode Au NP-LFIA, respectively. Thin plastic color light filters were integrated for filtering the excitation light source and, thereby, increasing the contrast of the emitted light for optimized visual detection. Integration of the OLED and color filters with the analytical membrane was achieved using adhesive techniques facilitated by the planar nature of the layers, which suggests possible large scale manufacturing using roll-to-roll processing. Gray scale analysis from digital images captured with a digital camera was used to quantify the visual sensitivity. The signal intensity, signal-to-noise ratio (SNR) and the limit of detection (LOD) of OLED integrated QD-LFIAs were compared to Au NP LFIAs. OLED QD-LFIA exhibited superior performance in all signal aspects: 7-8× higher signal intensity and SNR, and a 7× lower LOD of 3 nM (measured at S/N=3). These results demonstrate the potential of OLED-integrated in LFIA devices for obtaining sensitive, fast and low-cost POC diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Lateral flow devices for nucleic acid analysis exploiting quantum dots as reporters.

    PubMed

    Sapountzi, Eleni A; Tragoulias, Sotirios S; Kalogianni, Despina P; Ioannou, Penelope C; Christopoulos, Theodore K

    2015-03-15

    There is a growing interest in the development of biosensors in the form of simple lateral flow devices that enable visual detection of nucleic acid sequences while eliminating several steps required for pipetting, incubation and washing out the excess of reactants. In this work, we present the first dipstick-type nucleic acid biosensors based on quantum dots (QDs) as reporters. The biosensors enable sequence confirmation of the target DNA by hybridization and simple visual detection of the emitted fluorescence under a UV lamp. The 'diagnostic' membrane of the biosensor contains a test zone (TZ) and a control zone (CZ). The CZ always fluoresces in order to confirm the proper function of the biosensor. Fluorescence is emitted from the TZ, only when the specific nucleic acid sequence is present. We have developed two general types of QD-based nucleic acid biosensors, namely, Type I and Type II, in which the TZ consists of either immobilized streptavidin (Type I) or immobilized oligodeoxynucleotides (Type II). The control zone consists of immobilized biotinylated albumin. No purification steps are required prior to the application of the DNA sample on the strip. The QD-based nucleic acid biosensors performed accurately and reproducibly when applied to (a) the visual detection of PCR amplification products and (b) visual genotyping of single nucleotide polymorphisms (SNPs) in human genomic DNA from clinical samples. As low as 1.5 fmol of double-stranded DNA were clearly detected by naked eye and the dynamic range extended to 200 fmol. The %CV were estimated to be 4.3-8.2. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. High sensitive gold-nanoparticle based lateral flow Immunodevice for Cd2+ detection in drinking waters.

    PubMed

    López Marzo, Adaris M; Pons, Josefina; Blake, Diane A; Merkoçi, Arben

    2013-09-15

    In this work for first time a lateral flow immunosensor device (LFID) for Cd(2+) determination in drinking and tap waters using the Cd-EDTA-BSA-AuNP conjugate as signal producer tool is introduced. The principle of working is based on competitive reaction between the Cd-EDTA-BSA-AuNP conjugate deposited on the conjugation pad strip and the Cd-EDTA complex formed in the analysis sample for the same binding sites of the 2A81G5 monoclonal antibody, specific to Cd-EDTA but not Cd(2+) free, which is immobilized onto the test line. The device has a large response range within 0.4-2000ppb, being the linear response between 0.4 and 10ppb. The quantification and detection limits of 0.4 and 0.1ppb, respectively, represent the lowest ones reported so far for paper based metal sensors. The obtained detection limit is 50 times lower than the maximum contamination level required for drinking water. Here we also show a new option for increasing the sensibility in the LFDs with competitive format, through the decreasing in concentrations of the Cd-EDTA-BSA-AuNP conjugate deposited in the conjugation strip and the mAbs deposited in the test and control zones until to reach optimized concentrations. It is an important result take into account that the increase in sensibility is one of the challenges in the field of LFD sensors, where are focused many of the ongoing researches. In addition, a specificity study of the device for several metal interferences, where potential metal interferences are masked with the use of the EDTA and OVA optimized concentrations, is presented too.

  11. Tillage impact on herbicide loss by surface runoff and lateral subsurface flow.

    PubMed

    Potter, Thomas L; Bosch, David D; Strickland, Timothy C

    2015-10-15

    There is worldwide interest in conservation tillage practices because they can reduce surface runoff, and agrichemical and sediment losses from farm fields. Since these practices typically increase infiltration, their use may increase subsurface transport of water-soluble contaminants. Thus, to assess long-term environmental benefits of conservation tillage data may be needed that quantify both surface and subsurface contaminant fluxes. This study focused on the herbicide fluometuron (N,N-dimethyl-N'-[3-(trifluoromethyl)phenyl]-urea) and its soil degradate DMF (N-methyl-N'-[3-(trifluoromethyl) phenyl]-urea). Both compounds are classed as "leachable". They were measured for 10 years in surface runoff and lateral subsurface flow from paired fields located on a hill slope in the Atlantic Coastal Plain region of the southeastern USA. One group of fields was conventionally tilled incorporating all crop residues into soil prior to planting. The second was strip tilled, a common conservation tillage practice. Seven fluometuron applications were made to cotton (Gossypium hirsutum) produced in rotation with peanut (Arachis hypogea). Combined fluometuron and DMF surface and subsurface losses from the conventionally tilled fields were equivalent to 1.2% and 0.13% of fluometuron applied and 0.31% and 0.32% from the strip tilled fields. Annual surface runoff losses were significantly greater from the conventionally tilled fields while the strip tilled fields had significantly greater annual subsurface losses. Results demonstrated that shifting from conventional to conservation tillage management of farm fields in this landscape will reduce surface runoff losses of herbicides like fluometuron but subsurface losses will likely increase. The same trends can be expected in landscapes with similar soil and hydrologic properties. This should be considered when planning implementation of programs that promote conservation tillage use. Published by Elsevier B.V.

  12. Large-scale evaluation of the immuno-mycologics lateral flow and enzyme-linked immunoassays for detection of cryptococcal antigen in serum and cerebrospinal fluid.

    PubMed

    Hansen, Jessica; Slechta, E Susan; Gates-Hollingsworth, Marcellene A; Neary, Brandon; Barker, Adam P; Bauman, Sean; Kozel, Thomas R; Hanson, Kimberly E

    2013-01-01

    Cryptococcosis is a systemic infection caused by the pathogenic yeasts Cryptococcus neoformans and C. gattii. Detection of cryptococcal capsular antigen (CrAg) in serum and cerebrospinal fluid (CSF) plays an important diagnostic role. We prospectively compared the new Immuno-Mycologics Inc. (IMMY) lateral flow assay (LFA) and enzyme immunoassay (EIA) to our current CrAg test (Premier EIA; Meridian Bioscience Inc.). Discordant samples were retested with the latex-Cryptococcus antigen test (IMMY) and using serotype-specific monoclonal antibodies (MAbs). A total of 589 serum and 411 CSF specimens were tested in parallel. Qualitative agreement across assays was 97.7%. In all, 56 (41 serum and 15 CSF) samples were positive and 921 (527 serum and 394 CSF) samples were negative by all three assays. The 23 discrepant specimens were all Meridian EIA negative. Of 23 discordant specimens, 20 (87.0%) were positive by both the IMMY LFA and EIA, 2 were LFA positive only, and 1 was EIA positive only. Eleven discrepant specimens had adequate volume for latex agglutination (LA) testing; 8 were LA positive, and 3 were LA negative. LA-negative samples (2 CSF samples and 1 serum) had low IMMY LFA/EIA titers (≤1:10). Serotype-specific MAb analysis of the LA-positive samples suggested that these specimens contained CrAg epitopes similar to those of serotype C strains. In conclusion, the IMMY assays showed excellent overall concordance with the Meridian EIA. Assay performance differences were related to issues of analytic sensitivity and possible serotype bias. Incomplete access to patient-level data combined with low specimen volumes limited our ability to fully resolve discrepant results.

  13. Extended Result Reading Window in Lateral Flow Tests Detecting Exposure to Onchocerca volvulus: A New Technology to Improve Epidemiological Surveillance Tools

    PubMed Central

    Golden, Allison; Steel, Cathy; Yokobe, Lindsay; Jackson, Emily; Barney, Rebecca; Kubofcik, Joseph; Peck, Roger; Unnasch, Thomas R.; Nutman, Thomas B.; de los Santos, Tala; Domingo, Gonzalo J.

    2013-01-01

    Onchocerciasis is a neglected tropical disease caused by infection with the parasite Onchocerca volvulus (Ov). An estimated 180 million people are at risk for Ov infection, and 37 million people are infected, mostly in Africa. A lateral flow-based assay to detect human IgG4 antibodies to the Ov-specific antigen Ov-16 was developed as a rapid tool to detect exposure to Ov. The test, when performed on 449 sera specimens from patients with microfiladermia and Ov-negative patients, has a sensitivity of 89.1% (95% confidence interval: 86.2%–92.0%), and specificity of 97% (95% confidence interval: 95.4%–98.6%). Because the intended use of the test is for surveillance, it is highly desirable to have a stable, long-lasting result. An extended read window is thus desirable for a high-volume, busy workflow and facilitates post-surveillance quality assurance. The main restriction on achieving an extended read window for this assay was the erythrocyte lysis that can alter the signal-to-noise ratio, especially in those with low IgG4 levels (weak positives). We describe a test housing that incorporates a user-independent feature driven by assay fluid and an expanding wick that detaches the blood separation membrane from the nitrocellulose used in the assay, but before hemolysis occurs. We demonstrated material functionality at extreme operational conditions (37°C, 80% relative humidity) and a read window of a minimum of 70 days. The fluid-driven assay device performs equally as well with whole blood as with plasma, as demonstrated with 100 spiked clinical specimens (with a correlation coefficient of 0.96). We show a novel, inexpensive, and simple approach to actuating the detachment of the blood separation membrane from the nitrocellulose test with no impact on the performance characteristics of the test. PMID:23935960

  14. A CCD-based reader combined with CdS quantum dot-labeled lateral flow strips for ultrasensitive quantitative detection of CagA

    NASA Astrophysics Data System (ADS)

    Gui, Chen; Wang, Kan; Li, Chao; Dai, Xuan; Cui, Daxiang

    2014-02-01

    Immunochromatographic assays are widely used to detect many analytes. CagA is proved to be associated closely with initiation of gastric carcinoma. Here, we reported that a charge-coupled device (CCD)-based test strip reader combined with CdS quantum dot-labeled lateral flow strips for quantitative detection of CagA was developed, which used 365-nm ultraviolet LED as the excitation light source, and captured the test strip images through an acquisition module. Then, the captured image was transferred to the computer and was processed by a software system. A revised weighted threshold histogram equalization (WTHE) image processing algorithm was applied to analyze the result. CdS quantum dot-labeled lateral flow strips for detection of CagA were prepared. One hundred sera samples from clinical patients with gastric cancer and healthy people were prepared for detection, which demonstrated that the device could realize rapid, stable, and point-of-care detection, with a sensitivity of 20 pg/mL.

  15. A fast and sensitive immunoassay of avian influenza virus based on label-free quantum dot probe and lateral flow test strip.

    PubMed

    Li, Xuepu; Lu, Donglian; Sheng, Zonghai; Chen, Kun; Guo, Xuebo; Jin, Meilin; Han, Heyou

    2012-10-15

    A novel fluorescence immunoassay method for fast and ultrasensitive detection of avian influenza virus (AIV) was developed. The immunoassay method which integrated lateral flow test strip technique with fluorescence immunoassay used the label-free and high luminescent quantum dots (QDs) as signal output. By the sandwich immunoreaction performed on lateral flow test strip, the gold nanoparticle (NP) labels were captured in the test zone and further dissolved to release a large number of gold ions as a signal transduction bridge that was detected by the QDs-based fluorescence quenching method. Under the optimal conditions, the relative fluorescence intensity of QDs was linear over the range of 0.27-12 ng mL(-1) AIV, and the limit of detection was estimated to be 0.09 ng mL(-1) which was 100-fold greater than enzyme-linked immunosorbent assay (ELISA). The sensitive and specific response was also coupled with high reproducibility in the proposed method. A series of six parallel measurements produced reproducible fluorescent signals with a relative standard deviation of 4.7%. The proposed method can be used to directly detect clinical sample without any pretreatment, and showed high efficiency (90.0%), sensitivity (100.0%) and specificity (88.2%) compared with virus isolation (gold method). The new method shows great promise for rapid, sensitive, and quantitative detection of AIV in-field or point-of-care diagnosis.

  16. CFD simulation of an internal spin-filter: evidence of lateral migration and exchange flow through the mesh.

    PubMed

    Figueredo-Cardero, Alvio; Chico, Ernesto; Castilho, Leda R; Medronho, Ricardo A

    2009-11-01

    In the present work Computational Fluid Dynamics (CFD) was used to study the flow field and particle dynamics in an internal spin-filter (SF) bioreactor system. Evidence of a radial exchange flow through the filter mesh was detected, with a magnitude up to 130-fold higher than the perfusion flow, thus significantly contributing to radial drag. The exchange flow magnitude was significantly influenced by the filter rotation rate, but not by the perfusion flow, within the ranges evaluated. Previous reports had only given indirect evidences of this exchange flow phenomenon in spin-filters, but the current simulations were able to quantify and explain it. Flow pattern inside the spin-filter bioreactor resembled a typical Taylor-Couette flow, with vortices being formed in the annular gap and eventually penetrating the internal volume of the filter, thus being the probable reason for the significant exchange flow observed. The simulations also showed that cells become depleted in the vicinity of the mesh due to lateral particle migration. Cell concentration near the filter was approximately 50% of the bulk concentration, explaining why cell separation achieved in SFs is not solely due to size exclusion. The results presented indicate the power of CFD techniques to study and better understand spin-filter systems, aiming at the establishment of effective design, operation and scale-up criteria.

  17. Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test.

    PubMed

    Bowen, Damian E; Whitwell, James H; Lillford, Lucinda; Henderson, Debbie; Kidd, Darren; Mc Garry, Sarah; Pearce, Gareth; Beevers, Carol; Kirkland, David J

    2011-05-18

    With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals. Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow(®) flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays. A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45 h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays. All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint. The data generated from these

  18. Uncertainty Analysis of the Variable Parameter McCarthy-Muskingum (VPMM) Method with Presence of Lateral Flow

    NASA Astrophysics Data System (ADS)

    Yadav, B.; Bardossy, A.; Perumal, M.

    2014-12-01

    Uncertainty analysis of the estimate of a hydrological model is a required exercise for the risk management linked to the variable of interest. This study subjects the Variable Parameter McCarthy-Muskingum (VPMM) method recently proposed by Perumal and Price (2013) to uncertainty analysis. The VPMM method has been developed based on the assumption that there exists no lateral flow in the river stretch where it is employed for routing. But in this study this method is applied for the study of flood wave movement in a 24.2 km stretch between Rottweil and Oberndorf of Neckar River in Germany in the presence of lateral inflow. The study also proposes a general procedure for simulating flood events with the consideration of lateral flow in the reach. The cross sectioned information of the considered river stretch is estimated by the Robust Parameter Estimation (ROPE) algorithm. ROPE algorithm is used to get the best performing parameters set of bed width (Trapezoidal section) and side slope. As the evaluation of VPMM is done with the help of Nash-Sutcliffe efficiency criterion, this study uses it as an objective function to check the performance of the method with different data sets obtained using the ROPE algorithm. The uncertainty associated with parameter K and due to the presence of lateral flow is checked by the Jackknife method. All the 26 flood events observed from the Neckar catchment from 1999 to 2004 have been used for the analysis of the VPMM method. When inflow and outflow hydrographs for lateral flow estimation are used, performance of the VPMM method as per N-S efficiency criterion can be up to 97.061 %. By the analysis of all 27 available flood events, a relationship between total rainfall and total loss is obtained, and the value of loss obtained from the developed relationship can be used to simulate outflow hydrograph with the maximum N-S efficiency of 93.812 %.

  19. Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells.

    PubMed

    Karlsson, Annika C; Martin, Jeffrey N; Younger, Sophie R; Bredt, Barry M; Epling, Lorrie; Ronquillo, Rollie; Varma, Arjun; Deeks, Steven G; McCune, Joseph M; Nixon, Douglas F; Sinclair, Elizabeth

    2003-12-01

    The enumeration of antigen-specific T cell responses has been greatly facilitated in recent years by the development of methods based on the detection of cytokines. In particular, the enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) assays have become popular. Since both assays are likely to continue to be in widespread use, it is important to evaluate whether their results are comparable. In the current study, we compared the results obtained in the ELISPOT and CFC assays using peptide pools corresponding to CMV and HIV-1 proteins in chronically HIV-1-infected individuals. Analysis of T cell responses to peptide pools indicated that the CMV pp65 and HIV-1 Gag CFC and ELISPOT-derived results were statistically correlated. However, the results obtained with each assay differed in important ways: the magnitude of the response was consistently higher in the CFC assay while the CFC assay was less likely than the ELISPOT assay to detect low-level responses. Furthermore, there was a lack of numeric agreement between ELISPOT and CFC results. For studies that require the detection of low-level responses, or definition of responses as positive or negative, the ELISPOT assay may be preferable. In contrast, the CFC has a greater dynamic range and allows for phenotypic discrimination of responding cells, making it the assay of choice for most other applications.

  20. A low-cost flow cytometric assay for the detection and quantification of apoptosis using an anionic halogenated fluorescein dye.

    PubMed

    Meyer, Mervin; Essack, Magbubah; Kanyanda, Stonard; Rees, Jasper

    2008-09-01

    We describe here a technical improvement of an established colorimetric method used to detect and measure the occurrence of apoptosis in mammalian cells during in vitro cell culture. This assay uses an anionic halogenated fluorescein dye that is taken up by apoptotic cells at the stage of phosphatidylserine externalization. We demonstrate that apoptotic cells stained with this dye can be detected by flow cytometric analysis. Furthermore, we show that the modified method compares well with the standard annexin-V-based apoptosis assay and that it is significantly more cost-effective than the annexin-V assay.

  1. Classification of in vitro genotoxicants using a novel multiplexed biomarker assay compared to the flow cytometric micronucleus test.

    PubMed

    Wilde, Sabrina; Dambowsky, Miriam; Hempt, Claudia; Sutter, Andreas; Queisser, Nina

    2017-09-21

    Regulatory in vitro genotoxicity testing exhibits shortcomings in specificity and mode of action (MoA) information. Thus, the aim of this work was to evaluate the performance of the novel MultiFlow(®) assay composed of mechanistic biomarkers quantified in TK6 cells after treatment (4 and 24 hr): γH2AX (DNA double strand breaks), phosphorylated H3 (mitotic cells), translocated p53 (genotoxicity), and cleaved PARP1 (apoptosis). A reference dataset of 31 compounds with well-established MoA was studied using the MicroFlow(®) micronucleus assay. A positive call was raised following the earlier published criteria from Litron Laboratories. In the light of our data, these evaluation criteria should probably be adjusted since only 8/11 (73%) nongenotoxicants and 18/20 (90%) genotoxicants were correctly identified. Moreover, there is a need for new in vitro tools to delineate the predominant MoA as in the MicroFlow(®) assay only 5/9 (56%) aneugens and 4/11 (36%) clastogens were correctly classified. In contrast, the MultiFlow(®) assay provides more in-depth information about the MoA and therefore reliably discriminates clastogens, aneugens, and nongenotoxicants. By using a lab-specific, practical threshold for the aforementioned biomarkers, 10/11 (91%) nongenotoxicants and 19/20 genotoxicants (95%), 9/11 (82%) clastogens, and 8/9 (89%) aneugens were correctly categorized, suggesting a clear improvement over the MicroFlow(®) . Furthermore, the MultiFlow markers were benchmarked against established methods to assess the validity of the data. Altogether, these findings demonstrated good agreement between the MultiFlow(®) assay and the benchmarking methods. Finally, p21 may improve class discrimination given the correct identification of 4/4 (100%) aneugens and 2/5 (40%) clastogens. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. Lateral and vertical facies relationships of bedforms deposited by aggrading supercritical flows: From cyclic steps to humpback dunes

    NASA Astrophysics Data System (ADS)

    Lang, Jörg; Winsemann, Jutta

    2013-10-01

    The preservation of bedforms related to supercritical flows and hydraulic jumps is commonly considered to be rare in the geologic record, although these bedforms are known from a variety of depositional environments. This field-based study presents a detailed analysis of the sedimentary facies and stacking pattern of deposits of cyclic steps, chutes-and-pools, antidunes and humpback dunes from three-dimensional outcrops. The well exposed Middle Pleistocene successions from northern Germany comprise glacilacustrine ice-contact subaqueous fan and glacial lake-outburst flood deposits. The studied successions give new insights into the depositional architecture of bedforms related to supercritical flows and may serve as an analogue for other high-energy depositional environments such as fluvial settings, coarse-grained deltas or turbidite systems. Deposits of cyclic steps occur within the glacial lake-outburst flood succession and are characterised by lenticular scours infilled by gently to steeply dipping backsets. Cyclic steps formed due to acceleration and flow thinning when the glacial lake-outburst flood spilled over a push-moraine ridge. These bedforms are commonly laterally and vertically truncated and alternate with deposits of chutes-and-pools and antidunes. The subaqueous fan successions are dominated by laterally extensive sinusoidal waveforms, which are interpreted as deposits of aggrading stationary antidunes, which require quasi-steady flows at the lower limit of the supercritical flow stage and high rates of sedimentation. Humpback dunes are characterised by downflow divergent cross-stratification, displaying differentiation into topsets, foresets and bottomsets, and are interpreted as deposited at the transition from subcritical to supercritical flow conditions or vice versa. Gradual lateral and vertical transitions between humpback dunes and antidune deposits are very common. The absence of planar-parallel stratification in all studied successions

  3. Utility of a Lateral Flow Immunoassay (LFI) to Detect Burkholderia pseudomallei in Soil Samples

    PubMed Central

    Rongkard, Patpong; Hantrakun, Viriya; Dittrich, Sabine; Srilohasin, Prapaporn; Amornchai, Premjit; Langla, Sayan; Lim, Cherry; Day, Nicholas P. J.; AuCoin, David; Wuthiekanun, Vanaporn

    2016-01-01

    Background Culture is the gold standard for the detection of environmental B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples as a tool to detect B. pseudomallei in environmental samples. Methodology/Principal Findings First, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 μL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting orf2. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for B. pseudomallei, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for B. pseudomallei

  4. Enhancement of the detection limit for lateral flow immunoassays: evaluation and comparison of bioconjugates.

    PubMed

    Linares, Elisângela M; Kubota, Lauro T; Michaelis, Jens; Thalhammer, Stefan

    2012-01-31

    There is an increasing demand for convenient and accurate point-of-care tools that can detect and diagnose different stages of a disease in remote or impoverished settings. In recent years, lateral flow immunoassays (LFIA) have been indicated as a suitable medical diagnostic tool for these environments because they require little or no sample preparation, provide rapid and reliable results with no electronic components and thus can be manufactured at low costs and operated by unskilled personnel. However, even though they have been successfully applied to acute and chronic disease detection, LFIA based on gold nanoparticles, the standard marker, show serious limitations when high sensitivity is needed, such as early stage disease detection. Moreover, based on the lack of comparative information for label performance, significant optimization of the systems that are currently in use might be possible. To this end, in the presented work, we compare the detection limit between the four most used labels: colloidal-gold, silver enhanced gold, blue latex bead and carbon black nanoparticles. Preliminary results were obtained by using the biotin-streptavidin coupling as a model system and showed that carbon black had a remarkably low detection limit of 0.01 μg/mL in comparison to 0.1 μg/mL, 1 μg/mL and 1mg/mL for silver-coated gold nanoparticles, gold nanoparticles and polystyrene beads, respectively. Therefore, as a proof of concept, carbon black was used in a detection system for Dengue fever. This was achieved by immobilizing monoclonal antibodies for the nonstructural glycoprotein (NS1) of the Dengue virus to carbon black. We found that the colorimetric detection limit of 57 ng/mL for carbon black was ten times lower than the 575 ng/mL observed for standard gold nanoparticles; which makes it sensitive enough to diagnose a patient on the first days of infection. We therefore conclude that, careful screening of detection labels should be performed as a necessary step

  5. Utility of a Lateral Flow Immunoassay (LFI) to Detect Burkholderia pseudomallei in Soil Samples.

    PubMed

    Rongkard, Patpong; Hantrakun, Viriya; Dittrich, Sabine; Srilohasin, Prapaporn; Amornchai, Premjit; Langla, Sayan; Lim, Cherry; Day, Nicholas P J; AuCoin, David; Wuthiekanun, Vanaporn; Limmathurotsakul, Direk

    2016-12-01

    Culture is the gold standard for the detection of environmental B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples as a tool to detect B. pseudomallei in environmental samples. First, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 μL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting orf2. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for B. pseudomallei, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for B. pseudomallei. The LFI can be used to detect B. pseudomallei in

  6. Sources of Variability in Platelet Accumulation on Type 1 Fibrillar Collagen in Microfluidic Flow Assays

    PubMed Central

    Neeves, Keith B.; Onasoga, Abimbola A.; Hansen, Ryan R.; Lilly, Jessica J.; Venckunaite, Diana; Sumner, Meghan B.; Irish, Andrew T.; Brodsky, Gary; Manco-Johnson, Marilyn J.; Di Paola, Jorge A.

    2013-01-01

    Microfluidic flow assays (MFA) that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s−1 through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (LagT), the rate of platelet accumulation (VPLT), and platelet surface coverage (SC). A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF) levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to VPLT and SC at all wall shear rates. A longer LagT for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s−1 and 300 s−1. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI) resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104) and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay. PMID:23355889

  7. Sources of variability in platelet accumulation on type 1 fibrillar collagen in microfluidic flow assays.

    PubMed

    Neeves, Keith B; Onasoga, Abimbola A; Hansen, Ryan R; Lilly, Jessica J; Venckunaite, Diana; Sumner, Meghan B; Irish, Andrew T; Brodsky, Gary; Manco-Johnson, Marilyn J; Di Paola, Jorge A

    2013-01-01

    Microfluidic flow assays (MFA) that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s⁻¹ through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (Lag(T)), the rate of platelet accumulation (V(PLT)), and platelet surface coverage (SC). A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF) levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to V(PLT) and SC at all wall shear rates. A longer Lag(T) for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s⁻¹ and 300 s⁻¹. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI) resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104) and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay.

  8. Rapid Multiplexed Flow Cytometric Assay for Botulinum Neurotoxin Detection Using an Automated Fluidic Microbead-Trapping Flow Cell for Enhanced Sensitivity

    SciTech Connect

    Ozanich, Richard M.; Bruckner-Lea, Cindy J.; Warner, Marvin G.; Miller, Keith D.; Antolick, Kathryn C.; Marks, James D.; Lou, Jianlong; Grate, Jay W.

    2009-07-15

    A bead-based sandwich immunoassay for botulinum neurotoxin serotype A (BoNT/A) has been developed and demonstrated using a recombinant 50 kDa fragment (BoNT/A-HC-fragment) of the BoNT/A heavy chain (BoNT/A-HC) as a structurally valid simulant. Three different anti-BoNT/A antibodies were attached to three different fluorescent dye encoded flow cytometry beads for multiplexing. The assay was conducted in two formats: a manual microcentrifuge tube format and an automated fluidic system format. Flow cytometry detection was used for both formats. The fluidic system used a novel microbead-trapping flow cell to capture antibody-coupled beads with subsequent sequential perfusion of sample, wash, dye-labeled reporter antibody, and final wash solutions. After the reaction period, the beads were collected for analysis by flow cytometry. Sandwich assays performed on the fluidic system gave median fluorescence intensity signals on the flow cytometer that were 2-4 times higher than assays performed manually in the same amount of time. Limits of detection were estimated at 1 pM (~50 pg/mL for BoNT/A-HC-fragment) for the 15 minute fluidic assay.

  9. Automated enzymatic assays in a renewable fashion using the multisyringe flow injection scheme with soluble enzymes.

    PubMed

    Pizà, Nicolau; Miró, Manuel; Estela, José Manuel; Cerdà, Víctor

    2004-02-01

    In this paper, a novel flowing stream scheme based upon the multisyringe flow injection (MSFI) technique is presented as a powerful tool to perform automated enzymatic assays. The exploitation of enzymes in homogeneous phase circumvents typical drawbacks associated with the commonly used packed-bead or open tubular permanent columns, namely, malfunctions of the reactor, carryover effects, flow resistance, loss of binding sites, large reagent consumption, and use of harmful organic solvents during immobilization procedures. The proposed MSFI system is able to handle minute volumes of soluble enzymes and accommodate reactions with divergent kinetic and pH demands, as demonstrated via the indirect chemiluminescence determination of trace levels of glucose. The procedure is based on the on-line glucose oxidase-catalyzed oxidation of beta-glucose in homogeneous phase to beta-glucono-delta-lactone and hydrogen peroxide. Subsequently, the generated oxidant merges downstream with an alkaline slug of 3-aminopthalhydrazide and a metal-catalyst zone (viz., Co(II)) at a total flow rate as high as 72 mL/min aiming to warrant maximum light collection from the fast CL reaction. Under optimum conditions for both sequentially occurring reactions, a glucose concentration as low as 90 microg/L may be easily detected at a 1000-fold photomultiplier gain. A second-order polynomial regression equation of light emission versus substrate concentration is found over the range 90 microg/L-2.7 mg/L glucose, although a maximum concentration of 180 mg/L may be determined by suitable gain selection without requiring manifold reconfiguration. An injection throughput of 20 h(-1), a repeatability better than 2.5% at the 1 mg/L level, and a 3sigma detection limit of 72 microg/L are the analytical features of the designed analyzer. The proposed approach was applied to the analysis of ultralow glucose content soft drinks as well as fruit juices suitable for diabetic consumers. The accuracy was

  10. Assessing lateral flows and solute transport during floods in a conduit-flow-dominated karst system using the inverse problem for the advection-diffusion equation

    NASA Astrophysics Data System (ADS)

    Cholet, Cybèle; Charlier, Jean-Baptiste; Moussa, Roger; Steinmann, Marc; Denimal, Sophie

    2017-07-01

    The aim of this study is to present a framework that provides new ways to characterize the spatio-temporal variability of lateral exchanges for water flow and solute transport in a karst conduit network during flood events, treating both the diffusive wave equation and the advection-diffusion equation with the same mathematical approach, assuming uniform lateral flow and solute transport. A solution to the inverse problem for the advection-diffusion equations is then applied to data from two successive gauging stations to simulate flows and solute exchange dynamics after recharge. The study site is the karst conduit network of the Fourbanne aquifer in the French Jura Mountains, which includes two reaches characterizing the network from sinkhole to cave stream to the spring. The model is applied, after separation of the base from the flood components, on discharge and total dissolved solids (TDSs) in order to assess lateral flows and solute concentrations and compare them to help identify water origin. The results showed various lateral contributions in space - between the two reaches located in the unsaturated zone (R1), and in the zone that is both unsaturated and saturated (R2) - as well as in time, according to hydrological conditions. Globally, the two reaches show a distinct response to flood routing, with important lateral inflows on R1 and large outflows on R2. By combining these results with solute exchanges and the analysis of flood routing parameters distribution, we showed that lateral inflows on R1 are the addition of diffuse infiltration (observed whatever the hydrological conditions) and localized infiltration in the secondary conduit network (tributaries) in the unsaturated zone, except in extreme dry periods. On R2, despite inflows on the base component, lateral outflows are observed during floods. This pattern was attributed to the concept of reversal flows of conduit-matrix exchanges, inducing a complex water mixing effect in the saturated zone

  11. Au@Ag SERRS tags coupled to a lateral flow immunoassay for the sensitive detection of pneumolysin.

    PubMed

    Blanco-Covián, Lucía; Montes-García, Verónica; Girard, Alexandre; Fernández-Abedul, M Teresa; Pérez-Juste, Jorge; Pastoriza-Santos, Isabel; Faulds, Karen; Graham, Duncan; Blanco-López, M Carmen

    2017-02-02

    Establishing a definitive diagnosis of pneumonia using conventional tests is difficult and expensive. Lateral flow immunoassays (LFIAs) are an advantageous point of care (POC) test option, but they have some limitations in terms of detection and quantification. In this work we have developed a lateral flow immunoassay for the ultrasensitive detection of penumolysin employing plasmonic Surface-Enhanced Resonance Raman Scattering (SERRS) tag as labelled probe. The combination of Au@Ag core-shell nanoparticles as plasmonic platform and Rhodamine B Isothiocyanate as Raman reporter has allowed us to fabricate a SERRS tag with high efficiency and reliability. The limit of detection of the SERRS-based LFIA was 1 pg mL(-1). This could be a strong foundation for a pneumonia diagnosis test based on pneumolysin detection.

  12. Comparative sperm chromatin structure assay measurements on epiillumination and orthogonal axes flow cytometers

    SciTech Connect

    Evenson, D.; Jost, L.; Gandour, D.; Gandour, D.; Rhodes, L.

    1995-04-01

    The sperm chromatin structure assay (SCSA) measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ, and was developed on two Ortho flow cytometers, an FC200 and a cytofluorograf 30 (BDIS), both having orthogonal axes of fluorochrome excitation, emission, and sample flow. Sperm cells are first treated with a pH 1.4 buffer to denature DNA in situ and then stained with the metachromatic dye acridine orange (AO). The metachromatic fluorescence measured reflects relative amounts of denatured (red fluorescence) and native (green fluorescence) DNA present per cell. The extent of DNA denaturation is quantified by the calculated parameter alpha t [{alpha}{sub t} = red/(red + green) fluorescence]. Alpha t variables important for correlations with fertility and toxicant-induced chromatin damage include mean (X{alpha}{sub t}), standard deviation (SD{alpha}{sub t}), and cells outside the main population (COMP{alpha}{sub t}). This study showed that the SCSA can be successfully run on two epiillumination-type instruments, an Ortho ICP22A and Skatron Argus {trademark}, and two additional orthogonal axes instruments, a Becton Dickinson FACScan {trademark} and a Coulter Elite {trademark}. Epiillumination instruments produced a different fluorescence distribution than orthogonal instruments, but the resulting {alpha}{sub t} values showed strong conformity and interpretation of results was the same. SCSA values obtained on the Coultier Elite {trademark} were most similar to the Cytofluorograf 30; the FACScan {trademark} green fluorescence distribution was narrower and allowed resolution of cell doublets. Neither orthogonal instrument has the ability to directly calculate {alpha}{sub t} values. Listmode data from these instruments were transferred to an off-line personal computer (PC) for calculation of {alpha}{sub t} values using LIST-VIEW {trademark} software. 28 refs., 5 figs., 2 tabs.

  13. A novel flow-based procedure for automation of respirometric assays in soils.

    PubMed

    Silva, Claudineia R; Oliveira, Eliezer; Zagatto, Elias A G; Henriquez, Camelia

    2016-09-01

    A flow-based strategy involving a gas-diffusion sampling probe was proposed for evaluating the respiration rate in soils. The amount of CO2 collected after a pre-defined time interval was proportional to the free CO2 released by the soil ecosystem. The 500-mL incubation flasks typically used for soil respirometric assays were adapted and a special cover was designed for connecting a tubular gas diffusion membrane, a fan, and a septum for adding the CO2(g) standards required for calibration. The method relied on the pH-dependent absorbance variations resulting from the CO2 collection. A 1.3mmolL(-1) bromothymol blue solution (pH 7.0) acted as both acceptor and carrier streams. In order to widen the dynamical working range to 0.003-0.2mmol CO2, two analytical curves were obtained, each related to a different time interval for the CO2 collection. Kinetic curves related to CO2 release by the soil samples were straightforwardly attained. Repeatability and detection limit were estimated as 2.0% and 0.001mmol CO2 (n=10), and accuracy was assessed in relation to a recommended titrimetric procedure.

  14. Flow Cytometry-based Assay for the Monitoring of NK Cell Functions.

    PubMed

    Tognarelli, Sara; Jacobs, Benedikt; Staiger, Nina; Ullrich, Evelyn

    2016-10-30

    Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

  15. Development of a lateral flow immunoassay for rapid field detection of the red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae).

    PubMed

    Valles, Steven M; Strong, Charles A; Callcott, Anne-Marie A

    2016-07-01

    The red imported fire ant, Solenopsis invicta, is an aggressive, highly invasive pest ant species from South America that has been introduced into North America, Asia, and Australia. Quarantine efforts have been imposed in the USA to minimize further spread of the ant. To aid the quarantine efforts, there remains an acute need for a rapid, field portable method for the identification of these ants. In this report, we describe two novel monoclonal antibodies that specifically bind the S. invicta venom protein 2 produced by S. invicta. Using these monoclonal antibodies we developed a lateral flow immunoassay that provides a rapid and portable method for the identification of S. invicta ants. The lateral flow immunoassay was validated against purified S. invicta venom protein 2 and 33 unique ant species (representing 15 % of the total species and 42 % of the Myrmicinae genera found in Florida), and only S. invicta and the S. invicta/richteri hybrid produced a positive result. These monoclonal antibodies were selective to S. invicta venom protein 2 and did not bind to proteins from congeners (i.e., S. geminata or S. richteri) known to produce a S. invicta venom protein 2 ortholog. This S. invicta lateral flow immunoassay provides a new tool for regulatory agencies in the USA to enforce quarantine protocols and limit the spread of this invasive ant. Graphical Abstract Field method to detect and identify the red imported fire ant, Solenopsis invicta.

  16. Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

    ERIC Educational Resources Information Center

    Ott, Laura E.; Carson, Susan

    2014-01-01

    Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

  17. Programmable flow system for automation of oxygen radical absorbance capacity assay using pyrogallol red for estimation of antioxidant reactivity.

    PubMed

    Ramos, Inês I; Gregório, Bruno J R; Barreiros, Luísa; Magalhães, Luís M; Tóth, Ildikó V; Reis, Salette; Lima, José L F C; Segundo, Marcela A

    2016-04-01

    An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540 nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5 µM of PGR and 125 mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90 ± 5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8 μM. RSD values were <3.4 and <4.9%, for intra and inter-assay precision, respectively. Compared to previous batch automated ORAC assays, the developed system also accounted for high sampling frequency (29 h(-1)), low operating costs and low generation of waste.

  18. Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

    ERIC Educational Resources Information Center

    Ott, Laura E.; Carson, Susan

    2014-01-01

    Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

  19. Evaluation of microparticles in whole blood by multicolour flow cytometry assay.

    PubMed

    Christersson, Christina; Johnell, Matilda; Siegbahn, Agneta

    2013-04-01

    To develop and evaluate a multicolour flow cytometry method for analysis of microparticles (MPs) in fresh whole blood without any centrifugation steps or freezing/thawing procedure. Flow cytometry was performed using a FC500 MPL cytometer. The compensation in the protocol was performed based on the platelet population. Polystyrene microspheres 0.50-1.27 μm were used for size position, and the MP gate was set as particles 0.5-1.0 μm. Whole blood was incubated with annexin V and antibodies to tissue factor (TF), platelets (CD41 and CD62P), monocyte (CD14) and endothelial cells (CD144). For comparison, MPs from platelet free supernatant was used. The TF activity was evaluated by Calibrated Automated Thrombogram. Annexin V was used to distinguish true events from background noise. For standardization, each analysis included 10,000 events in the gate of platelets. There were 622(462-1001) MP(annV+)/10,000 platelets and of these, 66 (49-82)/10,000 platelets expressed TF. After correction for the individual platelet counts, the amount of circulating MP(annV+) was 17.1 (12.1-24.9) × 10(9)/L in whole blood, and of these, 10% (6-12%) expressed TF. The majority of the MPs expressed CD41, and 5.6% (2.2-6.9%) of these co-expressed TF. The amount of CD41 + MP(annV+) tended to correlate to the TF activity in whole blood. There was no correlation between the MP(annV+) in whole blood and MPs derived from platelet free supernatant. Patients with pulmonary arterial hypertension and stable coronary artery disease had increased concentrations of CD41 + MP(annV+) in whole blood. This multicolour flow cytometry assay in whole blood mimics the in vivo situation by avoiding several procedure steps interfering with the MP count. By standardized quantification of MPs a reference interval of MPs can be created.

  20. Sound transmission loss through metamaterial plate with lateral local resonators in the presence of external mean flow.

    PubMed

    Wang, Ting; Sheng, Meiping; Qin, Qinghua

    2017-02-01

    In the context of sound incident upon a metamaterial plate, explicit formulas for sound transmission loss (STL) are derived in the presence of external mean flow. Metamaterial plate, consisting of homogeneous plate and lateral local resonators (LLRs), is homogenized by using effective medium method to obtain the effective mass density and facilitate the calculation of STL. Results show that (a) vigorously oscillating LLRs lead to higher STL compared with bare plate, (b) increasing Mach number of the external mean flow helps obtain higher STL below the coincidence frequency but decreases STL above the coincidence frequency due to the added mass effect of light fluid loading and aerodynamic damping effect, (c) the coincidence frequency shifts to higher frequency range for the refracted effect of the external mean flow. However, effects of the flow on STL within negative mass density range can be neglected because of the lateral local resonance occurring. Moreover, hysteretic damping from metamaterial can only smooth the transmission curves by lowering higher peaks and filling dips. Effects of incident angles on STL are also examined. It is demonstrated that increasing elevation angle can improve the sound insulation, while the azimuth angle does not.

  1. A summary of lateral-stability derivatives calculated for wing plan forms in supersonic flow

    NASA Technical Reports Server (NTRS)

    Jones, Arthur L; Alksne, Alberta

    1951-01-01

    A compilation of theoretical values of the lateral-stability derivatives for wings at supersonic speeds is presented in the form of design charts. The wing plan forms for which this compilation has been prepared include a rectangular, two trapezoidal, two triangular, a fully-tapered swept-back, a sweptback hexagonal, an unswept hexagonal, and a notched triangular plan form. A full set of results, that is, values for all nine of the lateral-stability derivatives for wings, was available for the first six of these plan forms only. The reasons for the incompleteness of the results available for other plan forms are discussed.

  2. Simulating shifting vertical and lateral flow path conditions in periglacial cover beds of a small-scale low mountainous catchment

    NASA Astrophysics Data System (ADS)

    Bestian, Konrad; Kraft, Philipp; Breuer, Lutz

    2017-04-01

    Periglacial cover beds are widely spread in European low mountain regions. This concept is based on three main types of sedimentary layers differing in texture properties: The main layer containing silty material (aeolian loess sedimentation), the basal layer containing gravel and decayed bedrock material (frost weathering of bedrock) and sometimes the intermediate layer in between containing mixed material from main and basal layer. Each layer type is characterized by specific hydraulic properties related to the climatic conditions during sedimentation. Recent research shows a shifting effect on runoff generation depending on the water content of the periglacial layers. Under low water content the basal layer impedes vertical flow whereas at high water content it becomes a preferential flow path for interflow. Reproducing these shifting vertical and lateral flow path effects will increase the credibility of rainfall-runoff models. The objective of this work was to implement these shifting effects in runoff modelling. We used the Catchment Modeling Framework (CMF) as modular toolkit. First we created a hillslope model to reproduce the effect of shifting flow path. Secondly, we built a semi-distributed catchment runoff model using Hydrological Response Units (HRU) defined by expert-knowledge based on topography, land use and groundwater information. The model was set up in a way that it provides the possibility to implement shifting vertical and lateral flow paths in later model runs. We performed several field experiments in the small-scale agricultural Schwingbach observatory (1.28 km2 AEO, Hessen, Germany) to gain expert-knowledge. For instance, we identified the spatial distribution of periglacial cover beds, measured hydraulic soil properties and installed 13 piezometers. We further ran conductivity tests of the groundwater body in the piezometer using slug and bail tests. Climate data were used as forcing data and discharge data for calibration and validation

  3. Prospective evaluation of a rapid nanoparticle-based lateral flow immunoassay (STic Expert(®) HIT) for the diagnosis of heparin-induced thrombocytopenia.

    PubMed

    Leroux, Dorothée; Hezard, Nathalie; Lebreton, Aurélien; Bauters, Anne; Suchon, Pierre; de Maistre, Emmanuel; Biron, Christine; Huisse, Marie-Genevieve; Ternisien, Catherine; Voisin, Sophie; Gruel, Yves; Pouplard, Claire

    2014-09-01

    A rapid lateral flow immunoassay (LFIA) (STic Expert(®) HIT), recently developed for the diagnosis of heparin-induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 28·7%, 61·7% and 9·6% of patients, respectively. Definite HIT was diagnosed in 40 patients (12·0%) with positive results on both enzyme-linked immunosorbent assay (Asserachrom(®) HPIA IgG) and serotonin release assay. The inter-reader reproducibility of results obtained was excellent (kappa ratio > 0·9). The negative predictive value of LFIA with plasma samples was 99·6% with a negative likelihood ratio (LR) of 0·03, and was comparable to those of the particle gel immunoassay (H/PF4-PaGIA(®) ) performed in 124 cases. Positive predictive value and positive LR were 44·4% and 5·87, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 11·2% to 0·4% when the LFIA result was negative and increased to 42·5% when it was positive. In conclusion, the STic Expert(®) HIT combined with the 4Ts score is a reliable tool to rule out the diagnosis of HIT.

  4. A novel colloidal gold-based lateral flow immunoassay for rapid simultaneous detection of cyromazine and melamine in foods of animal origin.

    PubMed

    Le, Tao; Yan, Peifeng; Xu, Jian; Hao, Youjing

    2013-06-01

    A rapid and sensitive lateral flow immunoassay (LFIA) based on competitive format was developed and validated for simultaneous detection of cyromazine (CA) and melamine (MA) in foods of animal origin. With this method, the cut-off value for the two test lines were achieved at 25 ng/g, which was lower than the maximum residue levels (MRLs) established for CA and MA. At three fortified levels (50, 100, and 150 ng/g), the recoveries for CA and MA ranged from 73.9% to 104.2% with the relative standard deviation (RSD) less than 11.9%, based on within day and interday analysis. The lower detection limit for CA and MA in matrix sample were 0.22 ng/ml and 0.26 ng/ml, respectively, which were lower than those of published literatures. A parallel analysis of CA and MA in real samples conducted by HPLC showed comparable results to those obtained from LFIA. The results of LFIA were in good agreement with those of high performance liquid chromatography (HPLC) in the analysis of CA and MA in foods of animal origin, demonstrating the practical applicability of the developed assay in real samples. Overall, to our knowledge, this is the first report of quantitative or semi-quantitative simultaneous detection for CA and MA by immunochromatographic assay.

  5. Rapid diagnosis of Theileria annulata by recombinase polymerase amplification combined with a lateral flow strip (LF-RPA) in epidemic regions.

    PubMed

    Yin, Fangyuan; Liu, Junlong; Liu, Aihong; Li, Youquan; Luo, Jianxun; Guan, Guiquan; Yin, Hong

    2017-04-15

    Rapid and accurate diagnosis of Theileria annulata infection contributes to the formulation of strategies to eradicate this parasite. A simple and efficient diagnostic tool, recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip, was used in detection of Theileria and compared to other methods that require expensive instruments and skilled personnel. Herein, we established and optimized an LF-RPA method to detect the cytochrome b gene of T. annulata mitochondrial DNA from experimentally infected and field-collected blood samples. This method has many unparalleled characteristics, including that it is rapid (clear detection in 5min at constant temperature), sensitive (the limitation of detection is at least 2pg genomic DNA), and specific (no cross-reaction with other piroplasms that infect cattle). The LF-RPA assay was evaluated via testing 17 field blood samples and comparing the results of that of a PCR, showing 100% agreement, which demonstrates the ability of the LF-RPA assay to detect T. annulata infections in small number of samples (n=17). Taken together, the results indicate that this method could be used as an ideal diagnostic tool for detecting T. annulata in endemic regions with limited to fewer and local resources and could also be a potential technique for the surveillance and control of blood protozoa.

  6. Loop-Mediated Isothermal Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Enterococcus faecalis and Staphylococcus aureus.

    PubMed

    Wang, Yi; Li, Hui; Wang, Yan; Zhang, Lu; Xu, Jianguo; Ye, Changyun

    2017-01-01

    The report describes a simple, rapid and sensitive assay for visual and multiplex detection of Enterococcus faecalis and Staphylococcus aureus based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow biosensor (LFB). Detection and differentiation of the Ef0027 gene (E. faecalis-specific gene) and nuc gene (S. aureus-specific gene) were determined using fluorescein (FITC)-and digoxin-modified primers in the mLAMP process. In the presence of biotin- and FITC-/digoxin-modified primers, the mLAMP yielded numerous biotin- and FITC-/digoxin-attached duplex products, which were detected by LFB through biotin/streptavidin interaction (biotin on the duplex and streptavidin on the gold nanoparticle) and immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the LFB test line). The accumulation of gold nanoparticles generated a characteristic red line, enabling visual and multiplex detection of target pathogens without instrumentation. The limit of detection (LoD), analytical specificity and feasibility of LAMP-LFB technique were successfully examined in pure culture and blood samples. The entire procedure, including specimen (blood samples) processing (30 min), isothermal reaction (40 min) and result reporting (within 2 min), could be completed within 75 min. Thus, this assay offers a simple, rapid, sensitive and specific test for multiplex detection of E. faecalis and S. aureus strains. Furthermore, the LAMP-LFB strategy is a universal technique, which can be extended to detect various target sequences by re-designing the specific LAMP primers.

  7. Resolving the lateral component of blood flow velocity based on ultrasound speckle size change with scan direction and speed.

    PubMed

    Xu, Tiantian; Bashford, Gregory R

    2009-01-01

    Conventional blood flow velocity measurement using ultrasound is capable of resolving the axial component (i.e., that aligned with the ultrasound propagation direction) of the blood flow velocity vector. However, these Doppler-based methods are incapable of detecting blood flow in the direction normal to the ultrasound beam. In addition, these methods require repeated pulse-echo interrogation at the same spatial location. In this paper, we introduce a method which estimates the lateral component of blood flow within a single image frame using the observation that the speckle pattern corresponding to the blood reflectors (typically red blood cells) stretches (i.e., is "smeared") if the blood is moving in the same direction as the electronically-controlled transducer line selection in a 2D image. The situation is analogous to the observed elongation of a subject photographed with a moving camera. Here, we develop a relationship between speckle size, scan speed, and blood flow velocity. Experiments were performed with a blood flow phantom and high-frequency transducer of a commercially available ultrasound machine. Data was captured through an interface allowing access to the raw beam formed data. Blood flow with velocities ranging from 15 to 40 cm/s were investigated in this paper. Results show that there is a linear relationship between the reciprocal of the stretch factor and blood flow velocity. Two scan speeds were used in our experiments. When the scan velocity is 64.8 cm/s, compared with the theoretical model, fitting results based on experimental data gave us a linear relationship with average flow estimation error of 1.74+/-1.48 cm/s. When the scan velocity is 37.4 cm/s, the average estimation error is 0.65+/-0.45 cm/s.

  8. Mesoscopic lateral S/N/S weak links: Josephson effects and Josephson-like vortex flow

    NASA Astrophysics Data System (ADS)

    Carapella, G.; Sabatino, P.; Gombos, M.

    2017-02-01

    We report an experimental and numerical study of magneto-transport properties of mesoscopic lateral S/N/S superconducting weak links where the N region is made of the same material as the S banks, though with strongly reduced critical temperature. Magnetoresistance oscillations and clear dc and ac Josephson effects are observed. Experimental results are analyzed in the framework of the time-dependent Ginzburg-Landau model for mesoscopic type II superconductors with an inhomogeneous critical temperature. The analysis suggests that dissipative branches of the current-voltage curve of the weak link in the presence of a magnetic field are accounted for by moving ‘Josephson-like’ vortices. These relatively fast excitations are anisotropic as per the ordinary Josephson vortex in tunnel junctions, but have a normal core like the ordinary Abrikosov vortex in plain superconducting strips. Moreover, unlike the vortex in tunneling junctions, in the lateral S/N/S weak link, the extension of the moving vortex is larger than the extension of the static one. Further, we report in some detail on the lateral proximity effect, and the deviations from the ideality of the current-phase relation of this kind of lateral weak link in the Josephson regime.

  9. Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.

    PubMed

    Ayers, Lisa; Kohler, Malcolm; Harrison, Paul; Sargent, Ian; Dragovic, Rebecca; Schaap, Marianne; Nieuwland, Rienk; Brooks, Susan A; Ferry, Berne

    2011-04-01

    Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

  10. Quantification of mucosal mononuclear cells in tissues with a fluorescent bead-based polychromatic flow cytometry assay.

    PubMed

    Reeves, R Keith; Evans, Tristan I; Gillis, Jacqueline; Wong, Fay E; Connole, Michelle; Carville, Angela; Johnson, R Paul

    2011-03-31

    Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P<0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.

  11. Development of an automated analysis system for data from flow cytometric intracellular cytokine staining assays from clinical vaccine trials

    PubMed Central

    Shulman, Nick; Bellew, Matthew; Snelling, George; Carter, Donald; Huang, Yunda; Li, Hongli; Self, Steven G.; McElrath, M. Juliana; De Rosa, Stephen C.

    2008-01-01

    Background Intracellular cytokine staining (ICS) by multiparameter flow cytometry is one of the primary methods for determining T cell immunogenicity in HIV-1 clinical vaccine trials. Data analysis requires considerable expertise and time. The amount of data is quickly increasing as more and larger trials are performed, and thus there is a critical need for high throughput methods of data analysis. Methods A web based flow cytometric analysis system, LabKey Flow, was developed for analyses of data from standardized ICS assays. A gating template was created manually in commercially-available flow cytometric analysis software. Using this template, the system automatically compensated and analyzed all data sets. Quality control queries were designed to identify potentially incorrect sample collections. Results Comparison of the semi-automated analysis performed by LabKey Flow and the manual analysis performed using FlowJo software demonstrated excellent concordance (concordance correlation coefficient >0.990). Manual inspection of the analyses performed by LabKey Flow for 8-color ICS data files from several clinical vaccine trials indicates that template gates can appropriately be used for most data sets. Conclusions The semi-automated LabKey Flow analysis system can analyze accurately large ICS data files. Routine use of the system does not require specialized expertise. This high-throughput analysis will provide great utility for rapid evaluation of complex multiparameter flow cytometric measurements collected from large clinical trials. PMID:18615598

  12. Breakdown of Burton Prim Slichter approach and lateral solute segregation in radially converging flows

    NASA Astrophysics Data System (ADS)

    Priede, J.; Gerbeth, G.

    2005-11-01

    A theoretical study is presented of the effect of a radially converging melt flow, which is directed away from the solidification front, on the radial solute segregation in simple solidification models. We show that the classical Burton-Prim-Slichter (BPS) solution describing the effect of a diverging flow on the solute incorporation into the solidifying material breaks down for the flows converging along the solidification front. The breakdown is caused by a divergence of the integral defining the effective boundary layer thickness which is the basic concept of the BPS theory. Although such a divergence can formally be avoided by restricting the axial extension of the melt to a layer of finite height, radially uniform solute distributions are possible only for weak melt flows with an axial velocity away from the solidification front comparable to the growth rate. There is a critical melt velocity for each growth rate at which the solution passes through a singularity and becomes physically inconsistent for stronger melt flows. To resolve these inconsistencies we consider a solidification front presented by a disk of finite radius R0 subject to a strong converging melt flow and obtain an analytic solution showing that the radial solute concentration depends on the radius r as ˜ln(R0/r) and ˜ln(R0/r) close to the rim and at large distances from it. The logarithmic increase of concentration is limited in the vicinity of the symmetry axis by the diffusion becoming effective at a distance comparable to the characteristic thickness of the solute boundary layer. The converging flow causes a solute pile-up forming a logarithmic concentration peak at the symmetry axis which might be an undesirable feature for crystal growth processes.

  13. Flow cytometry-based analysis of artemisinin-resistant Plasmodium falciparum in the ring-stage survival assay.

    PubMed

    Amaratunga, Chanaki; Neal, Aaron T; Fairhurst, Rick M

    2014-08-01

    The ring-stage survival assay (RSA) is a powerful tool for phenotyping artemisinin-resistant Plasmodium falciparum but requires experienced microscopists to count viable parasites among 10,000 erythrocytes in Giemsa-stained thin blood smears. Here we describe a rapid flow cytometric assay that accurately counts viable parasites among 250,000 erythrocytes in suspension. This method performs as well as light microscopy and can be used to standardize the collection of RSA data between research groups in laboratory and field settings.

  14. Lateral migration of a two-dimensional vesicle in unbounded Poiseuille flow

    NASA Astrophysics Data System (ADS)

    Kaoui, B.; Ristow, G. H.; Cantat, I.; Misbah, C.; Zimmermann, W.

    2008-02-01

    The migration of a suspended vesicle in an unbounded Poiseuille flow is investigated numerically in the low Reynolds number limit. We consider the situation without viscosity contrast between the interior of the vesicle and the exterior. Using the boundary integral method we solve the corresponding hydrodynamic flow equations and track explicitly the vesicle dynamics in two dimensions. We find that the interplay between the nonlinear character of the Poiseuille flow and the vesicle deformation causes a cross-streamline migration of vesicles toward the center of the Poiseuille flow. This is in a marked contrast with a result [L. G. Leal, Annu. Rev. Fluid Mech. 12, 435 (1980)] according to which the droplet moves away from the center (provided there is no viscosity contrast between the internal and the external fluids). The migration velocity is found to increase with the local capillary number (defined by the time scale of the vesicle relaxation toward its equilibrium shape times the local shear rate), but reaches a plateau above a certain value of the capillary number. This plateau value increases with the curvature of the parabolic flow profile. We present scaling laws for the migration velocity.

  15. Lateral migration of a two-dimensional vesicle in unbounded Poiseuille flow.

    PubMed

    Kaoui, B; Ristow, G H; Cantat, I; Misbah, C; Zimmermann, W

    2008-02-01

    The migration of a suspended vesicle in an unbounded Poiseuille flow is investigated numerically in the low Reynolds number limit. We consider the situation without viscosity contrast between the interior of the vesicle and the exterior. Using the boundary integral method we solve the corresponding hydrodynamic flow equations and track explicitly the vesicle dynamics in two dimensions. We find that the interplay between the nonlinear character of the Poiseuille flow and the vesicle deformation causes a cross-streamline migration of vesicles toward the center of the Poiseuille flow. This is in a marked contrast with a result [L. G. Leal, Annu. Rev. Fluid Mech. 12, 435 (1980)] according to which the droplet moves away from the center (provided there is no viscosity contrast between the internal and the external fluids). The migration velocity is found to increase with the local capillary number (defined by the time scale of the vesicle relaxation toward its equilibrium shape times the local shear rate), but reaches a plateau above a certain value of the capillary number. This plateau value increases with the curvature of the parabolic flow profile. We present scaling laws for the migration velocity.

  16. Ethacrynic acid rapidly and selectively abolishes blood flow in vessels supplying the lateral wall of the cochlea.

    PubMed

    Ding, Dalian; McFadden, Sandra L; Woo, Jenifer M; Salvi, Richard J

    2002-11-01

    The mechanisms underlying the ototoxicity of ethacrynic acid (EA) are not fully understood. Previous studies have focused on morphologic and enzymatic changes in the stria vascularis. The current experiment shows that one of the earliest effects of EA is ischemia, resulting from impaired blood flow in vessels supplying the lateral wall of the cochlea. Inner ear microcirculation, endocochlear potentials, compound action potentials (CAP), cochlear microphonics (CM) and summating potentials (SP) were monitored over time in chinchillas following a single injection of EA (40 mg/kg i.v.). At all times after EA injection, blood vessels supplying the spiral lamina, modiolus, and vestibular end organs appeared normal. In contrast, lateral wall (spiral ligament and stria vascularis) vessels were poorly stained with eosin 2 min after EA injection, and devoid of red blood cells at 30 min post EA. Decline, but not recovery, of CAP, CM and SP followed the microcirculation changes in the lateral wall. Reperfusion was delayed in stria vascularis arterioles relative to other lateral wall vessels. The ischemia-reperfusion caused by EA would be expected to generate large quantities of free radicals, which may trigger or contribute to the cellular, enzymatic, and functional pathologies that have been described in detail previously.

  17. Redistribution of blood flow and lung volume between lungs in lateral decubitus postures during unilateral atelectasis and PEEP.

    PubMed

    Chang, Hung; Lai-Fook, Stephen J; Domino, Karen B; Schimmel, Carmel; Hildebrandt, Jack; Lee, Shih-Chun; Kao, Chung-Cheng; Hsu, Jane-Yi; Robertson, H Thomas; Glenny, Robb W; Hlastala, Michael P

    2006-04-30

    The effect of left lung atelectasis on the regional distribution of blood flow (Q), ventilation (V(A)) and gas exchange on the right lung ventilated with 100% O2 was studied in anesthetized dogs in the lateral decubitus posture. Q and V(A) were measured in 1.7 ml lung volume pieces using injected and aerosolized fluorescent microspheres, respectively. Hypoxic pulmonary vasoconstriction (HPV) in the atelectatic lung shifted flow to the ventilated lung. The increased flow in the ventilated lung ensured adequate gas exchange, compensating for the hypoxemia due to shunt contributed by the atelectatic lung. Left lung atelectasis caused a compensatory increase in the ventilated lung FRC that was smaller in the right (RLD) than left (LLD) lateral posture, the effect of lung compression by the atelectatic lung and mediastinal contents in the RLD posture. The O2 deficit measured by (A-a)DO2 increased with left lung atelectasis and was exacerbated in the LLD posture by 10 cm H2O PEEP, a result of increased shunt caused by a shift in Q from the ventilated to the atelectatic lung. The PEEP-induced O2 deficit was eliminated with inversion to the RLD posture.

  18. Inferring common cognitive mechanisms from brain blood-flow lateralization data: a new methodology for fTCD analysis.

    PubMed

    Meyer, Georg F; Spray, Amy; Fairlie, Jo E; Uomini, Natalie T

    2014-01-01

    Current neuroimaging techniques with high spatial resolution constrain participant motion so that many natural tasks cannot be carried out. The aim of this paper is to show how a time-locked correlation-analysis of cerebral blood flow velocity (CBFV) lateralization data, obtained with functional TransCranial Doppler (fTCD) ultrasound, can be used to infer cerebral activation patterns across tasks. In a first experiment we demonstrate that the proposed analysis method results in data that are comparable with the standard Lateralization Index (LI) for within-task comparisons of CBFV patterns, recorded during cued word generation (CWG) at two difficulty levels. In the main experiment we demonstrate that the proposed analysis method shows correlated blood-flow patterns for two different cognitive tasks that are known to draw on common brain areas, CWG, and Music Synthesis. We show that CBFV patterns for Music and CWG are correlated only for participants with prior musical training. CBFV patterns for tasks that draw on distinct brain areas, the Tower of London and CWG, are not correlated. The proposed methodology extends conventional fTCD analysis by including temporal information in the analysis of cerebral blood-flow patterns to provide a robust, non-invasive method to infer whether common brain areas are used in different cognitive tasks. It complements conventional high resolution imaging techniques.

  19. Inferring common cognitive mechanisms from brain blood-flow lateralization data: a new methodology for fTCD analysis

    PubMed Central

    Meyer, Georg F.; Spray, Amy; Fairlie, Jo E.; Uomini, Natalie T.

    2014-01-01

    Current neuroimaging techniques with high spatial resolution constrain participant motion so that many natural tasks cannot be carried out. The aim of this paper is to show how a time-locked correlation-analysis of cerebral blood flow velocity (CBFV) lateralization data, obtained with functional TransCranial Doppler (fTCD) ultrasound, can be used to infer cerebral activation patterns across tasks. In a first experiment we demonstrate that the proposed analysis method results in data that are comparable with the standard Lateralization Index (LI) for within-task comparisons of CBFV patterns, recorded during cued word generation (CWG) at two difficulty levels. In the main experiment we demonstrate that the proposed analysis method shows correlated blood-flow patterns for two different cognitive tasks that are known to draw on common brain areas, CWG, and Music Synthesis. We show that CBFV patterns for Music and CWG are correlated only for participants with prior musical training. CBFV patterns for tasks that draw on distinct brain areas, the Tower of London and CWG, are not correlated. The proposed methodology extends conventional fTCD analysis by including temporal information in the analysis of cerebral blood-flow patterns to provide a robust, non-invasive method to infer whether common brain areas are used in different cognitive tasks. It complements conventional high resolution imaging techniques. PMID:24982641

  20. Using an aqueous two-phase polymer-salt system to rapidly concentrate viruses for improving the detection limit of the lateral-flow immunoassay.

    PubMed

    Jue, Erik; Yamanishi, Cameron D; Chiu, Ricky Y T; Wu, Benjamin M; Kamei, Daniel T

    2014-12-01

    The development of point-of-need (PON) diagnostics for viruses has the potential to prevent pandemics and protects against biological warfare threats. Here we discuss the approach of using aqueous two-phase systems (ATPSs) to concentrate biomolecules prior to the lateral-flow immunoassay (LFA) for improved viral detection. In this paper, we developed a rapid PON detection assay as an extension to our previous proof-of-concept studies which used a micellar ATPS. We present our investigation of a more rapid polymer-salt ATPS that can drastically improve the assay time, and show that the phase containing the concentrated biomolecule can be extracted prior to macroscopic phase separation equilibrium without affecting the measured biomolecule concentration in that phase. We could therefore significantly decrease the time of the diagnostic assay with an early extraction time of just 30 min. Using this rapid ATPS, the model virus bacteriophage M13 was concentrated between approximately 2 and 10-fold by altering the volume ratio between the two phases. As the extracted virus-rich phase contained a high salt concentration which destabilized the colloidal gold indicator used in LFA, we decorated the gold nanoprobes with polyethylene glycol (PEG) to provide steric stabilization, and used these nanoprobes to demonstrate a 10-fold improvement in the LFA detection limit. Lastly, a MATLAB script was used to quantify the LFA results with and without the pre-concentration step. This approach of combining a rapid ATPS with LFA has great potential for PON applications, especially as greater concentration-fold improvements can be achieved by further varying the volume ratio. Biotechnol. Bioeng. 2014;111: 2499-2507. © 2014 Wiley Periodicals, Inc.

  1. Development of a recombinase polymerase amplification lateral flow dipstick (RPA-LFD) for the field diagnosis of caprine arthritis-encephalitis virus (CAEV) infection.

    PubMed

    Tu, Po-An; Shiu, Jia-Shian; Lee, Shu-Hwae; Pang, Victor Fei; Wang, De-Chi; Wang, Pei-Hwa

    2017-05-01

    Caprine arthritis-encephalitis (CAE) in goats is a complex disease syndrome caused by a lentivirus. This persistent viral infection results in arthritis in adult goats and encephalitis in lambs. The prognosis for the encephalitic form is normally poor, and this form of the disease has caused substantial economic losses for goat farmers. Hence, a more efficient detection platform based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) was developed in the present study for detecting the proviral DNA of caprine arthritis-encephalitis virus (CAEV). Under the optimal incubation conditions, specifically, 30min at 37°C for RPA followed by 5min at room temperature for LFD, the assay was found to be sensitive to a lower limit of 80pg of total DNA and 10 copies of plasmid DNA. Furthermore, there was no cross-reaction with other tested viruses, including goat pox virus and bovine leukemia virus. Given its simplicity and portability, this RPA-LFD protocol can serve as an alternative tool to ELISA for the primary screening of CAEV, one that is suitable for both laboratory and field application. When the RPA-LFD was applied in parallel with serological ELISA for the detection of CAEV in field samples, the RPA-LFD assay exhibited a higher sensitivity than the traditional method, and 82% of the 200 samples collected in Taiwan were found to be positive. To our knowledge, this is the first report providing evidence to support the use of an RPA-LFD assay as a specific and sensitive platform for detecting CAEV proviral DNA in goats in a faster manner, one that is also applicable for on-site utilization at farms and that should be useful in both eradication programs and epidemiological studies.

  2. Near-infrared fluorescence-based multiplex lateral flow immunoassay for the simultaneous detection of four antibiotic residue families in milk.

    PubMed

    Chen, Yiqiang; Chen, Qian; Han, Miaomiao; Liu, Jiangyang; Zhao, Peng; He, Lidong; Zhang, Yuan; Niu, Yiming; Yang, Wenjun; Zhang, Liying

    2016-05-15

    In this study, we developed a novel near-infrared fluorescence based multiplex lateral flow immunoassay by conjugating a near-infrared label to broad-specificity monoclonal antibody/receptor as detection complexes. Different antigens were dispensed onto separate test zones of nitrocellulose membrane to serve as capture reagents. This assay format allowed the simultaneous detection of four families of antibiotics (β-lactams, tetracyclines, quinolones and sulfonamides) in milk within 20 min. Qualitative and quantitative analysis of target antibiotics were realized by imaging the fluorescence intensity of the near-infrared label captured on respective test lines. For qualitative analysis, the cut-off values of β-lactams, tetracyclines, quinolones and sulfonamides were determined to be 8 ng/mL, 2 ng/mL, 4 ng/mL and 8 ng/mL respectively, which were much lower than the conventional gold nanoparticle based lateral flow immunoassay. For quantitative analysis, the detection ranges were 0.26-3.56 ng/mL for β-lactams, 0.04-0.98 ng/mL for tetracyclines, 0.08-2.0 ng/mL for quinolones, and 0.1-3.98 ng/mL for sulfonamides, with linear correlation coefficients higher than 0.97. The mean spiked recoveries ranged from 93.7% to 108.2% with coefficient of variations less than 16.3%. These results demonstrated that this novel immunoassay is a promising approach for rapidly screening the four families of antibiotic residues in milk. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Lateral migration of a microdroplet under optical forces in a uniform flow

    SciTech Connect

    Cho, Hyunjun; Chang, Cheong Bong; Jung, Jin Ho; Sung, Hyung Jin

    2014-12-15

    The behavior of a microdroplet in a uniform flow and subjected to a vertical optical force applied by a loosely focused Gaussian laser beam was studied numerically. The lattice Boltzmann method was applied to obtain the two-phase flow field, and the dynamic ray tracing method was adopted to calculate the optical force. The optical forces acting on the spherical droplets agreed well with the analytical values. The numerically predicted droplet migration distances agreed well with the experimentally obtained values. Simulations of the various flow and optical parameters showed that the droplet migration distance nondimensionalized by the droplet radius is proportional to the S number (z{sub d}/r{sub p} = 0.377S), which is the ratio of the optical force to the viscous drag. The effect of the surface tension was also examined. These results indicated that the surface tension influenced the droplet migration distance to a lesser degree than the flow and optical parameters. The results of the present work hold for the refractive indices of the mean fluid and the droplet being 1.33 and 1.59, respectively.

  4. Exploiting green analytical procedures for acidity and iron assays employing flow analysis with simple natural reagent extracts.

    PubMed

    Grudpan, Kate; Hartwell, Supaporn Kradtap; Wongwilai, Wasin; Grudpan, Supara; Lapanantnoppakhun, Somchai

    2011-06-15

    Green analytical methods employing flow analysis with simple natural reagent extracts have been exploited. Various formats of flow based analysis systems including a single line FIA, a simple lab on chip with webcam camera detector, and a newly developed simple lab on chip system with reflective absorption detection and the simple extracts from some available local plants including butterfly pea flower, orchid flower, and beet root were investigated and shown to be useful as alternative self indicator reagents for acidity assay. Various tea drinks were explored to be used for chromogenic reagents in iron determination. The benefit of a flow based system, which allows standards and samples to go through the analysis process in exactly the same conditions, makes it possible to employ simple natural extracts with minimal or no pretreatment or purification. The combinations of non-synthetic natural reagents with minimal processed extracts and the low volume requirement flow based systems create some unique green chemical analyses.

  5. Development of a lateral flow immunoassay strip for rapid detection of CagA antigen of Helicobacter pylori.

    PubMed

    Karakus, Cebrail

    2015-01-01

    About half of the world populations are known to be infected with Helicobacter pylori. The CagA antigen secreting strains provoke severe mucosal damages and act as a risk factor for the development of peptic ulceration and gastric cancer. A lateral flow immunoassay (LFIA) strip was developed based on sandwich format for rapid detection of CagA antigen of H. pylori using gold conjugated monoclonal antibody. This LFIA strip will provide a good aid in the diagnosis of CagA-secreting H. pylori within 10 min instead of time consuming, expensive and laborious invasive approaches.

  6. An eDNA Assay to Monitor a Globally Invasive Fish Species from Flowing Freshwater.

    PubMed

    Adrian-Kalchhauser, Irene; Burkhardt-Holm, Patricia

    2016-01-01

    Ponto-Caspian gobies are a flock of five invasive fish species that have colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby Neogobius melanostomus, figures among the 100 worst invaders in Europe. Current methods to detect the presence of Ponto-Caspian gobies involve catching or sighting the fish. These approaches are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile. To improve monitoring, we developed an assay based on the detection of DNA traces (environmental DNA, or eDNA) of Ponto-Caspian gobies in river water. The assay specifically detects invasive goby DNA and does not react to any native fish species. We apply the assay to environmental samples and demonstrate that parameters such as sampling depth, sampling location, extraction protocol, PCR protocol and PCR inhibition greatly impact detection. We further successfully outline the invasion front of Ponto-Caspian gobies in a large river, the High Rhine in Switzerland, and thus demonstrate the applicability of the assay to lotic environments. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than the conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by untrained individuals, and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen's' reports and fish community monitorings.

  7. Demonstration and properties of a planar heterojunction bipolar transistor with lateral current flow

    NASA Astrophysics Data System (ADS)

    Thornton, Robert L.; Mosby, William J.; Chung, Harlan F.

    1989-10-01

    The authors present fabrication techniques and device performance for a novel transistor structure, the lateral heterojunction bipolar transistor. The lateral heterojunctions are formed by impurity-induced disordering of a GaAs base layer sandwiched between two AlGaAs layers. These transistor structures exhibit current gains of 14 for base widths of 0.74 micron. Transistor action in this device occurs parallel to the surface of the device structure. The active base region of the structure is completely submerged, resulting in a reduction of surface recombination as a mechanism for gain reduction in the device. Impurity-induced disordering is used to widen the bandgap of the alloy in the emitter and collector, resulting in an improvement of the emitter injection efficiency. Since the device is based entirely on a surface diffusion process, the device is completely planar and has no steps involving etching of the III-V alloy material. These advantages lead this device to be considered as a candidate for optoelectronic integration applications. The transistor device functions as a buried heterostructure laser, with a threshold current as low as 6 mA for a 1.4-micron stripe.

  8. Development and evaluation of a flow cytometry microsphere assay to detect anti-histone antibody in dogs.

    PubMed

    Paul, Shoma; Wilkerson, Melinda J; Shuman, Wilma; Harkin, Kenneth R

    2005-09-15

    Anti-nuclear antibody (ANA) is one of the diagnostic parameters that support a diagnosis of autoimmune disorders in humans, dogs, and horses, particularly the condition systemic lupus erythematosus (SLE). The most commonly used method for detecting ANA in canine serum is the indirect immunofluorescence antibody assay (IFA) that detects dog IgG with reactivity towards mammalian cell nuclei. Interpretation of the IFA results is very subjective and dependent on the source of tissue/cellular substrate. We have developed a flow cytometry based assay to detect canine serum antibodies specific to histones. Histones were chosen as the target antigen because these nuclear proteins are the most common nuclear substrate for ANA in dogs with SLE. Microsphere beads were coated with histones and incubated with canine sera. Bound anti-histone antibodies were detected by FITC-conjugated rabbit F(ab')2 anti-dog IgG. Sera from four groups of dogs (47 dogs total) were tested for anti-histone antibodies and compared with the traditional IFA assay. The groups included 15 healthy dogs, 15 dogs with noninflammatory diseases, 9 dogs with polyarthritis and positive ANA, and 8 German shepherds with perianal fistulas. The microsphere assay results indicated that only one dog in the noninflammatory group and four out of nine dogs in the polyarthritis group had mean fluorescent intensity values above our established cut-off (defined as 2 S.D. above the mean of healthy controls). There was moderate agreement between the anti-histone assay and the traditional ANA (kappa statistic=0.54). Absorption of ANA positive serum with total histones dramatically diminished the fluorescent signal detected by flow cytometry and the speckled nuclear pattern observed by IFA, whereas preabsorption did not change the diffuse nuclear staining pattern. These findings indicate that the anti-histone assay will not replace the ANA test and that other nuclear proteins, such as ribonucleoproteins may contribute to the

  9. A simplified flow cytometric assay identifies children with acute lymphoblastic leukemia who have a superior clinical outcome

    PubMed Central

    Coustan-Smith, Elaine; Ribeiro, Raul C.; Stow, Patricia; Zhou, Yinmei; Pui, Ching-Hon; Rivera, Gaston K.; Pedrosa, Francisco; Campana, Dario

    2006-01-01

    Bone marrow normal lymphoid progenitors (CD19+, CD10+, and/or CD34+) are exquisitely sensitive to corticosteroids and other antileukemic drugs. We hypothesized that, in patients with B-lineage acute lymphoblastic leukemia (ALL), cells with this phenotype detected early in treatment should be leukemic rather than normal. We therefore developed a simple and inexpensive flow cytometric assay for such cells and prospectively applied it to bone marrow samples collected on day 19 from 380 children with B-lineage ALL. In 211 patients (55.5%), these cells represented 0.01% or more of the mononuclear cells; results correlated remarkably well with those of more complex flow cytometric and molecular minimal residual disease (MRD) evaluations. Among 84 uniformly treated children, the 10-year incidence of relapse or remission failure was 28.8% ± 7.1% (SE) for the 42 patients with 0.01% or more leukemic cells on day 19 detected by the simplified assay versus 4.8% ± 3.3% for the 42 patients with lower levels (P = .003). These assay results were the strongest predictor of outcome, even after adjustment for competing clinicobiologic variables. Thus, this new assay would enable most treatment centers to identify a high proportion of children with ALL who have an excellent early treatment response and a high likelihood of cure. (Blood. 2006;108:97-102) PMID:16537802

  10. Lateral migration and equilibrium shape and position of a single red blood cell in bounded Poiseuille flows.

    PubMed

    Shi, Lingling; Pan, Tsorng-Whay; Glowinski, Roland

    2012-11-01

    Lateral migration and equilibrium shape and position of a single red blood cell (RBC) in bounded two-dimensional Poiseuille flows are investigated by using an immersed boundary method. An elastic spring model is applied to simulate the skeleton structure of a RBC membrane. We focus on studying the properties of lateral migration of a single RBC in Poiseuille flows by varying the initial position, the initial angle, the swelling ratio (s), the membrane bending stiffness of RBC (k{b}), the maximum velocity of fluid flow (u{max}), and the degree of confinement. The combined effect of the deformability, the degree of confinement, and the shear gradient of the Poiseuille flow make the RBCs migrate toward a certain cross-sectional equilibrium position, which lies either on the center line of the channel or off center line. For s>0.8, the speed of the migration at the beginning decreases as one increases the swelling ratio s. But for s<0.8, the speed of the migration at the beginning is an increasing function of the swelling ratio s. Two motions of oscillation and vacillating breathing (swing) of RBCs are observed. The distance Y{d} between the cell mass center of the equilibrium position and the center line of the channel increases with increasing the Reynolds number Re and reaches a peak, then decreases with increasing Re. The peak of Re is a decreasing function of the swelling ratio (s<1.0). The cell membrane energy of the equilibrium position is an increasing function as Re increases. The slipper-shaped cell is more stable than the parachute-shaped one in the sense that the energy stored in the former is lower than that in the latter. For a given Re, the bigger the swelling ratio (s<1.0), the lower the cell membrane energy.

  11. Lateral migration and equilibrium shape and position of a single red blood cell in bounded Poiseuille flows

    NASA Astrophysics Data System (ADS)

    Shi, Lingling; Pan, Tsorng-Whay; Glowinski, Roland

    2012-11-01

    Lateral migration and equilibrium shape and position of a single red blood cell (RBC) in bounded two-dimensional Poiseuille flows are investigated by using an immersed boundary method. An elastic spring model is applied to simulate the skeleton structure of a RBC membrane. We focus on studying the properties of lateral migration of a single RBC in Poiseuille flows by varying the initial position, the initial angle, the swelling ratio (s*), the membrane bending stiffness of RBC (kb), the maximum velocity of fluid flow (umax), and the degree of confinement. The combined effect of the deformability, the degree of confinement, and the shear gradient of the Poiseuille flow make the RBCs migrate toward a certain cross-sectional equilibrium position, which lies either on the center line of the channel or off center line. For s*>0.8, the speed of the migration at the beginning decreases as one increases the swelling ratio s*. But for s*<0.8, the speed of the migration at the beginning is an increasing function of the swelling ratio s*. Two motions of oscillation and vacillating breathing (swing) of RBCs are observed. The distance Yd between the cell mass center of the equilibrium position and the center line of the channel increases with increasing the Reynolds number Re and reaches a peak, then decreases with increasing Re. The peak of Re is a decreasing function of the swelling ratio (s*<1.0). The cell membrane energy of the equilibrium position is an increasing function as Re increases. The slipper-shaped cell is more stable than the parachute-shaped one in the sense that the energy stored in the former is lower than that in the latter. For a given Re, the bigger the swelling ratio (s*<1.0), the lower the cell membrane energy.

  12. Rapid and sensitive detection of novel avian-origin influenza A (H7N9) virus by reverse transcription loop-mediated isothermal amplification combined with a lateral-flow device.

    PubMed

    Ge, Yiyue; Wu, Bin; Qi, Xian; Zhao, Kangchen; Guo, Xiling; Zhu, Yefei; Qi, Yuhua; Shi, Zhiyang; Zhou, Minghao; Wang, Hua; Cui, Lunbiao

    2013-01-01

    A severe disease in humans caused by a novel avian-origin influenza A (H7N9) virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD) assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9) virus infection.

  13. Rapid and Sensitive Detection of Novel Avian-Origin Influenza A (H7N9) Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Lateral-Flow Device

    PubMed Central

    Qi, Xian; Zhao, Kangchen; Guo, Xiling; Zhu, Yefei; Qi, Yuhua; Shi, Zhiyang; Zhou, Minghao; Wang, Hua; Cui, Lunbiao

    2013-01-01

    A severe disease in humans caused by a novel avian-origin influenza A (H7N9) virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD) assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9) virus infection. PMID:23936359

  14. Lateral Flow of Carbon From U.S. Agricultural Lands: Carbon Uptake, Consumption, and Respiration

    NASA Astrophysics Data System (ADS)

    Sabesan, A.; West, T. O.; Roddy, A. B.; Marland, G.; Bhaduri, B. L.

    2005-12-01

    Net carbon exchange between biomass and the atmosphere can be estimated and modeled on a regional basis to understand the effects of land-use change on the carbon cycle and on net CO2 emissions to the atmosphere. However, within ecosystems that are managed to produce commodities for consumption (i.e., agriculture and forest lands), carbon can be transported laterally when crops or timber are harvested, in addition to being transported vertically between plants and the atmosphere. The spatial and temporal domain over which carbon uptake, transport, and release occur has implications for regional carbon studies. For example, carbon may be taken up by crops in one region, but released through human consumption in another region. Estimates of lateral transport and release of carbon may therefore contribute another dimension to bottom-up carbon modeling, and may also be used as input for comparison to top-down atmospheric modeling. Our research to date has focused on the uptake, consumption, and respiration of CO2 associated with agricultural crops and related food commodities. We estimate a net uptake of 495 Tg C on U.S. croplands in 2000. This uptake occurs primarily in the Midwestern U.S. Human respiration of CO2 contributed about 31 Tg C and livestock emitted about 77 Tg C as CO2 and CH4 in 2000. Estimates of CO2 from food wastes in municipal landfills and from human excrement in wastewater treatment plants are currently being developed. The spatial distribution of CO2 uptake and release are mapped, respectively, at the county level and at 1km resolution that is commensurate with Landscan USA population data.

  15. Establishment of experimental conditions for preserving samples of fish blood for analysis with both comet assay and flow cytometry.

    PubMed

    Ramsdorf, Wanessa A; Guimarães, Fernando de S F; Ferraro, Marcos V M; Gabardo, Juarez; Trindade, Edvaldo da Silva; Cestari, Marta Margarete

    2009-02-19

    When environmental analysis is performed, the high number of samples required and handling conditions during the transport of these samples to the laboratory are common problems. The comet assay is a useful, highly sensitive tool in biomonitoring. Some studies in the literature aim to preserve slides in lysis solution for use in the comet assay. Until now, however, no efficient methodology for preserving blood samples for this assay has been described. Because of this, the present report aimed to establish the proper conditions for samples maintenance prior to comet