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Sample records for pathogen immunoassay sensing

  1. The antibody-based magnetic microparticle immunoassay using p-FET sensing platform for Alzheimer's disease pathogenic factor

    Science.gov (United States)

    Kim, Chang-Beom; Kim, Kwan-Soo; Song, Ki-Bong

    2013-05-01

    The importance of early Alzheimer's disease (AD) detection has been recognized to diagnose people at high risk of AD. The existence of intra/extracellular beta-amyloid (Aβ) of brain neurons has been regarded as the most archetypal hallmark of AD. The existing computed-image-based neuroimaging tools have limitations on accurate quantification of nanoscale Aβ peptides due to optical diffraction during imaging processes. Therefore, we propose a new method that is capable of evaluating a small amount of Aβ peptides by using photo-sensitive field-effect transistor (p-FET) integrated with magnetic force-based microbead collecting platform and selenium(Se) layer (thickness ~700 nm) as an optical filter. This method demonstrates a facile approach for the analysis of Aβ quantification using magnetic force and magnetic silica microparticles (diameter 0.2~0.3 μm). The microbead collecting platform mainly consists of the p-FET sensing array and the magnet (diameter ~1 mm) which are placed beneath each sensing region of the p-FET, which enables the assembly of the Aβ antibody conjugated microbeads, captures the Aβ peptides from samples, measures the photocurrents generated by the Q-dot tagged with Aβ peptides, and consequently results in the effective Aβ quantification.

  2. Multiplex detection of plant pathogens using a microsphere immunoassay technology.

    Directory of Open Access Journals (Sweden)

    Ratthaphol Charlermroj

    Full Text Available Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac, chilli vein-banding mottle virus (CVbMV, potyvirus, watermelon silver mottle virus (WSMoV, tospovirus serogroup IV and melon yellow spot virus (MYSV, tospovirus. An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour was much shorter than that of ELISA (4 hours. This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.

  3. Multiplex detection of plant pathogens using a microsphere immunoassay technology.

    Science.gov (United States)

    Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Kumpoosiri, Mallika; Warin, Nuchnard; Oplatowska, Michalina; Gajanandana, Oraprapai; Grant, Irene R; Karoonuthaisiri, Nitsara; Elliott, Christopher T

    2013-01-01

    Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.

  4. Fluorescence immunoassay for detecting periodontal bacterial pathogens in plaque.

    OpenAIRE

    Wolff, L F; Anderson, L; Sandberg, G P; Aeppli, D M; Shelburne, C E

    1991-01-01

    A particle concentration fluorescence immunoassay has been modified into a bacterial concentration fluorescence immunoassay (BCFIA) to rapidly detect periodontopathic bacteria in human plaque samples. The BCFIA utilizes fluorescently tagged monoclonal antibodies (MAbs) directed against the lipopolysaccharide of selected gram-negative plaque bacteria. Microorganisms closely associated with periodontal disease that can be identified in plaque with the BCFIA include Porphyromonas gingivalis, Bac...

  5. Quorum Sensing of Periodontal Pathogens

    Directory of Open Access Journals (Sweden)

    Darije Plančak

    2015-01-01

    Full Text Available The term ‘quorum sensing’ describes intercellular bacterial communication which regulates bacterial gene expression according to population cell density. Bacteria produce and secrete small molecules, named autoinducers, into the intercellular space. The concentration of these molecules increases as a function of population cell density. Once the concentration of the stimulatory threshold is reached, alteration in gene expression occurs. Gram-positive and Gram-negative bacteria possess different types of quorum sensing systems. Canonical LuxI/R-type/acyl homoserine lactone mediated quorum sensing system is the best studied quorum sensing circuit and is described in Gram-negative bacteria which employ it for inter-species communication mostly. Grampositive bacteria possess a peptide-mediated quorum sensing system. Bacteria can communicate within their own species (intra-species but also between species (inter-species, for which they employ an autoinducer-2 quorum sensing system which is called the universal language of the bacteria. Periodontal pathogenic bacteria possess AI-2 quorum sensing systems. It is known that they use it for regulation of biofilm formation, iron uptake, stress response and virulence factor expression. A better understanding of bacterial communication mechanisms will allow the targeting of quorum sensing with quorum sensing inhibitors to prevent and control disease.

  6. Development of a chip-based multiplexed immunoassay using liposomal nanovesicles and its application in the detection of pathogens causing female lower genital tract infections

    Directory of Open Access Journals (Sweden)

    Wen-Hsiang Su

    2013-03-01

    Conclusion: This microarray chip was a rapid, easy, inexpensive and sensitive tool for detecting female lower genital tract Candida infection in a one-time vaginal sampling process, although the data on the four other pathogens were still unavailable. A larger population study is encouraged to test the validity of this multiplexed immunoassay chip.

  7. Quorum sensing and bacterial pathogenicity: From molecules to disease

    Directory of Open Access Journals (Sweden)

    Antariksh Deep

    2011-01-01

    Full Text Available Quorum sensing in prokaryotic biology refers to the ability of a bacterium to sense information from other cells in the population when they reach a critical concentration (i.e. a Quorum and communicate with them. The "language" used for this intercellular communication is based on small, self-generated signal molecules called as autoinducers. Quorum sensing is thought to afford pathogenic bacteria a mechanism to minimize host immune responses by delaying the production of tissue-damaging virulence factors until sufficient bacteria have amassed and are prepared to overwhelm host defense mechanisms and establish infection. Quorum sensing systems are studied in a large number of gram-negative bacterial species belonging to α, β, and γ subclasses of proteobacteria. Among the pathogenic bacteria, Pseudomonas aeruginosa is perhaps the best understood in terms of the virulence factors regulated and the role the Quorum sensing plays in pathogenicity. Presently, Quorum sensing is considered as a potential novel target for antimicrobial therapy to control multi/all drug-resistant infections. This paper reviews Quorum sensing in gram positive and gram negative bacteria and its role in biofilm formation.

  8. Functionalization of single-walled carbon nanotubes with protein by click chemistry as sensing platform for sensitized electrochemical immunoassay

    International Nuclear Information System (INIS)

    Qi Honglan; Ling Chen; Huang Ru; Qiu Xiaoying; Shangguan Li; Gao Qiang; Zhang Chengxiao

    2012-01-01

    Highlights: ► Single-walled carbon nanotubes were functionalized with protein by click chemistry. ► The SWNTs conjugated with protein showed excellent dispersion in water and kept good bioacitvity. ► A competitive electrochemical immunoassay for the determination of anti-IgG was developed with high sensitivity and good stability. - Abstract: The application of the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition to the functionalization of single-walled carbon nanotubes (SWNTs) with the protein and the use of the artificial SWNTs as a sensing platform for sensitive immunoassay were reported. Covalent functionalization of azide decorated SWNTs with alkyne modified protein was firstly accomplished by the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition. FT-IR spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and transmission electron micrograph were used to characterize the protein-functionalized SWNTs. It was found that the SWNTs conjugated with the proteins showed excellent dispersion in water and kept good bioacitivity when immunoglobulin (IgG) and horseradish peroxidase (HRP) were chosen as model proteins. As a proof-of-concept, IgG-functionalized SWNTs were immobilized onto the surface of a glassy carbon electrode by simple casting method as immunosensing platform and a sensitive competitive electrochemical immunoassay was developed for the determination of anti-immunoglobulin (anti-IgG) using HRP as enzyme label. The fabrication of the immunosensor were characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the redox probe [Fe(CN) 6 ] 3−/4− . The SWNTs as immobilization platform showed better sensitizing effect, a detection limit of 30 pg mL −1 (S/N = 3) was obtained for anti-IgG. The proposed strategy provided a stable immobilization method and sensitized recognition platform for analytes. This work demonstrated that the click coupling of SWNTs with protein was an effective

  9. Mechanisms of quorum sensing and strategies for quorum sensing disruption in aquaculture pathogens.

    Science.gov (United States)

    Zhao, J; Chen, M; Quan, C S; Fan, S D

    2015-09-01

    In many countries, infectious diseases are a considerable threat to aquaculture. The pathogenicity of micro-organisms that infect aquaculture systems is closely related to the release of virulence factors and the formation of biofilms, both of which are regulated by quorum sensing (QS). Thus, QS disruption is a potential strategy for preventing disease in aquaculture systems. QS inhibitors (QSIs) not only inhibit the expression of virulence-associated genes but also attenuate the virulence of aquaculture pathogens. In this review, we discuss QS systems in important aquaculture pathogens and focus on the relationship between QS mechanisms and bacterial virulence in aquaculture. We further elucidate QS disruption strategies for targeting aquaculture pathogens. Four main types of QSIs that target aquaculture pathogens are discussed based on their mechanisms of action. © 2014 John Wiley & Sons Ltd.

  10. Actuation of chitosan-aptamer nanobrush borders for pathogen sensing.

    Science.gov (United States)

    Hills, Katherine D; Oliveira, Daniela A; Cavallaro, Nicholas D; Gomes, Carmen L; McLamore, Eric S

    2018-03-26

    We demonstrate a sensing mechanism for rapid detection of Listeria monocytogenes in food samples using the actuation of chitosan-aptamer nanobrush borders. The bio-inspired soft material and sensing strategy mimic natural symbiotic systems, where low levels of bacteria are selectively captured from complex matrices. To engineer this biomimetic system, we first develop reduced graphene oxide/nanoplatinum (rGO-nPt) electrodes, and characterize the fundamental electrochemical behavior in the presence and absence of chitosan nanobrushes during actuation (pH-stimulated osmotic swelling). We then characterize the electrochemical behavior of the nanobrush when receptors (antibodies or DNA aptamers) are conjugated to the surface. Finally, we test various techniques to determine the most efficient capture strategy based on nanobrush actuation, and then apply the biosensors in a food product. Maximum cell capture occurs when aptamers conjugated to the nanobrush bind cells in the extended conformation (pH 6). The aptamer-nanobrush hybrid material was more efficient than the antibody-nanobrush material, which was likely due to the relatively high adsorption capacity for aptamers. The biomimetic material was used to develop a rapid test (17 min) for selectively detecting L. monocytogenes at concentrations ranging from 9 to 107 CFU mL-1 with no pre-concentration, and in the presence of other Gram-positive cells (Listeria innocua and Staphylococcus aureus). Use of this bio-inspired material is among the most efficient for L. monocytogenes sensing to date, and does not require sample pretreatment, making nanobrush borders a promising new material for rapid pathogen detection in food.

  11. Hydrogel nanoparticle based immunoassay

    Science.gov (United States)

    Liotta, Lance A; Luchini, Alessandra; Petricoin, Emanuel F; Espina, Virginia

    2015-04-21

    An immunoassay device incorporating porous polymeric capture nanoparticles within either the sample collection vessel or pre-impregnated into a porous substratum within fluid flow path of the analytical device is presented. This incorporation of capture particles within the immunoassay device improves sensitivity while removing the requirement for pre-processing of samples prior to loading the immunoassay device. A preferred embodiment is coreshell bait containing capture nanoparticles which perform three functions in one step, in solution: a) molecular size sieving, b) target analyte sequestration and concentration, and c) protection from degradation. The polymeric matrix of the capture particles may be made of co-polymeric materials having a structural monomer and an affinity monomer, the affinity monomer having properties that attract the analyte to the capture particle. This device is useful for point of care diagnostic assays for biomedical applications and as field deployable assays for environmental, pathogen and chemical or biological threat identification.

  12. DMPD: Innate immune sensing of pathogens and danger signals by cell surface Toll-likereceptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17275324 Innate immune sensing of pathogens and danger signals by cell surface Toll... Show Innate immune sensing of pathogens and danger signals by cell surface Toll-likereceptors. PubmedID 172...75324 Title Innate immune sensing of pathogens and danger signals by cell surface

  13. Quorum sensing Inhibitors as anti-pathogenic drugs

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bovbjerg; Givskov, Michael Christian

    2006-01-01

    as well as elevated tolerance to the activity of the innate immune system. Gram-negative bacteria commonly use N-acyl homoserine lactones (AHL) as QS signal molecules. The use of signal molecule based drugs to attenuate bacterial pathogenecity rather than bacterial growth is attractive for several reasons......, particularly considering the emergence of increasingly antibiotic-resistant bacteria. Compounds capable of this type of interference have been termed anti-pathogenic drugs. A large variety of synthetic AHL analogues and natural products libraries have been screened and a number of QS inhibitors (QSI) have been...

  14. Novel redox species polyaniline derivative-Au/Pt as sensing platform for label-free electrochemical immunoassay of carbohydrate antigen 199

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Liyuan; Shan, Jiao; Feng, Feng; Ma, Zhanfang, E-mail: mazhanfang@cnu.edu.cn

    2016-03-10

    A novel electrochemical redox-active nanocomposite was synthesized by a one-pot method using N,N′-diphenyl-p-phenylediamine as monomer, and HAuCl{sub 4} and K{sub 2}PtCl{sub 4} as co-oxidizing agents. The as-prepared poly(N,N′-diphenyl-p-phenylediamine)-Au/Pt exhibited admirable electrochemical redox activity at 0.15 V, excellent H{sub 2}O{sub 2} electrocatalytic ability and favorable electron transfer ability. Based on these, the evaluation of the composite as sensing substrate for label-free electrochemical immunosensing to the sensitive detection of carbohydrate antigen 199 was described. This technique proved to be a prospective detection tool with a wide liner range from 0.001 U mL{sup −1} to 40 U mL{sup −1}, and a low detection limit of 2.3 × 10{sup −4} U mL{sup −1} (S/N = 3). In addition, this method was used for the analysis of human serum sample, and good agreement was obtained between the values and those of enzyme-linked immunosorbent assay, implying the potential application in clinical research. Importantly, the strategy of the present substrate could be extended to other polymer-based nanocomposites such as polypyrrole derivatives or polythiophene derivatives, and this could be of great significance for the electrochemical immunoassay. - Highlights: • A novel electrochemical redox composite PPPD-Au/Pt was synthesized by one-pot method. • PPPD-Au/Pt was used as sensing substrate for label-free electrochemical immunosensor. • The immunosensor showed wide detection range and ultralow detection limit for the detection of CA199.

  15. Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics

    DEFF Research Database (Denmark)

    Arevalo-Ferro, C.; Hentzer, Morten; Reil, G.

    2003-01-01

    The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely......-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and Has......Ap, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type...

  16. From deep-sea volcanoes to human pathogens: a conserved quorum-sensing signal in Epsilonproteobacteria.

    Science.gov (United States)

    Pérez-Rodríguez, Ileana; Bolognini, Marie; Ricci, Jessica; Bini, Elisabetta; Vetriani, Costantino

    2015-05-01

    Chemosynthetic Epsilonproteobacteria from deep-sea hydrothermal vents colonize substrates exposed to steep thermal and redox gradients. In many bacteria, substrate attachment, biofilm formation, expression of virulence genes and host colonization are partly controlled via a cell density-dependent mechanism involving signal molecules, known as quorum sensing. Within the Epsilonproteobacteria, quorum sensing has been investigated only in human pathogens that use the luxS/autoinducer-2 (AI-2) mechanism to control the expression of some of these functions. In this study we showed that luxS is conserved in Epsilonproteobacteria and that pathogenic and mesophilic members of this class inherited this gene from a thermophilic ancestor. Furthermore, we provide evidence that the luxS gene is expressed--and a quorum-sensing signal is produced--during growth of Sulfurovum lithotrophicum and Caminibacter mediatlanticus, two Epsilonproteobacteria from deep-sea hydrothermal vents. Finally, we detected luxS transcripts in Epsilonproteobacteria-dominated biofilm communities collected from deep-sea hydrothermal vents. Taken together, our findings indicate that the epsiloproteobacterial lineage of the LuxS enzyme originated in high-temperature geothermal environments and that, in vent Epsilonproteobacteria, luxS expression is linked to the production of AI-2 signals, which are likely produced in situ at deep-sea vents. We conclude that the luxS gene is part of the ancestral epsilonproteobacterial genome and represents an evolutionary link that connects thermophiles to human pathogens.

  17. Proximal Sensing of Plant-Pathogen Interactions in Spring Barley with Three Fluorescence Techniques

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    Georg Leufen

    2014-06-01

    Full Text Available In the last years fluorescence spectroscopy has come to be viewed as an essential approach in key research fields of applied plant sciences. However, the quantity and particularly the quality of information produced by different equipment might vary considerably. In this study we investigate the potential of three optical devices for the proximal sensing of plant-pathogen interactions in four genotypes of spring barley. For this purpose, the fluorescence lifetime, the image-resolved multispectral fluorescence and selected indices of a portable multiparametric fluorescence device were recorded at 3, 6, and 9 days after inoculation (dai from healthy leaves as well as from leaves inoculated with powdery mildew (Blumeria graminis or leaf rust (Puccinia hordei. Genotype-specific responses to pathogen infections were revealed already at 3 dai by higher fluorescence mean lifetimes in the spectral range from 410 to 560 nm in the less susceptible varieties. Noticeable pathogen-induced modifications were also revealed by the ‘Blue-to-Far-Red Fluorescence Ratio’ and the ‘Simple Fluorescence Ratio’. Particularly in the susceptible varieties the differences became more evident in the time-course of the experiment i.e., following the pathogen development. The relevance of the blue and green fluorescence to exploit the plant-pathogen interaction was demonstrated by the multispectral fluorescence imaging system. As shown, mildewed leaves were characterized by exceptionally high blue fluorescence, contrasting the values observed in rust inoculated leaves. Further, we confirm that the intensity of green fluorescence depends on the pathogen infection and the stage of disease development; this information might allow a differentiation of both diseases. Moreover, our results demonstrate that the detection area might influence the quality of the information, although it had a minor impact only in the current study. Finally, we highlight the relevance of

  18. Role and regulation of the orphan AphA protein of quorum sensing in pathogenic Vibrios.

    Science.gov (United States)

    Lu, Renfei; Osei-Adjei, George; Huang, Xinxiang; Zhang, Yiquan

    2018-03-01

    Quorum sensing (QS), a cell-to-cell communication process, is widely distributed in the bacterial kingdom. Bacteria use QS to control gene expression in response to cell density by detecting the signal molecules called autoinducers. AphA protein is the master QS regulator of vibrios operating at low cell density. It regulates the expression of a variety of genes, especially those encoding virulence factors, flagella/motility and biofilm formation. The role and regulation of AphA in vibrios, especially in human pathogenic vibrios, are summarized in this review. Clarification of the roles of AphA will help us to understand the pathogenesis of vibrios.

  19. Andrographolide interferes quorum sensing to reduce cell damage caused by avian pathogenic Escherichia coli.

    Science.gov (United States)

    Guo, Xun; Zhang, Li-Yan; Wu, Shuai-Cheng; Xia, Fang; Fu, Yun-Xing; Wu, Yong-Li; Leng, Chun-Qing; Yi, Peng-Fei; Shen, Hai-Qing; Wei, Xu-Bin; Fu, Ben-Dong

    2014-12-05

    Avian pathogenic Escherichia coli (APEC) induce septicemia in chickens by invading type II pneumocytes to breach the blood-air barrier. The virulence of APEC can be regulated by quorum sensing (QS). Andrographolide is a QS inhibitor of Pseudomonas aeruginosa (P. aeruginosa). Therefore, we investigate whether andrographolide inhibits the injury of chicken type II pneumocytes by avian pathogenic E. coli O78 (APEC-O78) by disrupting the bacterial QS system. The results showed that sub-MIC of andrographolide significantly reduced the release of lactate dehydrogenase (LDH), F-actin cytoskeleton polymerization, and the degree of the adherence to chicken type II pneumocytes induced by APEC-O78. Further, we found that andrographolide significantly decreased the autoinducer-2 (AI-2) activity and the expression of virulence factors of APEC-O78. These results suggest that andrographolide reduce the pathogenicity of APEC-O78 in chicken type II pneumocytes by interfering QS and decreasing virulence. These results provide new evidence for colibacillosis prevention methods in chickens. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Luminescence, virulence and quorum sensing signal production by pathogenic Vibrio campbellii and Vibrio harveyi isolates.

    Science.gov (United States)

    Defoirdt, T; Verstraete, W; Bossier, P

    2008-05-01

    To study the relationship between luminescence, autoinducer production and virulence of pathogenic vibrios. Luminescence, quorum sensing signal production and virulence towards brine shrimp nauplii of 13 Vibrio campbellii and Vibrio harveyi strains were studied. Although only two of the tested strains were brightly luminescent, all of them were shown to produce the three different types of quorum sensing signals known to be produced by Vibrio harveyi. Cell-free culture fluids of all strains significantly induced bioluminescence in the cholerae autoinducer 1, autoinducer 2 and harveyi autoinducer 1 reporter strains JAF375, JMH597 and JMH612, respectively. There was no relation between luminescence and signal production and virulence towards brine shrimp. There is a large difference between different strains of Vibrio campbellii and Vibrio harveyi with respect to bioluminescence. However, this is not reflected in signal production and virulence towards gnotobiotic brine shrimp. Moreover, there seems to be no relation between quorum sensing signal production and virulence towards brine shrimp. The results presented here indicate that strains that are most brightly luminescent are not necessarily the most virulent ones and that the lower virulence of some of the strains is not due to a lack of autoinducer production.

  1. Quorum sensing signal molecules (acylated homoserine lactones) in Gram-negative fish pathogenic bacteria

    DEFF Research Database (Denmark)

    Bruhn, Jesper Bartholin; Dalsgaard, Inger; Nielsen, K.F.

    2005-01-01

    The aim of the present study was to investigate the production of quorum sensing signals (specifically acylated homoserine lactones, AHLs) among a selection of strains of Gram-negative fish bacterial pathogens. These signals are involved in the regulation of virulence factors in some human...... salmonicida and Vibrio splendidus were also positive. Aeromonas species produced N-butanoyl homoserine lactone (BHL) and N-hexanoyl homoserine lactone (HHL) and 1 additional product, whereas N-3-oxo-hexanoyl homoserine lactone (OHHL) and HHL were detected in Vibrio salmonicida. N-3-oxo-octanoyl homoserine...... lactone (OOHL) and N-3-octanoyl homoserine lactone (OHL) were detected in Y. ruckeii. AHLs were not detected from strains of Photobacterium damselae, Flavobacterium psychrophilum or Moritella viscosa. AHLs were extracted from fish infected with Y. ruckeri but not from fish infected with A. salmonicida...

  2. Quorum Sensing Signaling and Quenching in the Multidrug-Resistant Pathogen Stenotrophomonas maltophilia

    Directory of Open Access Journals (Sweden)

    Pol Huedo

    2018-04-01

    Full Text Available Stenotrophomonas maltophilia is an opportunistic Gram-negative pathogen with increasing incidence in clinical settings. The most critical aspect of S. maltophilia is its frequent resistance to a majority of the antibiotics of clinical use. Quorum Sensing (QS systems coordinate bacterial populations and act as major regulatory mechanisms of pathogenesis in both pure cultures and poly-microbial communities. Disruption of QS systems, a phenomenon known as Quorum Quenching (QQ, represents a new promising paradigm for the design of novel antimicrobial strategies. In this context, we review the main advances in the field of QS in S. maltophilia by paying special attention to Diffusible Signal Factor (DSF signaling, Acyl Homoserine Lactone (AHL responses and the controversial Ax21 system. Advances in the DSF system include regulatory aspects of DSF synthesis and perception by both rpf-1 and rpf-2 variant systems, as well as their reciprocal communication. Interaction via DSF of S. maltophilia with unrelated organisms including bacteria, yeast and plants is also considered. Finally, an overview of the different QQ mechanisms involving S. maltophilia as quencher and as object of quenching is presented, revealing the potential of this species for use in QQ applications. This review provides a comprehensive snapshot of the interconnected QS network that S. maltophilia uses to sense and respond to its surrounding biotic or abiotic environment. Understanding such cooperative and competitive communication mechanisms is essential for the design of effective anti QS strategies.

  3. Modulation of Quorum Sensing in a Gram Positive Pathogen by Linear Imprinted Copolymers with anti-Infective Properties

    NARCIS (Netherlands)

    Motib, Anfal; Guerreiro, Antonio; Al-Bayati, Firas; Piletska, Elena; Manzoor, Irfan; Shafeeq, Sulman; Kadam, Anagha; Kuipers, Oscar; Hiller, Luisa; Cowen, Todd; Piletsky, Sergey; Andrew, Peter; Yesilkaya, Hasan

    2017-01-01

    Here we describe the development, characterization and biological testing of a new type of linear molecularly imprinted polymer (LMIP) designed to act as anti-infective by blocking the quorum sensing (QS) mechanism and so preventing virulence of the pathogen Streptococcus pneumoniae. The LMIP is

  4. Immunoassays in Biotechnology

    Science.gov (United States)

    Immunoassays have broad applications for a wide variety of important biological compounds and environmental contaminants. Immunoassays can detect the presence of an antigen in the human body, a pollutant in the environment, or a critical antibody in a patient’s serum to develop a...

  5. Advanced techniques in immunoassay

    International Nuclear Information System (INIS)

    Toth, G.

    1982-01-01

    A brief overview of the development history of radioimmunoassay and related techniques with their theory and practice are given. A comparison of radioimmunoassay (RIA), enzyme immunoassay (EIA), spin immunoassay (SIA), sequential saturation analysis (SSA) etc., based on their main parameters, and their fields of application and recent trends are presented. (Sz.J.)

  6. Interference of quorum sensing in urinary pathogen Serratia marcescens by Anethum graveolens.

    Science.gov (United States)

    Salini, Ramesh; Pandian, Shunmugiah Karutha

    2015-08-01

    Serratia marcescens is an opportunistic turned obligate pathogen frequently associated with urinary tract infections (UTI) and are multidrug resistant at most instances. Quorum sensing (QS) system, a population-dependent global regulatory system, controls the pathogenesis machinery of S. marcescens as it does in other pathogens. In the present study, methanol extract of a common herb and spice, Anethum graveolens (AGME) was assessed for its anti-QS potential against the clinical isolate of S. marcescens. AGME notably reduced the biofilm formation and QS-dependent virulence factors production in a concentration-dependent manner (64-1024 μg mL(-1)). The light and confocal microscopic images clearly evidenced the antibiofilm activity of AGME (256 μg mL(-1)) at its minimal biofilm inhibitory concentration. Besides, in support of biochemical assays, the expression analysis of QS-regulated genes fimC, bsmA and flhD which are crucial for initial adhesion and motility confirmed their downregulation upon exposure to AGME. LC-MS analysis of AGME revealed 3-O-methyl ellagic acid (3-O-ME) as one of its active principles having nearly similar antibiofilm activity and a reduced inhibition of prodigiosin (27%) and protease (15%) compared to AGME [prodigiosin (47%) and protease (50%)]. UFLC analysis revealed that 0.355 mg g(-1) of 3-O-ME was present in the AGME. AGME and the 3-O-ME significantly interfered the QS system of a QS model strain S. marcescens MG1 and its mutant S. marcescens MG44 which in turn corroborates the anti-QS mechanism of AGME. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Identification of anti-virulence compounds that disrupt quorum-sensing regulated acute and persistent pathogenicity.

    Directory of Open Access Journals (Sweden)

    Melissa Starkey

    2014-08-01

    Full Text Available Etiological agents of acute, persistent, or relapsing clinical infections are often refractory to antibiotics due to multidrug resistance and/or antibiotic tolerance. Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen that causes recalcitrant and severe acute chronic and persistent human infections. Here, we target the MvfR-regulated P. aeruginosa quorum sensing (QS virulence pathway to isolate robust molecules that specifically inhibit infection without affecting bacterial growth or viability to mitigate selective resistance. Using a whole-cell high-throughput screen (HTS and structure-activity relationship (SAR analysis, we identify compounds that block the synthesis of both pro-persistence and pro-acute MvfR-dependent signaling molecules. These compounds, which share a benzamide-benzimidazole backbone and are unrelated to previous MvfR-regulon inhibitors, bind the global virulence QS transcriptional regulator, MvfR (PqsR; inhibit the MvfR regulon in multi-drug resistant isolates; are active against P. aeruginosa acute and persistent murine infections; and do not perturb bacterial growth. In addition, they are the first compounds identified to reduce the formation of antibiotic-tolerant persister cells. As such, these molecules provide for the development of next-generation clinical therapeutics to more effectively treat refractory and deleterious bacterial-human infections.

  8. Radiolabelling for immunoassay

    International Nuclear Information System (INIS)

    Chapman, R.S.

    1998-01-01

    Since the early 1960s labelled compounds employed in immunoassay techniques, both radioimmunoassay and immunoradiometric assay, have involved radioisotopes typically 3 H (tritium) and 125 Iodine. With the advent of increasingly stringent governmental regulations regarding usage and disposal of radioisotopes and the impetus of research towards improved immunoassay sensitivity following the discovery of monoclonal antibodies and their application to excess reagent immunometric assay methodology, radioisotopic labels are gradually being replaced by non-isotopic labels: enzyme, fluorescence and chemiluminescence

  9. Quorum sensing coordinates brute force and stealth modes of infection in the plant pathogen Pectobacterium atrosepticum.

    Directory of Open Access Journals (Sweden)

    Hui Liu

    2008-06-01

    Full Text Available Quorum sensing (QS in vitro controls production of plant cell wall degrading enzymes (PCWDEs and other virulence factors in the soft rotting enterobacterial plant pathogen Pectobacterium atrosepticum (Pba. Here, we demonstrate the genome-wide regulatory role of QS in vivo during the Pba-potato interaction, using a Pba-specific microarray. We show that 26% of the Pba genome exhibited differential transcription in a QS (expI- mutant, compared to the wild-type, suggesting that QS may make a greater contribution to pathogenesis than previously thought. We identify novel components of the QS regulon, including the Type I and II secretion systems, which are involved in the secretion of PCWDEs; a novel Type VI secretion system (T6SS and its predicted substrates Hcp and VgrG; more than 70 known or putative regulators, some of which have been demonstrated to control pathogenesis and, remarkably, the Type III secretion system and associated effector proteins, and coronafacoyl-amide conjugates, both of which play roles in the manipulation of plant defences. We show that the T6SS and a novel potential regulator, VirS, are required for full virulence in Pba, and propose a model placing QS at the apex of a regulatory hierarchy controlling the later stages of disease progression in Pba. Our findings indicate that QS is a master regulator of phytopathogenesis, controlling multiple other regulators that, in turn, co-ordinately regulate genes associated with manipulation of host defences in concert with the destructive arsenal of PCWDEs that manifest the soft rot disease phenotype.

  10. Electrochemical sensing platform based on tris(2,2'-bipyridyl)cobalt(III) and multiwall carbon nanotubes-Nafion composite for immunoassay of carcinoma antigen-125

    International Nuclear Information System (INIS)

    Chen Shihong; Yuan Ruo; Chai Yaqin; Min Ligen; Li Wenjuan; Xu Yang

    2009-01-01

    A new strategy for constructing a sensitive mediator-type electrochemical immunosensor for the detection of carcinoma antigen-125 (CA125) was developed. In this strategy, mediator tris(2,2'-bipyridyl)cobalt(III) (Co(bpy) 3 3+ ) was incoporated into the multiwall carbon nanotubes-Nafion (MWNTs-Nafion) composite film via a simple ion-exchange route. Then, gold colloidal nanoparticles (nano-Au) were attached onto Co(bpy) 3 3+ /MWNTs-Nafion film through electrostatic interaction between negatively charged nano-Au and positively charged Co(bpy) 3 3+ . Finally, CA125 monoclonal antibody (anti-CA125), used as a model antibody, was assembled onto the surface of nano-Au to achieve an immunosensor for the determination of CA125 antigen. X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the assembly process of the modified electrode. The resulting immunosensor showed a high sensitivity, wide dynamic range consisting of two linear parts from 1.0 to 30 U mL -1 and 30 to 150 U mL -1 with a low detection limit of 0.36 U mL -1 at 3 times the background noise. Moreover, it displayed good reproducibility and stability, and would be potentially attractive for clinical immunoassay of CA125. The integration of mediator Co(bpy) 3 3+ and MWNTs-Nafion composite would offer potential promise for the fabrication of biosensors and biocatalysts.

  11. Panax ginseng has anti-infective activity against opportunistic pathogen Pseudomonas aeruginosa by inhibiting quorum sensing, a bacterial communication process critical for establishing infection

    DEFF Research Database (Denmark)

    Song, Z; Kong, K F; Wu, H

    2010-01-01

    Virulent factors produced by pathogens play an important role in the infectious process, which is regulated by a cell-to-cell communication mechanism called quorum sensing (QS). Pseudomonas aeruginosa is an important opportunistic human pathogen, which causes infections in patients with compromis...

  12. Quorum Sensing: A Transcriptional Regulatory System Involved in the Pathogenicity of Burkholderia mallei

    National Research Council Canada - National Science Library

    Ulrich, Ricky L; DeShazer, David; Hines, Harry B; Jeddeloh, Jeffrey A

    2004-01-01

    .... To determine if QS is involved in the virulence of B. mallei, we generated mutations in each putative luxIR homologue and tested the pathogenicities of the derivative strains in aerosol BALB/c mouse and intraperitoneal hamster models...

  13. Quorum Sensing: A transcriptional Regulatory System Involved in the Pathogenicity of Burkholderia mallei

    National Research Council Canada - National Science Library

    Ulrich, Ricky

    2004-01-01

    .... To determine if QS is involved in the virulence of B. mallei, we generated mutations in each putative luxIR homologue and tested the pathogenicities of the derivative strains in aerosol BALB/c mouse and intraperitoneal hamster models...

  14. Bead-based immunoassays

    NARCIS (Netherlands)

    Wal, van der F.J.; Bergervoet, J.H.W.; Achterberg, R.P.; Haasnoot, W.

    2014-01-01

    Since the first immunoassay with (radioactive) labeled antibodies in the middle of the 20th century [1], many different formats on various platforms have been developed, using antibodies for capture and/or detection. If antibodies are used to capture compounds, a support, such as the wall of a

  15. The immunoassay handbook

    National Research Council Canada - National Science Library

    Wild, David (David G.)

    2001-01-01

    ... importantly, enabling them to keep current on the basic theory behind immunoassay. Since the publication of the previous edition in 1994, the field has continued to evolve rapidly, and the need for a fully updated version of this book is now paramount. The second edition has been comprehensively updated and new chapters have been added to each section" [publisher's web site].

  16. Novel immunoassay formats for integrated microfluidic circuits: diffusion immunoassays (DIA)

    Science.gov (United States)

    Weigl, Bernhard H.; Hatch, Anson; Kamholz, Andrew E.; Yager, Paul

    2000-03-01

    Novel designs of integrated fluidic microchips allow separations, chemical reactions, and calibration-free analytical measurements to be performed directly in very small quantities of complex samples such as whole blood and contaminated environmental samples. This technology lends itself to applications such as clinical diagnostics, including tumor marker screening, and environmental sensing in remote locations. Lab-on-a-Chip based systems offer many *advantages over traditional analytical devices: They consume extremely low volumes of both samples and reagents. Each chip is inexpensive and small. The sampling-to-result time is extremely short. They perform all analytical functions, including sampling, sample pretreatment, separation, dilution, and mixing steps, chemical reactions, and detection in an integrated microfluidic circuit. Lab-on-a-Chip systems enable the design of small, portable, rugged, low-cost, easy to use, yet extremely versatile and capable diagnostic instruments. In addition, fluids flowing in microchannels exhibit unique characteristics ('microfluidics'), which allow the design of analytical devices and assay formats that would not function on a macroscale. Existing Lab-on-a-chip technologies work very well for highly predictable and homogeneous samples common in genetic testing and drug discovery processes. One of the biggest challenges for current Labs-on-a-chip, however, is to perform analysis in the presence of the complexity and heterogeneity of actual samples such as whole blood or contaminated environmental samples. Micronics has developed a variety of Lab-on-a-Chip assays that can overcome those shortcomings. We will now present various types of novel Lab- on-a-Chip-based immunoassays, including the so-called Diffusion Immunoassays (DIA) that are based on the competitive laminar diffusion of analyte molecules and tracer molecules into a region of the chip containing antibodies that target the analyte molecules. Advantages of this

  17. A Nod to disease vectors: mitigation of pathogen sensing by arthropod saliva

    Czech Academy of Sciences Publication Activity Database

    Sakhon, O. S.; Severo, M. S.; Kotsyfakis, Michalis; Pedra, J. H. F.

    2013-01-01

    Roč. 4, OCT 2013 (2013), a308 ISSN 1664-302X Institutional support: RVO:60077344 Keywords : nod-like receptors * inflammasome * vector-borne pathogens * vector-borne diseases * arthropod saliva * salivary proteins Subject RIV: EC - Immunology Impact factor: 3.941, year: 2013

  18. Chemiluminescence immunoassay for chloramphenicol

    International Nuclear Information System (INIS)

    Lin Si; Xu Wenge; Liu Yibing

    2007-06-01

    A simple, solid-phase chemiluminescence immunoassay (CLIA) for the measurement of Chloramphenicol(CAP) in foodstuffs is described. A rabbit anti-CAP IgG is passively adsorbed onto the walls of polypropylene plates. The labeled conjugant is horseradish peroxidase(HRP) conjugate of CAP. Luminol solution is used as the substrate of HRP. The light yield is inversely proportional to the concentration of CAP. The method has a similar sensitivity (0.05 ng/mL), specificity, precision, and accuracy to a conventional enzyme immunoassay (EIA). The intra-assay and inter-assay CVs of ten samples were <8 and <20%, respectively, and the analytical recovery of the method was 87% 100%. The experimental correlation coefficient of dilution was found to be 0.999 using milk supernatant as buffer. The assay range for the method was 0.1-10 ng/mL, and it displayed good linearity. (authors)

  19. The role of quorum sensing in the pathogenicity of the cunning aggressor Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Givskov, Michael Christian

    2007-01-01

    that bacteria preferentially live in communities in the form of primitive organisms in which the behavior of individual cells is coordinated by cell-cell communication, known as quorum sensing (QS). Bacteria use QS for regulation of the processes involved in their interaction with each other, their environment...... in the protective mechanisms of P. aeruginosa and show how disruption of the QS can be used as an approach to control this cunning aggressor....

  20. Specificity of immunoassays. Pt. 2

    International Nuclear Information System (INIS)

    Pratt, J.J.; Woldring, M.G.; Boonman, R.; Kittikool, J.

    1979-01-01

    Practical aspects of the measurement of the specificity of immunoassay are reviewed. Antibody heterogeneity in an antiserum makes a pragmatic rather than a theoretical approach necessary. A new method for the measurement of immunoassay specificity is described. This method is based on the errors caused by the cross-reacting antigens and is directly relevant to the validity of results obtained by immunoassay methods. The effect of selectively blocking the least specific antibodies in antisera raised against steroid haptens is tested. The practical consequences of these considerations are tested using steroid radioimmunoassay and enzyme-immunoassay. (orig.) [de

  1. MCL and mincle: C-type lectin receptors that sense damaged self and pathogen associated molecular patterns

    Directory of Open Access Journals (Sweden)

    Mark B Richardson

    2014-06-01

    Full Text Available MCL (macrophage C-type lectin and mincle (macrophage inducible C-type lectin comprise part of an extensive repertoire of pattern recognition receptors with the ability to sense damage associated and pathogen associated molecular patterns. In this review we cover the discovery and molecular characterization of these C-type lectin receptors, and highlight recent advances in the understanding of their roles in orchestrating the response of the immune system to bacterial and fungal infection, and damaged self. We also discuss the identification and structure-activity relationships of activating ligands, particularly trehalose dimycolate (TDM and related mycobacterial glycolipids, which have significant potential in the development of TH1/TH17 vaccination strategies.

  2. Characterization of additional components of the environmental pH-sensing complex in the pathogenic fungus Cryptococcus neoformans.

    Science.gov (United States)

    Pianalto, Kaila M; Ost, Kyla S; Brown, Hannah E; Alspaugh, J Andrew

    2018-05-16

    Pathogenic microorganisms must adapt to changes in their immediate surroundings, including alterations in pH, to survive the shift from the external environment to that of the infected host. In the basidiomycete fungal pathogen Cryptococcus neoformans , these pH changes are primarily sensed by the fungal-specific, alkaline pH-sensing Rim/Pal pathway. The C. neoformans Rim pathway has diverged significantly from that described in ascomycete fungi. We recently identified the C. neoformans putative pH sensor Rra1, which activates the Rim pathway in response to elevated pH. In this study, we probed the function of Rra1 by analyzing its cellular localization and performing protein co-immunoprecipitation to identify potential Rra1 interactors. We found that Rra1 does not strongly colocalize or interact with immediate downstream Rim pathway components. However, these experiments identified a novel Rra1 interactor, the previously uncharacterized C. neoformans nucleosome assembly protein 1 (Nap1), which was required for Rim pathway activation. We observed that Nap1 specifically binds to the C-terminal tail of the Rra1 sensor, likely promoting Rra1 protein stability. This function of Nap1 is conserved in fungi closely related to C. neoformans that contain Rra1 orthologs, but not in the more distantly-related ascomycete fungus Saccharomyces cerevisiae In conclusion, our findings have revealed the sophisticated, yet distinct, molecular mechanisms by which closely and distantly related microbial phyla rapidly adapt to environmental signals and changes such as alterations in pH. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Luminescence, virulence and quorum sensing signal production by pathogenic Vibrio campbellii and Vibrio harveyi isolates

    OpenAIRE

    Defoirdt, T.; Verstraete, W.; Bossier, P.

    2008-01-01

    Aims: To study the relationship between luminescence, autoinducer production and virulence of pathogenic vibrios.Methods and Results: Luminescence, quorum sensing signal production and virulence towards brine shrimp nauplii of 13 Vibrio campbellii and Vibrio harveyi strains were studied. Although only two of the tested strains were brightly luminescent, all of them were shown to produce the three different types of quorum sensing signals known to be produced by Vibrio harveyi. Cell-free cultu...

  4. Modulation of Quorum Sensing in a Gram-Positive Pathogen by Linear Molecularly Imprinted Polymers with Anti-infective Properties.

    Science.gov (United States)

    Motib, Anfal; Guerreiro, Antonio; Al-Bayati, Firas; Piletska, Elena; Manzoor, Irfan; Shafeeq, Sulman; Kadam, Anagha; Kuipers, Oscar; Hiller, Luisa; Cowen, Todd; Piletsky, Sergey; Andrew, Peter W; Yesilkaya, Hasan

    2017-12-22

    We describe the development, characterization, and biological testing of a new type of linear molecularly imprinted polymer (LMIP) designed to act as an anti-infective by blocking the quorum sensing (QS) mechanism and so abrogating the virulence of the pathogen Streptococcus pneumoniae. The LMIP is prepared (polymerized) in presence of a template molecule, but unlike in traditional molecular imprinting approaches, no cross-linker is used. This results in soluble low-molecular-weight oligomers that can act as a therapeutic agent in vitro and in vivo. The LMIP was characterized by mass spectrometry to determine its monomer composition. Fragments identified were then aligned along the peptide template by computer modeling to predict the possible monomer sequence of the LMIP. These findings provide a proof of principle that LMIPs can be used to block QS, thus setting the stage for the development of LMIPs a novel drug-discovery platform and class of materials to target Gram-positive pathogens. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Interference in immunoassay

    International Nuclear Information System (INIS)

    Chapman, R.S.

    1998-01-01

    Interfering factors are evident in both limited reagent (radioimmunoassay) and excess reagent (immunometric assay) technologies and should be suspected whenever there is a discrepancy between analytical results and clinical findings in the investigation of particular diseases. The overall effect of interference in immunoassay is analytical bias in result, either positive or negative of variable magnitude. The interference maybe caused by a wide spectrum of factors from poor sample collection and handling to physiological factors e.g. lipaemia, heparin treatment, binding protein abnormalities, autoimmunity and drug treatments. The range of interfering factors is extensive and difficult to discuss effectively in a short review

  6. Rhodococcus erythropolis and Its γ-Lactone Catabolic Pathway: An Unusual Biocontrol System That Disrupts Pathogen Quorum Sensing Communication

    Directory of Open Access Journals (Sweden)

    Xavier Latour

    2013-12-01

    Full Text Available Rhodococcus erythropolis is an environmental Gram-positive Actinobacterium with a versatile metabolism involved in various bioconversions and degradations. Rhodococci are best known for their great potential in numerous decontamination and industrial processes. However, they can also prevent plant disease by disrupting quorum sensing-based communication of Gram-negative soft-rot bacteria, by degrading N-acyl-homoserine lactone signaling molecules. Such biocontrol activity results partly from the action of the γ-lactone catabolic pathway. This pathway is responsible for cleaving the lactone bond of a wide range of compounds comprising a γ-butyrolactone ring coupled to an alkyl or acyl chain. The aliphatic products of this hydrolysis are then activated and enter fatty acid metabolism. This short pathway is controlled by the presence of the γ-lactone, presumably sensed by a TetR-like transcriptional regulator, rather than the presence of the pathogen or the plant-host in the environment of the Rhodococci. Both the density and biocontrol activity of R. erythropolis may be boosted in crop systems. Treatment with a cheap γ-lactone stimulator, for example, the food flavoring γ-caprolactone, induces the activity in the biocontrol agent, R. erythropolis, of the pathway degrading signaling molecules; such treatments thus promote plant protection.

  7. Diffusible signal factor-dependent quorum sensing in pathogenic bacteria and its exploitation for disease control.

    Science.gov (United States)

    Dow, J M

    2017-01-01

    Cell-to-cell signals of the diffusible signal factor (DSF) family are cis-2-unsaturated fatty acids of differing chain length and branching pattern. DSF signalling has been described in diverse bacteria to include plant and human pathogens where it acts to regulate functions such as biofilm formation, antibiotic tolerance and the production of virulence factors. DSF family signals can also participate in interspecies signalling with other bacteria and interkingdom signalling such as with the yeast Candida albicans. Interference with DSF signalling may afford new opportunities for the control of bacterial disease. Such strategies will depend in part on detailed knowledge of the molecular mechanisms underlying the processes of signal synthesis, perception and turnover. Here, I review both recent progress in understanding DSF signalling at the molecular level and prospects for translating this knowledge into approaches for disease control. © 2016 The Society for Applied Microbiology.

  8. Biosensors for plant pathogen detection.

    Science.gov (United States)

    Khater, Mohga; de la Escosura-Muñiz, Alfredo; Merkoçi, Arben

    2017-07-15

    Infectious plant diseases are caused by pathogenic microorganisms such as fungi, bacteria, viruses, viroids, phytoplasma and nematodes. Worldwide, plant pathogen infections are among main factors limiting crop productivity and increasing economic losses. Plant pathogen detection is important as first step to manage a plant disease in greenhouses, field conditions and at the country boarders. Current immunological techniques used to detect pathogens in plant include enzyme-linked immunosorbent assays (ELISA) and direct tissue blot immunoassays (DTBIA). DNA-based techniques such as polymerase chain reaction (PCR), real time PCR (RT-PCR) and dot blot hybridization have also been proposed for pathogen identification and detection. However these methodologies are time-consuming and require complex instruments, being not suitable for in-situ analysis. Consequently, there is strong interest for developing new biosensing systems for early detection of plant diseases with high sensitivity and specificity at the point-of-care. In this context, we revise here the recent advancement in the development of advantageous biosensing systems for plant pathogen detection based on both antibody and DNA receptors. The use of different nanomaterials such as nanochannels and metallic nanoparticles for the development of innovative and sensitive biosensing systems for the detection of pathogens (i.e. bacteria and viruses) at the point-of-care is also shown. Plastic and paper-based platforms have been used for this purpose, offering cheap and easy-to-use really integrated sensing systems for rapid on-site detection. Beside devices developed at research and development level a brief revision of commercially available kits is also included in this review. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Procedures for Sensitive Immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Givol, D. [Department of Chemical Immunology, Weizmann Institute of Science, Rehovot (Israel)

    1970-02-15

    Sensitive immunoassay methods should be applied to small molecules of biological importance, which are non-immunogenic by themselves, such as small peptide hormones (e.g. bradykinin), plant hormones (e.g. indoleacetic acid), nucleotides and other small molecules. Methods of binding these small molecules, as haptens, to immunogenic carriers by various cross-linking agents are described (dicyclohexylcarbodiimide, tolylene-diisocyanate and glutaraldehyde), and the considerations involved in relation to the methods of binding and the specificity of the antibodies formed are discussed. Some uses of antibody bound to bromoacetyl cellulose as an immuno adsorbent convenient for assay of immunoglobulins are described. Finally, the sensitive immunoassay method of chemically modified phage is described. This includes methods of binding small molecules (such as the dinitrophenyl group, penicillin, indoleacetic acid) or proteins (such as insulin, immunoglobulins) to phages. Methods of direct chemical conjugation, or an indirect binding via anti-phage Fab, are described. The phage inactivation method by direct plating and its modifications (such as decision technique and complex inactivation) are compared with the more simple end-point titration method. The inhibition of phage inactivation has some advantages as it does not require radioactive material, or expensive radioactive counters, and avoids the need for separation between bound and unbound antigen. Hence, if developed, it could be used as an alternative to radioimmunoassay. (author)

  10. AsrR is an oxidative stress sensing regulator modulating Enterococcus faecium opportunistic traits, antimicrobial resistance, and pathogenicity.

    Directory of Open Access Journals (Sweden)

    François Lebreton

    Full Text Available Oxidative stress serves as an important host/environmental signal that triggers a wide range of responses in microorganisms. Here, we identified an oxidative stress sensor and response regulator in the important multidrug-resistant nosocomial pathogen Enterococcus faecium belonging to the MarR family and called AsrR (antibiotic and stress response regulator. The AsrR regulator used cysteine oxidation to sense the hydrogen peroxide which results in its dissociation to promoter DNA. Transcriptome analysis showed that the AsrR regulon was composed of 181 genes, including representing functionally diverse groups involved in pathogenesis, antibiotic and antimicrobial peptide resistance, oxidative stress, and adaptive responses. Consistent with the upregulated expression of the pbp5 gene, encoding a low-affinity penicillin-binding protein, the asrR null mutant was found to be more resistant to β-lactam antibiotics. Deletion of asrR markedly decreased the bactericidal activity of ampicillin and vancomycin, which are both commonly used to treat infections due to enterococci, and also led to over-expression of two major adhesins, acm and ecbA, which resulted in enhanced in vitro adhesion to human intestinal cells. Additional pathogenic traits were also reinforced in the asrR null mutant including greater capacity than the parental strain to form biofilm in vitro and greater persistance in Galleria mellonella colonization and mouse systemic infection models. Despite overexpression of oxidative stress-response genes, deletion of asrR was associated with a decreased oxidative stress resistance in vitro, which correlated with a reduced resistance to phagocytic killing by murine macrophages. Interestingly, both strains showed similar amounts of intracellular reactive oxygen species. Finally, we observed a mutator phenotype and enhanced DNA transfer frequencies in the asrR deleted strain. These data indicate that AsrR plays a major role in antimicrobial

  11. Immunoassay separation technique

    International Nuclear Information System (INIS)

    1977-01-01

    A method for effecting the immunoassay of a multiplicity of samples, each possibly containing an antigen or an antibody to be assayed, is discussed. Each sample is incubated with a solution containing a detectable antigen or antibody to form a multiplicity of mixtures, each mixture containing as components antigen-antibody, non-complexed antigen and non-complexed antibody. At least one of the components of the said mixture is separated by adsorption. There after, quantity of detectable antigen or antibody is detected in one of the non-adsorbed portions of the mixture. An improvement, compared to other techniques, is the continuous and sequential separation of at least one component, which is intended to be separated from each said multiplicity of mixtures

  12. Monoclonal antibody-based immunoassays.

    Science.gov (United States)

    Appleby, P; Reischl, U

    1998-01-01

    An immunoassay may be defined as an assay that employs an immunological reagent, usually an antibody, to confer specificity for the ligand being measured. As a corollary to this, the discovery, and subsequent development, of monoclonal antibodies (MAbs) has greatly expanded the application and use of immunoassays. Polyclonal reagents, with their associated problems of specificity and quality control, have now been largely replaced by readily available MAbs of potential immortality and well-defined specificity and affinity. This has resulted, in the last two decades, in a great expansion in the range of immunoassays available and also a significant improvement in their reproducibility and reliability.

  13. Immunoassay for thymopoietin

    International Nuclear Information System (INIS)

    Goldstein, G.

    1979-01-01

    The patent describes the development of a radio-immunoassay for thymopoietin in biological samples. The method of raising antibodies to this polypeptide hormone is described. This is achieved by injecting a host animal with an antigen consisting of thymopoietin covalently bonded by glutaraldehyde to a carrier protein such as bovine serum albumin and equine globulin. Different methods of radiolabelling thymopoietin with 125 I for use as the tracer antigen are described. The Bolton-Hunter procedure was preferred to the chloramine-T method since direct iodination of the tyrosyl moieties of thymopoietin resulted in some loss of immunoreactivity. Systems for separating the antigen-antibody complex and unbound antigen are compared. Binding-inhibition curves for unlabelled thymopoietin in the assay employing polyethylene glycol separation showed a sensitivity of 5 ng thymopoietin/ml. However, using the double antibody or dextran coated charcoal separation techniques, the sensitivity of thymopoietin was 0.1 ng/ml. Thus these latter two procedures are thus especially suitable for measuring thymopoietin levels in serum or plasma samples. The assay was shown to be specific for thymopoietin, no significant displacement being produced by control polypeptides. (U.K.)

  14. Cross-species induction of antimicrobial compounds, biosurfactants and quorum-sensing inhibitors in tropical marine epibiotic bacteria by pathogens and biofouling microorganisms.

    Science.gov (United States)

    Dusane, Devendra H; Matkar, Pratiek; Venugopalan, Valayam P; Kumar, Ameeta Ravi; Zinjarde, Smita S

    2011-03-01

    Enhancement or induction of antimicrobial, biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions was investigated. Four marine epibiotic bacteria (Bacillus sp. S3, B. pumilus S8, B. licheniformis D1, and Serratia marcescens V1) displaying antimicrobial activity against pathogenic or biofouling fungi (Candida albicans CA and Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA and Bacillus pumilus BP) were chosen for this study. The marine epibiotic bacteria when co-cultivated with the aforementioned fungi or bacteria showed induction or enhancement in antimicrobial activity, biosurfactant production, and quorum-sensing inhibition. Antifungal activity against Y. lipolytica YL was induced by co-cultivation of the pathogens or biofouling strains with the marine Bacillus sp. S3, B. pumilus S8, or B. licheniformis D1. Antibacterial activity against Ps. aeruginosa PA or B. pumilus BP was enhanced in most of the marine isolates after co-cultivation. Biosurfactant activity was significantly increased when cells of B. pumilus BP were co-cultivated with S. marcescens V1, B. pumilus S8, or B. licheniformis D1. Pigment reduction in the quorum-sensing inhibition indicator strain Chromobacterium violaceum 12472 was evident when the marine strain of Bacillus sp. S3 was grown in the presence of the inducer strain Ps. aeruginosa PA, suggesting quorum-sensing inhibition. The study has important ecological and biotechnological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite production.

  15. Rapid micromotor-based naked-eye immunoassay.

    Science.gov (United States)

    de Ávila, Berta Esteban-Fernández; Zhao, Mingjiao; Campuzano, Susana; Ricci, Francesco; Pingarrón, José M; Mascini, Marcello; Wang, Joseph

    2017-05-15

    A dynamic micromotor-based immunoassay, exemplified by cortisol detection, based on the use of tubular micromotors functionalized with a specific antibody is described. The use of antibody-functionalized micromotors offers huge acceleration of both direct and competitive cortisol immunoassays, along with greatly enhanced sensitivity of direct and competitive immunoassays. The dramatically improved speed and sensitivity reflect the greatly increased likelihood of antibody-cortisol contacts and fluid mixing associated with the dynamic movement of these microtube motors and corresponding bubble generation that lead to a highly efficient and rapid recognition process. Rapid naked-eye detection of cortisol in the sample is achieved in connection to use of horseradish peroxidase (HRP) tag and TMB/H 2 O 2 system. Key parameters of the competitive immunoassay (e.g., incubation time and reaction volume) were optimized. This fast visual micromotor-based sensing approach enables "on the move" specific detection of the target cortisol down to 0.1μgmL -1 in just 2min, using ultrasmall (50µL) sample volumes. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. AuNP-RF sensor: An innovative application of RF technology for sensing pathogens electrically in liquids (SPEL) within the food supply chain.

    Science.gov (United States)

    Matta, Leann Lerie; Karuppuswami, Saranraj; Chahal, Premjeet; Alocilja, Evangelyn C

    2018-07-15

    Rapid detection techniques of pathogenic bacteria in the liquid food supply chain are of significant research interest due to their pivotal role in preventing foodborne outbreaks, and in maintaining high standards of public health and safety. Milk and dairy products are of particular interest due to their widespread consumption across the globe. In this paper, a biosensor for detecting pathogenic bacteria in milk using dextrin-capped gold nanoparticles (d-AuNP) as labels decoded at microwave frequencies is presented. The SPEL (sensing pathogens electrically in liquids) biosensor consists of a 3D printed vial and uses an RF reader and an RFID (radio-frequency identification) compatible Split Ring Resonator (SRR) based tag. The SPEL biosensor is capable of detecting bacteria at 5 log CFU/mL within 75 min, with the possibility of testing multiple concurrent samples. Detection is based on impedance loading of SRR by d-AuNP bound to pathogenic bacteria. Spectrophotometry, along with carbohydrate-functionalized magnetic nanoparticle (MNP) cell capture, is used to verify the sensitivity of the SPEL biosensor with respect to d-AuNP presence. The proof-of-concept device, along with challenges and opportunities for commercialization, are also outlined. Copyright © 2018. Published by Elsevier B.V.

  17. Pathogen intelligence

    Directory of Open Access Journals (Sweden)

    Michael eSteinert

    2014-01-01

    Full Text Available Different species inhabit different sensory worlds and thus have evolved diverse means of processing information, learning and memory. In the escalated arms race with host defense, each pathogenic bacterium not only has evolved its individual cellular sensing and behaviour, but also collective sensing, interbacterial communication, distributed information processing, joint decision making, dissociative behaviour, and the phenotypic and genotypic heterogeneity necessary for epidemiologic success. Moreover, pathogenic populations take advantage of dormancy strategies and rapid evolutionary speed, which allow them to save co-generated intelligent traits in a collective genomic memory. This review discusses how these mechanisms add further levels of complexity to bacterial pathogenicity and transmission, and how mining for these mechanisms could help to develop new anti-infective strategies.

  18. Immunoassay for determination of trilobolide

    Czech Academy of Sciences Publication Activity Database

    Huml, L.; Jurášek, M.; Mikšátková, P.; Zimmermann, T.; Tomanová, P.; Buděšínský, Miloš; Rottnerová, Z.; Šimková, M.; Harmatha, Juraj; Kmoníčková, Eva; Lapčík, O.; Drašar, P. B.

    2017-01-01

    Roč. 117, Jan (2017), s. 105-111 ISSN 0039-128X. [Conference on Isoprenoids /23./. Minsk, 04.09.2016-07.09.2016] Institutional support: RVO:61388963 ; RVO:68378041 Keywords : trilobolide * avidin-biotin * ELISA * Laser trilobum * synthesis * immunoassay Subject RIV: CE - Biochemistry; FR - Pharmacology ; Medidal Chemistry (UEM-P) OBOR OECD: Biochemical research methods; Pharmacology and pharmacy (UEM-P) Impact factor: 2.282, year: 2016

  19. The Biochemistry of Sensing: Enteric Pathogens Regulate Type III Secretion in Response to Environmental and Host Cues.

    Science.gov (United States)

    De Nisco, Nicole J; Rivera-Cancel, Giomar; Orth, Kim

    2018-01-16

    Enteric pathogens employ sophisticated strategies to colonize and infect mammalian hosts. Gram-negative bacteria, such as Escherichia coli , Salmonella , and Campylobacter jejuni , are among the leading causes of gastrointestinal tract infections worldwide. The virulence strategies of many of these Gram-negative pathogens rely on type III secretion systems (T3SSs), which are macromolecular syringes that translocate bacterial effector proteins directly into the host cytosol. However, synthesis of T3SS proteins comes at a cost to the bacterium in terms of growth rate and fitness, both in the environment and within the host. Therefore, expression of the T3SS must be tightly regulated to occur at the appropriate time and place during infection. Enteric pathogens have thus evolved regulatory mechanisms to control expression of their T3SSs in response to specific environmental and host cues. These regulatory cascades integrate multiple physical and chemical signals through complex transcriptional networks. Although the power of bacterial genetics has allowed elucidation of many of these networks, the biochemical interactions between signal and sensor that initiate the signaling cascade are often poorly understood. Here, we review the physical and chemical signals that Gram-negative enteric pathogens use to regulate T3SS expression during infection. We highlight the recent structural and functional studies that have elucidated the biochemical properties governing both the interaction between sensor and signal and the mechanisms of signal transduction from sensor to downstream transcriptional networks. Copyright © 2018 De Nisco et al.

  20. The Biochemistry of Sensing: Enteric Pathogens Regulate Type III Secretion in Response to Environmental and Host Cues

    Directory of Open Access Journals (Sweden)

    Nicole J. De Nisco

    2018-01-01

    Full Text Available Enteric pathogens employ sophisticated strategies to colonize and infect mammalian hosts. Gram-negative bacteria, such as Escherichia coli, Salmonella, and Campylobacter jejuni, are among the leading causes of gastrointestinal tract infections worldwide. The virulence strategies of many of these Gram-negative pathogens rely on type III secretion systems (T3SSs, which are macromolecular syringes that translocate bacterial effector proteins directly into the host cytosol. However, synthesis of T3SS proteins comes at a cost to the bacterium in terms of growth rate and fitness, both in the environment and within the host. Therefore, expression of the T3SS must be tightly regulated to occur at the appropriate time and place during infection. Enteric pathogens have thus evolved regulatory mechanisms to control expression of their T3SSs in response to specific environmental and host cues. These regulatory cascades integrate multiple physical and chemical signals through complex transcriptional networks. Although the power of bacterial genetics has allowed elucidation of many of these networks, the biochemical interactions between signal and sensor that initiate the signaling cascade are often poorly understood. Here, we review the physical and chemical signals that Gram-negative enteric pathogens use to regulate T3SS expression during infection. We highlight the recent structural and functional studies that have elucidated the biochemical properties governing both the interaction between sensor and signal and the mechanisms of signal transduction from sensor to downstream transcriptional networks.

  1. Phytosynthesized silver nanoparticles as antiquorum sensing and antibiofilm agent against the nosocomial pathogen Serratia marcescens: an in vitro study.

    Science.gov (United States)

    Ravindran, D; Ramanathan, S; Arunachalam, K; Jeyaraj, G P; Shunmugiah, K P; Arumugam, V R

    2018-02-12

    Serratia marcescens is an important multidrug-resistant human pathogen. The pathogenicity of S. marcescens mainly depends on the quorum sensing (QS) mechanism, which regulates the virulence factors production and biofilm formation. Hence, targeting QS mechanism in S. marcescens will ultimately pave the way to combat its pathogenicity. Thus, the present study is intended to evaluate the efficacy of Vetiveria zizanioides root extract-mediated silver nanoparticles (AgNPs) as a potent anti-QS and antibiofilm agent against S. marcescens. The AgNPs were synthesized using V. zizanioides aqueous root extract and the physiochemical properties of V. zizanioides-based AgNPs (VzAgNPs) were evaluated using analytical techniques such as ultraviolet-visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, dynamic light scattering and scanning and transmission electron microscopic techniques. VzAgNPs were found to attenuate the QS-dependent virulence factors, namely prodigiosin, protease, lipase, exopolysaccharide productions and biofilm formation of S. marcescens, without inhibiting its growth. Further, the transcriptomic analysis confirmed the down-regulation of QS-dependent genes, which encode for the production of virulence factors and biofilm formation. The current study confirms VzAgNPs as an ideal anti-QS and antibiofilm agent against S. marcescens. This is the first approach that validates the anti-QS and antibiofilm potential of phytosynthesized VzAgNPs against the nosocomial pathogen, S. marcescens. As VzAgNPs exhibits potent antivirulent activities, it could be used to treat hospital-acquired S. marcescens infections. © 2018 The Society for Applied Microbiology.

  2. [Automated analyzer of enzyme immunoassay].

    Science.gov (United States)

    Osawa, S

    1995-09-01

    Automated analyzers for enzyme immunoassay can be classified by several points of view: the kind of labeled antibodies or enzymes, detection methods, the number of tests per unit time, analytical time and speed per run. In practice, it is important for us consider the several points such as detection limits, the number of tests per unit time, analytical range, and precision. Most of the automated analyzers on the market can randomly access and measure samples. I will describe the recent advance of automated analyzers reviewing their labeling antibodies and enzymes, the detection methods, the number of test per unit time and analytical time and speed per test.

  3. Abbott prism: a multichannel heterogeneous chemiluminescence immunoassay analyzer.

    Science.gov (United States)

    Khalil, O S; Zurek, T F; Tryba, J; Hanna, C F; Hollar, R; Pepe, C; Genger, K; Brentz, C; Murphy, B; Abunimeh, N

    1991-09-01

    We describe a multichannel heterogeneous immunoassay analyzer in which a sample is split between disposable reaction trays in a group of linear tracks. The system's pipettor uses noninvasive sensing of the sample volume and disposable pipet tips. Each assay track has (a) a conveyor belt for moving reaction trays to predetermined functional stations, (b) temperature-controlled tunnels, (c) noncontact transfer of the reaction mixture between incubation and detection wells, and (d) single-photon counting to detect a chemiluminescence (CL) signal from the captured immunochemical product. A novel disposable reaction tray, with separate reaction and detection wells and self-contained fluid removal, is used in conjunction with the transfer device on the track to produce a carryover-free system. The linear immunoassay track has nine predetermined positions for performing individual assay steps. Assay step sequence and timing is selected by changing the location of the assay modules between these predetermined positions. The assay methodology, a combination of microparticle capture and direct detection of a CL signal on a porous matrix, offers excellent sensitivity, specificity, and ease of automation. Immunoassay configurations have been tested for hepatitis B surface antigen and for antibodies to hepatitis B core antigen, hepatitis C virus, human immunodeficiency virus I and II, and human T-cell leukemia virus I and II.

  4. Survey of immunoassay techniques for biological analysis

    International Nuclear Information System (INIS)

    Burtis, C.A.

    1986-10-01

    Immunoassay is a very specific, sensitive, and widely applicable analytical technique. Recent advances in genetic engineering have led to the development of monoclonal antibodies which further improves the specificity of immunoassays. Originally, radioisotopes were used to label the antigens and antibodies used in immunoassays. However, in the last decade, numerous types of immunoassays have been developed which utilize enzymes and fluorescent dyes as labels. Given the technical, safety, health, and disposal problems associated with using radioisotopes, immunoassays that utilize the enzyme and fluorescent labels are rapidly replacing those using radioisotope labels. These newer techniques are as sensitive, are easily automated, have stable reagents, and do not have a disposal problem. 6 refs., 1 fig., 2 tabs

  5. Fast and sensitive detection of enteropathogenic Yersinia by immunoassays.

    Science.gov (United States)

    Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe; Simon, Stéphanie

    2015-01-01

    Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 10(3) CFU/ml to 8.8 × 10(4) CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 10(5) CFU/ml to 10(6) CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Immunoassay of β-endorphin

    International Nuclear Information System (INIS)

    Hoellt, V.; Gramsch, C.; Herz, A.

    1979-01-01

    The present paper describes the characteristics of a series of antisera against β-endorphin (β-E) developed in our laboratory which all recognize β-lipotropin and their use in (1) the determination of β-E in tissue and in body fluids and in (2) the immunocytochemical localization of β-E containing neurons in the rat brain and in (3) the study of the conversion of the β-E/β-LPH precursor into β-LPH and β-E in the pars intermedia/nervosa of the rat pituitary. It is the purpose of the present report to critically analyze the pitfalls and drawbacks, as well as the advantage, of the use of radioimmunoassay and other immunoassays in the determination of β-E. (Auth.)

  7. Materials for Microfluidic Immunoassays: A Review.

    Science.gov (United States)

    Mou, Lei; Jiang, Xingyu

    2017-08-01

    Conventional immunoassays suffer from at least one of these following limitations: long processing time, high costs, poor user-friendliness, technical complexity, poor sensitivity and specificity. Microfluidics, a technology characterized by the engineered manipulation of fluids in channels with characteristic lengthscale of tens of micrometers, has shown considerable promise for improving immunoassays that could overcome these limitations in medical diagnostics and biology research. The combination of microfluidics and immunoassay can detect biomarkers with faster assay time, reduced volumes of reagents, lower power requirements, and higher levels of integration and automation compared to traditional approaches. This review focuses on the materials-related aspects of the recent advances in microfluidics-based immunoassays for point-of-care (POC) diagnostics of biomarkers. We compare the materials for microfluidic chips fabrication in five aspects: fabrication, integration, function, modification and cost, and describe their advantages and drawbacks. In addition, we review materials for modifying antibodies to improve the performance of the reaction of immunoassay. We also review the state of the art in microfluidic immunoassays POC platforms, from the laboratory to routine clinical practice, and also commercial products in the market. Finally, we discuss the current challenges and future developments in microfluidic immunoassays. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora: the role of expR(Ecc).

    Science.gov (United States)

    Andersson, R A; Eriksson, A R; Heikinheimo, R; Mäe, A; Pirhonen, M; Kõiv, V; Hyytiäinen, H; Tuikkala, A; Palva, E T

    2000-04-01

    The production of the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the extracellular cell wall-degrading enzymes, is partly controlled by the diffusible signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). OHHL is synthesized by the product of the expI/carI gene. Linked to expI we found a gene encoding a putative transcriptional regulator of the LuxR-family. This gene, expR(Ecc), is transcribed convergently to the expI gene and the two open reading frames are partially overlapping. The ExpR(Ecc) protein showed extensive amino acid sequence similarity to the repressor EsaR from Pantoea stewartii subsp. stewartii (formerly Erwinia stewartii subsp. stewartii) and to the ExpR(Ech) protein of Erwinia chrysanthemi. Inactivation of the E. carotovora subsp. carotovora expR(Ecc) gene caused no decrease in virulence or production of virulence determinants in vitro. In contrast, there was a slight increase in the maceration capacity of the mutant strain. The effects of ExpR(Ecc) were probably mediated by changes in OHHL levels. Inactivation of expR(Ecc) resulted in increased OHHL levels during early logarithmic growth. In addition, overexpression of expR(Ecc) caused a clear decrease in the production of virulence determinants and part of this effect was likely to be caused by OHHL binding to ExpR(Ecc). ExpR(Ecc) did not appear to exhibit transcriptional regulation of expI, but the effect on OHHL was apparently due to other mechanisms.

  9. Variance function estimation for immunoassays

    International Nuclear Information System (INIS)

    Raab, G.M.; Thompson, R.; McKenzie, I.

    1980-01-01

    A computer program is described which implements a recently described, modified likelihood method of determining an appropriate weighting function to use when fitting immunoassay dose-response curves. The relationship between the variance of the response and its mean value is assumed to have an exponential form, and the best fit to this model is determined from the within-set variability of many small sets of repeated measurements. The program estimates the parameter of the exponential function with its estimated standard error, and tests the fit of the experimental data to the proposed model. Output options include a list of the actual and fitted standard deviation of the set of responses, a plot of actual and fitted standard deviation against the mean response, and an ordered list of the 10 sets of data with the largest ratios of actual to fitted standard deviation. The program has been designed for a laboratory user without computing or statistical expertise. The test-of-fit has proved valuable for identifying outlying responses, which may be excluded from further analysis by being set to negative values in the input file. (Auth.)

  10. Trends in interfacial design for surface plasmon resonance based immunoassays

    International Nuclear Information System (INIS)

    Shankaran, Dhesingh Ravi; Miura, Norio

    2007-01-01

    Immunosensors based on surface plasmon resonance (SPR) have become a promising tool in sensor technology for biomedical, food, environmental, industrial and homeland security applications. SPR is a surface sensitive optical technique, suitable for real-time and label-free analysis of biorecognition events at functional transducer surfaces. Fabrication of highly active and robust sensing surfaces is an important part in immunoassays because the quality, quantity, chemistry and topography of the interfacial biomembranes play a major role in immunosensor performance. Eventually, a variety of immobilization methods such as physical adsorption, covalent coupling, Langmuir-Blodgett film, polymer thin film, self-assembly, sol-gel, etc, have been introduced over the years for the immobilization of biomolecules (antibody or antigen) on the transducer surfaces. The selection of an immobilization method for an immunoassay is governed by several factors such as nature and stability of the biomolecules, target analyte, application, detection principle, mode of signal transduction, matrix complexity, etc. This paper provides an overview of the various surface modification methods for SPR based immunosensor fabrication. The preparation, structure and application of different functional interfacial surfaces have been discussed along with a brief introduction to the SPR technology, biomolecules and detection principles. (review article)

  11. Trends in interfacial design for surface plasmon resonance based immunoassays

    Energy Technology Data Exchange (ETDEWEB)

    Shankaran, Dhesingh Ravi [Art, Science and Technology Center for Cooperative Research, Kyushu University, Kasuga-shi, Fukuoka, 816-8580 (Japan); Miura, Norio [Art, Science and Technology Center for Cooperative Research, Kyushu University, Kasuga-shi, Fukuoka, 816-8580 (Japan)

    2007-12-07

    Immunosensors based on surface plasmon resonance (SPR) have become a promising tool in sensor technology for biomedical, food, environmental, industrial and homeland security applications. SPR is a surface sensitive optical technique, suitable for real-time and label-free analysis of biorecognition events at functional transducer surfaces. Fabrication of highly active and robust sensing surfaces is an important part in immunoassays because the quality, quantity, chemistry and topography of the interfacial biomembranes play a major role in immunosensor performance. Eventually, a variety of immobilization methods such as physical adsorption, covalent coupling, Langmuir-Blodgett film, polymer thin film, self-assembly, sol-gel, etc, have been introduced over the years for the immobilization of biomolecules (antibody or antigen) on the transducer surfaces. The selection of an immobilization method for an immunoassay is governed by several factors such as nature and stability of the biomolecules, target analyte, application, detection principle, mode of signal transduction, matrix complexity, etc. This paper provides an overview of the various surface modification methods for SPR based immunosensor fabrication. The preparation, structure and application of different functional interfacial surfaces have been discussed along with a brief introduction to the SPR technology, biomolecules and detection principles. (review article)

  12. Recent advancements in the immunoassay domain

    International Nuclear Information System (INIS)

    Pradelles, Ph.

    1997-01-01

    The two types of immunoassay techniques, the competition analysis and the immuno-metric analysis (sandwich type), are described; the tracers used with theses methods have high specific radioactivity levels in order to be traced at extremely low content. Non radioactive tracers have been also developed, such as enzymatic, fluorescent, luminescent tracers, which are simpler and may be used at home. The Cea has recently developed some innovative immunoassay formats, such as acetylcholinesterase as a new enzymatic tracer, and immuno-metric dosage for very small molecules such as haptenes

  13. The hydrocarbon-degrading marine bacterium Cobetia sp. strain MM1IDA2H-1 produces a biosurfactant that interferes with quorum sensing of fish pathogens by signal hijacking

    Science.gov (United States)

    Ibacache-Quiroga, C; Ojeda, J; Espinoza-Vergara, G; Olivero, P; Cuellar, M; Dinamarca, M A

    2013-01-01

    Summary Biosurfactants are produced by hydrocarbon-degrading marine bacteria in response to the presence of water-insoluble hydrocarbons. This is believed to facilitate the uptake of hydrocarbons by bacteria. However, these diffusible amphiphilic surface-active molecules are involved in several other biological functions such as microbial competition and intra-or inter-species communication. We report the isolation and characterization of a marine bacterial strain identified as Cobetia sp. MM1IDA2H-1, which can grow using the sulfur-containing heterocyclic aromatic hydrocarbon dibenzothiophene (DBT). As with DBT, when the isolated strain is grown in the presence of a microbial competitor, it produces a biosurfactant. Because the obtained biosurfactant was formed by hydroxy fatty acids and extracellular lipidic structures were observed during bacterial growth, we investigated whether the biosurfactant at its critical micelle concentration can interfere with bacterial communication systems such as quorum sensing. We focused on Aeromonas salmonicida subsp. salmonicida, a fish pathogen whose virulence relies on quorum sensing signals. Using biosensors for quorum sensing based on Chromobacterium violaceum and Vibrio anguillarum, we showed that when the purified biosurfactant was mixed with N-acyl homoserine lactones produced by A. salmonicida, quorum sensing was inhibited, although bacterial growth was not affected. In addition, the transcriptional activities of A. salmonicida virulence genes that are controlled by quorum sensing were repressed by both the purified biosurfactant and the growth in the presence of Cobetia sp. MM1IDA2H-1. We propose that the biosurfactant, or the lipid structures interact with the N-acyl homoserine lactones, inhibiting their function. This could be used as a strategy to interfere with the quorum sensing systems of bacterial fish pathogens, which represents an attractive alternative to classical antimicrobial therapies in fish

  14. The right environment for the immunoassay

    International Nuclear Information System (INIS)

    Emon, J.M. Van; Gerlach, C.L.

    1995-01-01

    For the US Environmental Protection Agency (EPA), the first in-house research effort began in 1987, when results of an early immunoassay field study verified the technology's potential for environmental applications. Looking at the fundamental features of immunochemical reactions from the clinical laboratories, analytical chemists realized the potential value of these methods for hazardous waste site characterization and pesticide monitoring. Immunoassays rely on the interaction between an antibody and a target analyte. For environmental purposes, enzyme immunoassays are generally used. After the target analyte binds to the antibody, an enzymatic reaction yields a colorimetric change. This change, read visually or by a spectrophotometer, indicates the concentration of the target analyte. Promising results with assays for compounds (such as paraquat and pentachlorophenol) and compound groups (such as total petroleum hydrocarbons and polychlorinated biphenyls) spurred interest among various entrepreneurs. The first target market for immunoassays was environmental engineers and field crews who needed quick answers on-site to determine the direction of further remediation efforts

  15. A Switchable Linker-Based Immunoassay for Ultrasensitive Visible Detection of Salmonella in Tomatoes.

    Science.gov (United States)

    Hahn, Jungwoo; Kim, Eunghee; You, Young Sang; Gunasekaran, Sundaram; Lim, Seokwon; Choi, Young Jin

    2017-10-01

    On-site detection for sensitive identification of foodborne pathogens on fresh produce with minimal use of specialized instrumentation is crucial to the food industry. A switchable linker (SL)-based immunoassay was designed for ultrasensitive on-site detection of Salmonella in tomato samples. The assay is based on large-scale aggregation of gold nanoparticles (GNPs), induced by a quantitative relationship among the biotinylated Salmonella polyclonal antibody (b-Ab) used as the SL, the functionalized GNPs, and Salmonella. Important factors such as the concentration of SLs, time required for large-scale aggregation, and selectivity of b-Ab were optimized to minimize the detection time (within 45 min with gentle agitation) and achieve the lowest limit of detection (LOD; 10 CFU/g in tomato samples) possible. This SL-based immunoassay with its relatively low LOD and short detection time may meet the need for rapid, simple, on-site analysis of pathogens in fresh produce. The novel switchable linker-based immunoassay is a rapid, specific, and sensitive method that has potential applications for routine diagnostics of Salmonella in tomato products. These advantages make it a practical approach for general use in the processing industry to detect Salmonella rapidly and to implement appropriate regulatory procedures. Furthermore, it could be applied to other fresh products including cantaloupe, strawberry, and cucumbers. © 2017 Institute of Food Technologists®.

  16. Dual-Mode SERS-Fluorescence Immunoassay Using Graphene Quantum Dot Labeling on One-Dimensional Aligned Magnetoplasmonic Nanoparticles.

    Science.gov (United States)

    Zou, Fengming; Zhou, Hongjian; Tan, Tran Van; Kim, Jeonghyo; Koh, Kwangnak; Lee, Jaebeom

    2015-06-10

    A novel dual-mode immunoassay based on surface-enhanced Raman scattering (SERS) and fluorescence was designed using graphene quantum dot (GQD) labels to detect a tuberculosis (TB) antigen, CFP-10, via a newly developed sensing platform of linearly aligned magnetoplasmonic (MagPlas) nanoparticles (NPs). The GQDs were excellent bilabeling materials for simultaneous Raman scattering and photoluminescence (PL). The one-dimensional (1D) alignment of MagPlas NPs simplified the immunoassay process and enabled fast, enhanced signal transduction. With a sandwich-type immunoassay using dual-mode nanoprobes, both SERS signals and fluorescence images were recognized in a highly sensitive and selective manner with a detection limit of 0.0511 pg mL(-1).

  17. Surface-Enhanced Raman Scattering (SERS) for Detection in Immunoassays: applications, fundamentals, and optimization

    International Nuclear Information System (INIS)

    Jeremy Daniel Driskell

    2006-01-01

    Immunoassays have been utilized for the detection of biological analytes for several decades. Many formats and detection strategies have been explored, each having unique advantages and disadvantages. More recently, surface-enhanced Raman scattering (SERS) has been introduced as a readout method for immunoassays, and has shown great potential to meet many key analytical figures of merit. This technology is in its infancy and this dissertation explores the diversity of this method as well as the mechanism responsible for surface enhancement. Approaches to reduce assay times are also investigated. Implementing the knowledge gained from these studies will lead to a more sensitive immunoassay requiring less time than its predecessors. This dissertation is organized into six sections. The first section includes a literature review of the previous work that led to this dissertation. A general overview of the different approaches to immunoassays is given, outlining the strengths and weaknesses of each. Included is a detailed review of binding kinetics, which is central for decreasing assay times. Next, the theoretical underpinnings of SERS is reviewed at its current level of understanding. Past work has argued that surface plasmon resonance (SPR) of the enhancing substrate influences the SERS signal; therefore, the SPR of the extrinsic Raman labels (ERLs) utilized in our SERS-based immunoassay is discussed. Four original research chapters follow the Introduction, each presented as separate manuscripts. Chapter 2 modifies a SERS-based immunoassay previously developed in our group, extending it to the low-level detection of viral pathogens and demonstrating its versatility in terms of analyte type, Chapter 3 investigates the influence of ERL size, material composition, and separation distance between the ERLs and capture substrate on the SERS signal. This chapter links SPR with SERS enhancement factors and is consistent with many of the results from theoretical treatments

  18. Surface-Enhanced Raman Scattering (SERS) for Detection in Immunoassays. Applications, fundamentals, and optimization

    Energy Technology Data Exchange (ETDEWEB)

    Driskell, Jeremy Daniel [Iowa State Univ., Ames, IA (United States)

    2006-08-09

    Immunoassays have been utilized for the detection of biological analytes for several decades. Many formats and detection strategies have been explored, each having unique advantages and disadvantages. More recently, surface-enhanced Raman scattering (SERS) has been introduced as a readout method for immunoassays, and has shown great potential to meet many key analytical figures of merit. This technology is in its infancy and this dissertation explores the diversity of this method as well as the mechanism responsible for surface enhancement. Approaches to reduce assay times are also investigated. Implementing the knowledge gained from these studies will lead to a more sensitive immunoassay requiring less time than its predecessors. This dissertation is organized into six sections. The first section includes a literature review of the previous work that led to this dissertation. A general overview of the different approaches to immunoassays is given, outlining the strengths and weaknesses of each. Included is a detailed review of binding kinetics, which is central for decreasing assay times. Next, the theoretical underpinnings of SERS is reviewed at its current level of understanding. Past work has argued that surface plasmon resonance (SPR) of the enhancing substrate influences the SERS signal; therefore, the SPR of the extrinsic Raman labels (ERLs) utilized in our SERS-based immunoassay is discussed. Four original research chapters follow the Introduction, each presented as separate manuscripts. Chapter 2 modifies a SERS-based immunoassay previously developed in our group, extending it to the low-level detection of viral pathogens and demonstrating its versatility in terms of analyte type, Chapter 3 investigates the influence of ERL size, material composition, and separation distance between the ERLs and capture substrate on the SERS signal. This chapter links SPR with SERS enhancement factors and is consistent with many of the results from theoretical treatments

  19. Microfluidic Platform for Enzyme-Linked and Magnetic Particle-Based Immunoassay

    Directory of Open Access Journals (Sweden)

    Dorota G. Pijanowska

    2013-06-01

    Full Text Available This article presents design and testing of a microfluidic platform for immunoassay. The method is based on sandwiched ELISA, whereby the primary antibody is immobilized on nitrocelluose and, subsequently, magnetic beads are used as a label to detect the analyte. The chip takes approximately 2 h and 15 min to complete the assay. A Hall Effect sensor using 0.35-μm BioMEMS TSMC technology (Taiwan Semiconductor Manufacturing Company Bio-Micro-Electro-Mechanical Systems was fabricated to sense the magnetic field from the beads. Furthermore, florescence detection and absorbance measurements from the chip demonstrate successful immunoassay on the chip. In addition, investigation also covers the Hall Effect simulations, mechanical modeling of the bead–protein complex, testing of the microfluidic platform with magnetic beads averaging 10 nm, and measurements with an inductor-based system.

  20. Development of national immunoassay reagent programmes

    International Nuclear Information System (INIS)

    Sufi, S.B.; Micallef, J.V.; Ahsan, R.; Goncharov, N.P.

    1992-01-01

    Despite the existence of networks of fully equipped laboratories with well-trained staff, the availability of immunodiagnostic services in developing countries is often limited by the high cost of imported kits. There are a number of ways of tackling this problem, ranging from bulk purchase of kits or reagents to local development and production of assay systems. Argentina/Chile, China, Cuba/Mexico, and Thailand are amongst the countries which have established local immunoassay reagent programmes to manufacture low cost, high quality immunoassay reagents. Kits from these projects are now beginning to become available, and it is hoped that they will promote national diagnostic services and research, as well as stimulating the development of reagent programmes for other analytes. (author). 4 refs, 1 tab

  1. Studies on direct and indirect electrochemical immunoassays

    OpenAIRE

    Buckley, Eileen

    1989-01-01

    Two approaches to electrochemical immunoassay are reported. The first approach was an indirect method, involving an electroactive, enzyme-catalysed, substrate to product reaction. Conditions were optimised for the amperometric detection of para-aminophenol, the electroactive product of the alkaline phosphatase catalysed hydrolysis of a new substrate, p-aminophenylphosphate, after separation by HPLC. The second approach involved the direct electrochemical detection of an immunoglo...

  2. Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays.

    Science.gov (United States)

    Chen, Hui; Hagström, Anna E V; Kim, Jinsu; Garvey, Gavin; Paterson, Andrew; Ruiz-Ruiz, Federico; Raja, Balakrishnan; Strych, Ulrich; Rito-Palomares, Marco; Kourentzi, Katerina; Conrad, Jacinta C; Atmar, Robert L; Willson, Richard C

    2016-04-14

    In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 10(6) virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings.

  3. Massively multi-parametric immunoassays using ICPMS

    International Nuclear Information System (INIS)

    Tanner, S.D.; Ornatsky, O.; Bandura, D.R.; Baranov, V.I.

    2009-01-01

    The use of stable isotopes as tags in immunoassays, and their determination by ICPMS, is poised to have a huge impact on multi-parametric bioanalysis. A new technology, which we term 'mass cytometry', enables high throughput, highly multiplexed individual cell analysis. Preliminary results for T-cell immunophenotyping in peripheral blood mononuclear cells (PBMC), agonist influence on concomitant phosphorylation pathways, and sub-classification of acute myeloid leukemia patients' samples will be presented. The significance of individual cell analysis is demonstrated by the identification of populations of rogue cells in PBMC samples through the use of multidimensional neural network cluster analysis. (author)

  4. Gliadin Detection in Food by Immunoassay

    Science.gov (United States)

    Grant, Gordon; Sporns, Peter; Hsieh, Y.-H. Peggy

    Immunoassays are very sensitive and efficient tests that are commonly used to identify a specific protein. Examples of applications in the food industry include identification of proteins expressed in genetically modified foods, allergens, or proteins associated with a disease, including celiac disease. This genetic disease is associated with Europeans and affects about one in every 200 people in North America. These individuals react immunologically to wheat proteins, and consequently their own immune systems attack and damage their intestines. This disease can be managed if wheat proteins, specifically "gliadins," are avoided in foods.

  5. Review of the biochemical basis of enzyme immunoassays

    International Nuclear Information System (INIS)

    Klingler, W.

    1982-01-01

    The ever increasing number of radioimmunological determination poses problems allied with the handling of radioactive substances. In recent years various non-radioactive methods have been developed, among which the enzyme immunoassay is already in routine use. Homogeneous and heterogeneous enzyme immunoassays are described. Criteria for enzymes, substrates and enzyme-substrate reactions are listed. (orig.) [de

  6. Fake news? Biotin interference in thyroid immunoassays.

    Science.gov (United States)

    Koehler, Viktoria F; Mann, Ulrike; Nassour, Ayham; Alexander Mann, W

    2018-05-29

    We report on a 47 year old male patient with multiple sclerosis (MS) presenting in our outpatient neurology clinic in Frankfurt/Main for therapy evaluation. Before change of treatment laboratory investigations were performed. Thyroid function tests (TFTs) with a streptavidin/biotin based immunoassay revealed severe hyperthyroidism with positive thyroid autoantibodies suggestive for Graves' disease. Clinical presentation and thyroid sonography were unremarkable. Due to the discordance between clinical presentation and TFTs, we repeated medical history, in which the patient reported taking high-doses of biotin (300 mg/day) for MS. Recent studies with patients suffering from primary and secondary progressive MS, indicated promising effects of high-dose biotin on MS-related disability. In immunoassays relaying on streptavidin-biotin interaction, biotin intake can cause falsely high or low results. Two weeks after withdrawing biotin, biotin/streptavidin dependant assays showed no longer the biochemical picture of severe hyperthyroidism. Biotin intake should be paused for at least two to five days prior to the use of biotin/streptavidin dependant assays. Alternatively, non-biotin/streptavidin dependant assays (radioimmunoassay, gas chromatography-mass spectrometry/liquid chromatography-mass spectrometry) may be used. Copyright © 2017. Published by Elsevier B.V.

  7. Universal quantum dot-based sandwich-like immunoassay strategy for rapid and ultrasensitive detection of small molecules using portable and reusable optofluidic nano-biosensing platform

    International Nuclear Information System (INIS)

    Zhou, Liping; Zhu, Anna; Lou, Xuening; Song, Dan; Yang, Rong; Shi, Hanchang; Long, Feng

    2016-01-01

    . - Highlights: • Sandwich-like immunoassay strategy was used for rapid detection of small molecule. • Optofluidic sensing platform investigated the sandwich-like immunoassay mechanism. • A versatile optical biosensor like ELISA was developed using QD-immunoprobe. • No modified primary antibody with highest activity improved biosensor performance. • QD-immunoprobe-based biosensor was highly sensitive for small molecule detection. • Reusable sensing surface was applied for multiple sensing events with low cost.

  8. Universal quantum dot-based sandwich-like immunoassay strategy for rapid and ultrasensitive detection of small molecules using portable and reusable optofluidic nano-biosensing platform

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Liping; Zhu, Anna; Lou, Xuening; Song, Dan; Yang, Rong [School of Environment and Natural Resources, Renmin University of China, Beijing (China); Shi, Hanchang [School of Environment, Tsinghua University, Beijing (China); Long, Feng, E-mail: longf04@ruc.edu.cn [School of Environment and Natural Resources, Renmin University of China, Beijing (China)

    2016-01-28

    . - Highlights: • Sandwich-like immunoassay strategy was used for rapid detection of small molecule. • Optofluidic sensing platform investigated the sandwich-like immunoassay mechanism. • A versatile optical biosensor like ELISA was developed using QD-immunoprobe. • No modified primary antibody with highest activity improved biosensor performance. • QD-immunoprobe-based biosensor was highly sensitive for small molecule detection. • Reusable sensing surface was applied for multiple sensing events with low cost.

  9. A novel plant ferredoxin-like protein and the regulator Hor are quorum-sensing targets in the plant pathogen Erwinia carotovora.

    Science.gov (United States)

    Sjöblom, Solveig; Harjunpää, Heidi; Brader, Günter; Palva, E Tapio

    2008-07-01

    Quorum sensing (QS), a population-density-sensing mechanism, controls the production of the main virulence determinants, the plant cell-wall-degrading enzymes (PCWDEs) of the soft-rot phytopathogen Erwinia carotovora subsp. carotovora. In this study, we used random transposon mutagenesis with a gusA reporter construct to identify two new QS-controlled genes encoding the regulator Hor and a plant ferredoxin-like protein, FerE. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and mediated by the global repressor RsmA. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production. Our results showed that FerE contributes to oxidative stress tolerance and in planta fitness of the bacteria and suggest that QS could be central to control of oxidative stress tolerance. The presence of the FerE protein appears to be rather unique in heterotrophic bacteria and suggests an acquisition of the corresponding gene from plant host by horizontal gene transfer.

  10. Chemiluminescence immunoassay for prostate-specific antigen

    International Nuclear Information System (INIS)

    Zhang Xuefeng; Liu Yibing; Jia Juanjuan; Xu Wenge; Li Ziying; Chen Yongli; Han Shiquan

    2008-01-01

    The chemiluminescence immunoassay (CLIA) for serum total prostate-specific antigen (T-PSA) was developed. The reaction of luminol with hydrogen peroxide was introduced into this chemiluminescence system. The detection limit is established as 0.12 μg/L (n=10, mean of zero standard + 2SD) and the analytical recovery of PSA is 83.8%-118.7%. The intra-assay and inter-assay CVs vary from 4.4%-5.0% and 6.2%-11.7%, respectively. The experimental correlation coefficient of dilution is found to be 0.999. Compared with immunoradiometric assay (IRMA) kits, the correlative equation is y=1.07x+0.68, and correlation coefficient r=0.97. The standard range for the method is 1.5-80 μg/L, and it presents good linearity. (authors)

  11. Simplified immunoassay for rapid Dengue serotype diagnosis, revealing insensitivity to non-specific binding interference

    Directory of Open Access Journals (Sweden)

    Fernanda C.C.L. Loureiro

    2017-04-01

    Full Text Available Proof of concept of an immunoassay, which is easy to implement, for rapid Dengue virus (DENV serotype diagnosis, in the early infection stage, is reported. The four-layer assay is immobilized onto a thin gold film and relies on a low cost, disposable polymer biochip for optical surface plasmon resonance sensing and detection. The protocol comprises Neutravidin-Biotin mediated monoclonal antibody (MAB attachment as the functionalized sensing element. Formation of the MAB-DENV complex results in a pronounced thickness change that is optically recorded in real time, employing a microfluidic set-up. Virus presence is confirmed by atomic force microscopy from the same sample. Serum samples were collected from a patient in acute febrile state. Simultaneous serological analysis by means of the reverse transcription polymerase chain reaction, independently, confirmed presence of DENV2 and DENV3. The protocol proved applicable in presence of strong non-specific binding interference that originates from, and is caused by, various blood, serum and other body fluid constituents. False positive indications for both, negative serum and blood control samples were not observed. The achievable limit of detection was estimated to be 2×104 particles/ml. Eventually, the method can be modified towards detection of other viruses by using the same protocol. Keywords: Immuno-assay, Dengue virus detection, Non-specific binding

  12. Enhanced Plasmonic Biosensors of Hybrid Gold Nanoparticle-Graphene Oxide-Based Label-Free Immunoassay

    Science.gov (United States)

    Chiu, Nan-Fu; Chen, Chi-Chu; Yang, Cheng-Du; Kao, Yu-Sheng; Wu, Wei-Ren

    2018-05-01

    In this study, we propose a modified gold nanoparticle-graphene oxide sheet (AuNP-GO) nanocomposite to detect two different interactions between proteins and hybrid nanocomposites for use in biomedical applications. GO sheets have high bioaffinity, which facilitates the attachment of biomolecules to carboxyl groups and has led to its use in the development of sensing mechanisms. When GO sheets are decorated with AuNPs, they introduce localized surface plasmon resonance (LSPR) in the resonance energy transfer of spectral changes. Our results suggest a promising future for AuNP-GO-based label-free immunoassays to detect disease biomarkers and rapidly diagnose infectious diseases. The results showed the detection of antiBSA in 10 ng/ml of hCG non-specific interfering protein with dynamic responses ranging from 1.45 nM to 145 fM, and a LOD of 145 fM. Considering the wide range of potential applications of GO sheets as a host material for a variety of nanoparticles, the approach developed here may be beneficial for the future integration of nanoparticles with GO nanosheets for blood sensing. The excellent anti-interference characteristics allow for the use of the biosensor in clinical analysis and point-of-care testing (POCT) diagnostics of rapid immunoassay products, and it may also be a potential tool for the measurement of biomarkers in human serum.

  13. Can LC and LC-MS ever replace immunoassays?

    Directory of Open Access Journals (Sweden)

    Timothy G. Cross

    2016-10-01

    Full Text Available Immunoassays have been the technology of choice for the analysis of biomolecules for many decades across a wide range of applications in research, diagnostics and infectious disease monitoring. There are good reasons for the wide adoption of immunoassays but even such a well established and characterised technique has limitations and as such investigators are looking at alternative technologies. One such alternative is liquid chromatography (LC and, more specifically, liquid chromatography coupled with mass spectrometry (LC-MS. This article will review both immunoassay and LC and LC-MS technologies and methodologies and discuss the advantages and limitations of both approaches. In addition, the next developments that will need to occur before there is widespread adoption of LC and LC-MS technology preferentially over immunoassays will be examined.

  14. Rivers and flooded areas identified by medium-resolution remote sensing improve risk prediction of the highly pathogenic avian influenza H5N1 in Thailand

    Directory of Open Access Journals (Sweden)

    Weerapong Thanapongtharm

    2013-11-01

    Full Text Available Thailand experienced several epidemic waves of the highly pathogenic avian influenza (HPAI H5N1 between 2004 and 2005. This study investigated the role of water in the landscape, which has not been previously assessed because of a lack of high-resolution information on the distribution of flooded land at the time of the epidemic. Nine Landsat 7- Enhanced Thematic Mapper Plus scenes covering 174,610 km2 were processed using k-means unsupervised classification to map the distribution of flooded areas as well as permanent lakes and reservoirs at the time of the main epidemic HPAI H5N1 wave of October 2004. These variables, together with other factors previously identified as significantly associated with risk, were entered into an autologistic regression model in order to quantify the gain in risk explanation over previously published models. We found that, in addition to other factors previously identified as associated with risk, the proportion of land covered by flooding along with expansion of rivers and streams, derived from an existing, sub-district level (administrative level no. 3 geographical information system database, was a highly significant risk factor in this 2004 HPAI epidemic. These results suggest that water-borne transmission could have partly contributed to the spread of HPAI H5N1 during the epidemic. Future work stemming from these results should involve studies where the actual distribution of small canals, rivers, ponds, rice paddy fields and farms are mapped and tested against farm-level data with respect to HPAI H5N1.

  15. Detection of narcotics with an immunoassay film badge

    International Nuclear Information System (INIS)

    Lukens, H.R.

    1993-01-01

    Efficient personnel performance, a major requirement for a safe nuclear industry, is jeopardized where personnel use narcotics. However, detection of narcotics at nuclear plants is a challenge. The unique specificity and sensitivity of an immunoassay has been implemented in the form of a small, dry immunoassay film badge (IFB) for the detection of vapors emitted by narcotics. The device is suitable as an area monitor, and its characteristics are suitable for use as a breath monitor for the detection of drug use

  16. Status of immunoassay as an analytical tool in environmental investigations

    International Nuclear Information System (INIS)

    Van Emon, J.M.

    2000-01-01

    Immunoassay methods were initially applied in clinical situations where their sensitivity and selectivity were utilized for diagnostic purposes. In the 1970s, pesticide chemists realized the potential benefits of immunoassay methods for compounds difficult to analyze by gas chromatography. This transition of the technology has extended to the analysis of soil, water, food and other matrices of environmental and human exposure significance particularly for compounds difficult to analyze by chromatographic methods. The utility of radioimmunoassays and enzyme immunoassays for environmental investigations was recognized in the 1980s by the U.S. Environmental Protection Agency (U.S. EPA) with the initiation of an immunoassay development programme. The U.S. Department of Agriculture (USDA) and the U.S. Food and Drug Administration (PDA) have investigated immunoassays for the detection of residues in food both from an inspection and a contamination prevention perspective. Environmental immunoassays are providing rapid screening information as well as quantitative information to fulfill rigorous data quality objectives for monitoring programmes

  17. Targeted deposition of antibodies on a multiplex CMOS microarray and optimization of a sensitive immunoassay using electrochemical detection.

    Directory of Open Access Journals (Sweden)

    John Cooper

    2010-03-01

    Full Text Available The CombiMatrix ElectraSense microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray; and, in this configuration, it has also been used to tether antibodies (Abs specifically on electrodes using complementary DNA sequences conjugated to the Abs.An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSense microarray based upon targeted deposition of polypyrrole (Ppy and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical (ECD reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B (SEB is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different.Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pg/ml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay.

  18. Piper betle and its bioactive metabolite phytol mitigates quorum sensing mediated virulence factors and biofilm of nosocomial pathogen Serratia marcescens in vitro.

    Science.gov (United States)

    Srinivasan, Ramanathan; Devi, Kannan Rama; Kannappan, Arunachalam; Pandian, Shunmugiah Karutha; Ravi, Arumugam Veera

    2016-12-04

    Piper betle, a tropical creeper plant belongs to the family Piperaceae. The leaves of this plant have been well known for their therapeutic, religious and ceremonial value in South and Southeast Asia. It has also been reported to possess several biological activities including antimicrobial, antioxidant, antinociceptive, antidiabetic, insecticidal and gastroprotective activities and used as a common ingredient in indigenous medicines. In Indian system of ayurvedic medicine, P. betle has been well recognized for its antiseptic properties and is commonly applied on wounds and lesions for its healing effects. To evaluate the anti-quorum sensing (anti-QS) and antibiofilm efficacy of P. betle and its bioactive metabolite phytol against Serratia marcescens. The P. betle ethyl acetate extract (PBE) was evaluated for its anti-QS efficacy against S. marcescens by assessing the prodigiosin and lipase production at 400 and 500µgml -1 concentrations. In addition, the biofilm biomass quantification assay was performed to evaluate the antibiofilm activity of PBE against S. marcescens. Besides, the influence of PBE on bacterial biofilm formation was assessed through microscopic techniques. The biofilm related phenomenons like exopolysaccharides (EPS) production, hydrophobicity and swarming motility were also examined to support the antibiofilm activity of PBE. Transcriptional analysis of QS regulated genes in S. marcescens was also done. Characterization of PBE was done by separation through column chromatography and identification of active metabolites by gas chromatography -mass spectrometry. The major compounds of active fractions such as hexadecanoic acid, eugenol and phytol were assessed for their anti-QS activity against S. marcescens. Further, the in vitro bioassays such as protease, biofilm and HI quantification were also carried out to confirm the anti-QS and antibiofilm potential of phytol in PBE. PBE inhibits QS mediated prodigiosin pigment production in S. marcescens

  19. Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing.

    Science.gov (United States)

    Aronoff-Spencer, Eliah; Venkatesh, A G; Sun, Alex; Brickner, Howard; Looney, David; Hall, Drew A

    2016-12-15

    Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a "renewable" capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Potential applications of immunoassays in studies of flatfish recruitment

    Science.gov (United States)

    Feller, Robert J.

    The fisheries recruitment-stock problem, a lack of correlation between measures of reproductive output of the parent stock and recruitment to the fishery, has several potential biotic and abiotic causes. Immunoassays may be useful in examining several aspects of this and several other problems in flatfish ecology: stock identification, parasitism and disease, and trophic interactions. Given stage-specific antisera capable of recognozing antigenic moieties of, for instance, eggs, larvae, or newly-settled juveniles, it is possible to screen stomach contents of many putative predators ( e.g., shrimp or crabs) rapidly for the presence and amounts of platfish prey. This trophic application of immunological methods has great promise for measuring loss of potential recruits to predation. All immunoassays are limited by the quality of antisera used and the researcher's ability to interpret quantitative data in an ecologically meaningful way. Key references for applications of immunoassays in fish-related questions are provided with recommendations for their utilization.

  1. Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.

    Science.gov (United States)

    Mecklenburg, M; Lindbladh, C; Li, H; Mosbach, K; Danielsson, B

    1993-08-01

    A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.

  2. Rapid detection of fungal alpha-amylase in the work environment with a lateral flow immunoassay

    NARCIS (Netherlands)

    Bogdanovic, J.; Koets, M.; Sander, I.; Wouters, I.; Meijster, T.; Heederik, D.J.J.; Amerongen, van A.; Doekes, G.

    2006-01-01

    Background Occupational allergen exposure assessment usually requires airborne dust sampling at the worksite followed by dust extraction and enzyme immunoassay (EIA) analysis at the laboratory. Use of semiquantitative lateral flow immunoassays (LFIAs) may allow a more rapid detection procedure with

  3. Preparation by irradiation of a solid support for enzyme immunoassay

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.

    1984-01-01

    Reagents (immobilized anti-α-fetoprotein discs) having a porous structure were prepared for enzyme immunoassay of α-fetoprotein by radiation polymerization at low temperatures. Discs were attached to sticks for easy handling. The activity (determined by absorbance at 492 nm) of the discs varied with the hydrophilic properties and size of the discs. The discs are sufficiently sensitive and precise for enzyme immunoassay of α-fetoprotein. Anti-AFP dissolved in PBS solution was mixed with a monomer solution of hydroxyethyl methacrylate and hydroxypropyl methacrylate. The mixture was frozen to -78 0 C and gamma irradiated. (Auth.)

  4. Multiplex Immunoassay Profiling of Hormones Involved in Metabolic Regulation.

    Science.gov (United States)

    Stephen, Laurie; Guest, Paul C

    2018-01-01

    Multiplex immunoassays are used for rapid profiling of biomarker proteins and small molecules in biological fluids. The advantages over single immunoassays include lower sample consumption, cost, and labor. This chapter details a protocol to develop a 5-plex assay for glucagon-like peptide 1, growth hormone, insulin, leptin, and thyroid-stimulating hormone on the Luminex ® platform. The results of the analysis of insulin in normal control subjects are given due to the important role of this hormone in nutritional programming diseases.

  5. Development of recombinant antibody technology for application in plant pathogen diagnosis

    NARCIS (Netherlands)

    Griep, R.

    1999-01-01

    This thesis describes the applicability of the novel phage display technique to select plant-pathogen-specific monoclonal antibodies (MAbs) from combinatorial antibody libraries. The retrieved MAbs are so specific that they can be used as diagnostic tools in sensitive immunoassays for the

  6. Biosensor immunoassay for flumequine in broiler serum and muscle

    NARCIS (Netherlands)

    Haasnoot, W.; Gercek, H.; Cazemier, G.; Nielen, M.W.F.

    2007-01-01

    Flumequine (Flu) is one of the fluoroquinolones most frequently applied for the treatment of broilers in The Netherlands. For the detection of residues of Flu in blood serum of broilers, a biosensor immunoassay (BIA) was developed which was fast (7.5 min per sample) and specific (no cross-reactivity

  7. Feasibility of a simple microsieve-based immunoassay platform

    NARCIS (Netherlands)

    Zweitzig, D.R.; Tibbe, Arjan G.J.; Nguyen, A.T.; van Rijn, C.J.M.; Kopnitsky, M.J.; Cichonski, K.; Terstappen, Leonardus Wendelinus Mathias Marie

    2016-01-01

    The intrinsic properties of silicon microsieves, such as an optically flat surface, high overall porosity, and low flow resistance have led to an increasing number of biotechnology applications. In this report, the feasibility of creating a microsieve-based immunoassay platform was explored.

  8. Feasibility of a simple microsieve-based immunoassay platform

    NARCIS (Netherlands)

    Zweitzig, Daniel R.; Tibbe, Arjan G.; Nguyen, Ai T.; Rijn, van Cees J.M.; Kopnitsky, Mark J.; Cichonski, Kathleen; Terstappen, Leon W.M.M.

    2016-01-01

    The intrinsic properties of silicon microsieves, such as an optically flat surface, high overall porosity, and low flow resistance have led to an increasing number of biotechnology applications. In this report, the feasibility of creating a microsieve-based immunoassay platform was explored.

  9. Kinase Activity Studied in Living Cells Using an Immunoassay

    Science.gov (United States)

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  10. IMMUNOASSAYS FOR THE DETECTION OF UROKINASE RECEPTOR FORMS

    DEFF Research Database (Denmark)

    2004-01-01

    amounts of uPAR forms in a sample with data indicative of the presence of the cancer disease. The method can be used for staging, prognosis or diagnosis of prostate cancer. Two novel monoclonal antibodies and a kit and immunoassays for detecting at least one uPAR form(s) are also described in the present...

  11. Direct salivary cortisol radio-immunoassay determination. Clinical applications

    International Nuclear Information System (INIS)

    Simon, C.; Cherfan, J.; Kurtz, F.; Vignon, F.; Schlienger, J.L.; Chabrier, G.

    1987-01-01

    Salivary cortisol levels reflect the biologically active free fraction of blood cortisol. The authors describe the results obtained with the aim of a radio-immunoassay commercial serum cortisol kit, without prealable extraction in different physiological and pathological situations. Salivary cortisol determination appears performant both in nycthemeral studies and in stimulation or freination tests [fr

  12. Evaluation of Six Different Immunoassays for Serum Thyrotropin

    International Nuclear Information System (INIS)

    Ma Donghong; Lu Hankui; Gao Yunchao; Ge Wenli; Xiong Jiang; Liu Qiaoping; Gu Qing

    2010-01-01

    To analyzes the discrepancy and association among six different thyrotropin (TSH) immunoassay methods and to study their impact on the clinical diagnoses of thyroid diseases, the 150 serum samples from three groups consisting of hyperthyroidism, hypothyroidism and healthy subjects, 50 samples in each group were included in this study. The serum TSH levels were measured simultaneously by radioimmunoassay (RIA), immunoradiometric assay (IRMA), three-type chemilumiminescence immunoassay (CLIA) and electrochemiluminescence immunoassay (ECLIA). The results showed that individual serum TSH level varied significantly from one assay to another. There was no correlation between TSH RIA and other five assays in groups of hyperthyroidism and healthy subjects(P>0.05). The correlations between TSH IRMA and four automatic assays in hyperthyroidism group were relatively low (r= 0.38∼0.41). However, among the four automatic assays, TSH levels were well correlated (r= 0.92∼0.99). For clinical diagnoses, TSH RIA alone was not useful in the differentiation of hyperthyroidism and normal subjects, and TSH IRMA was misleading in some hyperthyroidism. There were no significant differences for four TSH automatic immunoassays in differential diagnoses of thyroid diseases. (authors)

  13. Comparison of capillary electrophoresis-based immunoassay with fluorescence polarization immunoassay for the immunodetermination of methamphetamine using various methamphetamine antibodies.

    Science.gov (United States)

    Choi, J; Kim, C; Choi, M J

    1998-11-01

    An accurate and simple immunoassay using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) was performed for the detection of methamphetamine (MA) in urine. The CE-LIF was conducted with an untreated fused-silica column using antiserum and a tracer of fluorescein isothiocyanate (FITC)-labeled MA. This CE-LIF system was compared with fluorescence polarization immunoassay (FPIA) in a TDx analyzer in the photo-check mode using the same FITC-labeled tracer and the same antiserum. Various antibodies, not only those prepared by our own immunogens but also those from commercial sources, were screened and characterized in both assay systems with regard to sensitivity, precision, and cross-reactivity. Both systems satisfied analytical precision and gave similar cross-reactivity patterns. However, the CE-LIF-based immunoassay was approximately one order superior to FPIA in sensitivity, requiring less volume of sample, antiserum, and tracer for the assay. Considering that the FPIA system is well known to be a useful tool for screening antibodies and detecting drugs, the CE-LIF-based immunoassay system, which is seemingly more advantageous than the FPIA system, appears to have great power for the characterization of antibodies and for the detection of MA in urine.

  14. A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.

    Science.gov (United States)

    Chen, Rui; Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua; Xiong, Yonghua

    2016-12-01

    Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H 2 O 2 ) and H 2 O 2 -sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H 2 O 2 and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H 2 O 2 , as well as the incubation time between H 2 O 2 and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10 2  CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10 3  CFU/mL to 1.18 × 10 6  CFU/mL were in the range of 65.88%-105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Direct tissue blot immunoassay for detection of Xylella fastidiosa in olive trees

    Directory of Open Access Journals (Sweden)

    Khaled DJELOUAH

    2015-01-01

    Full Text Available A direct tissue blot immunoassay (DTBIA technique has been compared with ELISA and PCR for detection of Xylella fastidiosa in olive trees from Apulia (southern Italy. Fresh cross-sections of young twigs and leaf petioles were printed onto nitrocellulose membranes and analyzed in the laboratory. Analyses of a first group of 61 samples gave similar efficiency for the three diagnostic techniques for detection the bacterium (24 positive and 36 negative samples, except for a single sample which was positive only with DTBIA and PCR. Similar results were obtained by separately analyzing suckers and twigs collected from different sectors of tree canopies of a second group of 20 olive trees (ten symptomatic and ten symptomless. In this second test the three diagnostic techniques confirmed the irregular distribution of the bacterium in the tree canopies and erratic detectability of the pathogen in the young suckers. It is therefore necessary to analyse composite samples per tree which should be prepared with twigs collected from different sides of the canopy. The efficiency comparable to ELISA and PCR, combined with the advantages of easier handling, speed and cost, make DTBIA a valid alternative to ELISA in large-scale surveys for occurrence of X. fastidiosa. Moreover, the printing of membranes directly in the field prevents infections spreading to Xylella-free areas, through movement of plant material with pathogen vectors for laboratory testing.

  16. Foodborne pathogens

    Directory of Open Access Journals (Sweden)

    Thomas Bintsis

    2017-06-01

    Full Text Available Foodborne pathogens are causing a great number of diseases with significant effects on human health and economy. The characteristics of the most common pathogenic bacteria (Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cronobacter sakazakii, Esherichia coli, Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococccus aureus, Vibrio spp. and Yersinia enterocolitica, viruses (Hepatitis A and Noroviruses and parasites (Cyclospora cayetanensis, Toxoplasma gondii and Trichinella spiralis, together with some important outbreaks, are reviewed. Food safety management systems based on to classical hazard-based approach has been proved to be inefficient, and risk-based food safety approach is now suggested from leading researchers and organizations. In this context, a food safety management system should be designed in a way to estimate the risks to human health from food consumption and to identify, select and implement mitigation strategies in order to control and reduce these risks. In addition, the application of suitable food safety education programs for all involved people in the production and consumption of foods is suggested.

  17. Design of a surface plasmon resonance immunoassay for therapeutic drug monitoring of amikacin.

    Science.gov (United States)

    Losoya-Leal, Adrian; Estevez, M-Carmen; Martínez-Chapa, Sergio O; Lechuga, Laura M

    2015-08-15

    The therapeutic drug monitoring (TDM) of pharmaceutical drugs with narrow therapeutic ranges is of great importance in the clinical setting. It provides useful information towards the enhancement of drug therapies, aiding in dosage control and toxicity risk management. Amikacin is an aminoglycoside antibiotic commonly used in neonatal therapies that is indicated for TDM due to the toxicity risks inherent in its use. Current techniques for TDM such as high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are costly, time consuming, and cannot be performed at the site of action. Over the last decades, surface plasmon resonance (SPR) biosensors have become increasingly popular in clinical diagnostics due to their ability to detect biomolecular interactions in real-time. We present an SPR-based competitive immunoassay for the detection of the antibiotic amikacin, suitable for TDM in both adults and neonates. We have obtained high specificity and sensitivity levels with an IC50 value of 1.4ng/mL and a limit of detection of 0.13ng/mL, which comfortably comply with the drug's therapeutic range. Simple dilution of serum can therefore be sufficient to analyze low-volume real samples from neonates, increasing the potential of the methodology for TDM. Compared to current TDM conventional methods, this SPR-based immunoassay can provide advantages such as simplicity, potential portability, and label-free measurements with the possibility of high throughput. This work is the foundation towards the development of an integrated, simple use, highly sensitive, fast, and point-of-care sensing platform for the opportune TDM of antibiotics and other drugs in a clinical setting. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Design and Fabrication of a PDMS Microchip Based Immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Guocheng; Wang, Wanjun; Wang, Jun; Lin, Yuehe

    2010-07-01

    In this paper, we describe the design and fabrication process of a polydimethylsiloxane (PDMS) microchip for on-chip multiplex immunoassay application. The microchip consists of a PDMS microfluidic channel layer and a micro pneumatic valve control layer. By selectively pressurizing the pneumatic microvalves, immuno reagents were controlled to flow and react in certain fluidic channel sites. Cross contamination was prevented by tightly closed valves. Our design was proposed to utilize PDMS micro channel surface as the solid phase immunoassay substrate and simultaneously detect four targets antigens on chip. Experiment result shows that 20psi valve pressure is sufficient to tightly close a 200µm wide micro channel with flow rate up to 20µl/min.

  19. Microfluidic "Pouch" Chips for Immunoassays and Nucleic Acid Amplification Tests.

    Science.gov (United States)

    Mauk, Michael G; Liu, Changchun; Qiu, Xianbo; Chen, Dafeng; Song, Jinzhao; Bau, Haim H

    2017-01-01

    Microfluidic cassettes ("chips") for processing and analysis of clinical specimens and other sample types facilitate point-of-care (POC) immunoassays and nucleic acid based amplification tests. These single-use test chips can be self-contained and made amenable to autonomous operation-reducing or eliminating supporting instrumentation-by incorporating laminated, pliable "pouch" and membrane structures for fluid storage, pumping, mixing, and flow control. Materials and methods for integrating flexible pouch compartments and diaphragm valves into hard plastic (e.g., acrylic and polycarbonate) microfluidic "chips" for reagent storage, fluid actuation, and flow control are described. We review several versions of these pouch chips for immunoassay and nucleic acid amplification tests, and describe related fabrication techniques. These protocols thus offer a "toolbox" of methods for storage, pumping, and flow control functions in microfluidic devices.

  20. Evaluation of four immunoassays for diagnosis of brucellosis in Cuba

    International Nuclear Information System (INIS)

    Peraza, C.; Valdes, O.; Fonseca, N.; Garcia, M.; Alvarez, M.; Izquierdo, D.L.

    1998-01-01

    Four immunoassays (two indirect and two competitive ones) were evaluated by samples from areas free of disease, free by vaccination and affected areas using as reference techniques the Bengal Rose Tests, the Antigen in Buffered Plate Tests and the Complement Fixation Reaction Test. The evaluated samples demonstrated that the competitive assays (ELISAC-1 and ELISAC-2) detected less false positives than the indirect ones (ELISAI-1 and ELISAI-2). Of the competitive ELISAS, version 2 presented better sensitivity and specificity results in affected areas for 95% confidence: 80.9 - 96.9% and 97.5 - 99.4% respectively with positive predictive value in the range of 76 to 94% and negative predictive one between 98.1 and 99.7%. It was concluded that this assay can be used for brucellosis control because it gives higher assurance than the other evaluated immunoassays and it can discriminate infected from vaccinated animals. (author)

  1. Label-Free Electrochemical Immunoassay for C-Reactive Protein

    Directory of Open Access Journals (Sweden)

    Madasamy Thangamuthu

    2018-03-01

    Full Text Available C-reactive protein (CRP is one of the most expressed proteins in blood during acute phase inflammation, and its minute level increase has also been recognized for the clinical diagnosis of cardio vascular diseases. Unfortunately, the available commercial immunoassays are labour intensive, require large sample volumes, and have practical limitations, such as low stability and high production costs. Hence, we have developed a simple, cost effective, and label-free electrochemical immunoassay for the measurement of CRP in a drop of serum sample using an immunosensor strip made up of a screen printed carbon electrode (SPE modified with anti-CRP functionalized gold nanoparticles (AuNPs. The measurement relies on the decrease of the oxidation current of the redox indicator Fe3+/Fe2+, resulting from the immunoreaction between CRP and anti-CRP. Under optimal conditions, the present immunoassay measures CRP in a linear range from 0.4–200 nM (0.047–23.6 µg mL−1, with a detection limit of 0.15 nM (17 ng mL−1, S/N = 3 and sensitivity of 90.7 nA nM−1, in addition to a good reproducibility and storage stability. The analytical applicability of the presented immunoassay is verified by CRP measurements in human blood serum samples. This work provides the basis for a low-priced, safe, and easy-to-use point-of-care immunosensor assay to measure CRP at clinically relevant concentrations.

  2. Determination of digoxin by enzyme immunoassay and radioimmunoassay

    International Nuclear Information System (INIS)

    Mueller, H.; Braeuer, H.; Foerster, G.; Reinhardt, M.

    1978-01-01

    The results of parallel determinations of digoxin in the sera of non selected patients (n=104) by enzyme immunoassay (EMIT.EIA) and radioimmunoassay (J-125 labeled RIA) were compared with each other. The determinations revealed considerably different concentrations; the values determined by EIA were statistical lower (for EIA 1,09+'0,99ng/ml, for RIA 1,34+'1,01ng/ml, p [de

  3. Towards the development of a radioenzyme-immunoassay

    International Nuclear Information System (INIS)

    Schuurs, A.H.W.M.; Waart, M. v. d.

    1976-01-01

    We have tried to develop a very sensitive enzyme-immunoassay. For this purpose, a very sensitive radiochemical enzyme assay was used. HCG was chosen as test model and AChE as labelling enzyme. The test appeared to be much more sensitive than the normal enzymeimmunoassay. And, in comparison with RIA, it was about as sensitive but less time-consuming, and it makes use, in principle, of stable reagents. (orig./GSE) [de

  4. History of inductively coupled plasma mass spectrometry-based immunoassays

    International Nuclear Information System (INIS)

    Giesen, Charlotte; Waentig, Larissa; Panne, Ulrich; Jakubowski, Norbert

    2012-01-01

    The analysis of biomolecules requires highly sensitive and selective detection methods capable of tolerating a complex, biological matrix. First applications of biomolecule detection by ICP-MS relied on the use of heteroelements as a label for quantification. However, the combination of immunoassays and ICP-MS facilitates multiparametric analyses through elemental tagging, and provides a powerful alternative to common bioanalytical methods. This approach extends the detection of biomarkers in clinical diagnosis, and has the potential to provide a deeper understanding of the investigated biological system. The results might lead to the detection of diseases at an early stage, or guide treatment plans. Immunoassays are well accepted and established for diagnostic purposes, albeit ICP-MS is scarcely applied for the detection of immune-based assays. However, the screening of biomarkers demands high throughput and multiplex/multiparametric techniques, considering the variety of analytes to be queried. Finally, quantitative information on the expression level of biomarkers is highly desirable to identify abnormalities in a given organism. Thus, it is the aim of this review to introduce the fundamentals, and to discuss the enormous strength of ICP-MS for the detection of different immunoassays on the basis of selected applications, with a special focus on LA‐ICP‐MS. - Highlights: ► We discuss the fundamentals of elemental tagging for ICP‐MS applications. ► We propose a definition for the expressions “label” and “tag”. ► We highlight LA‐ICP‐MS‐based heteroelement detection. ► We give an historic overview on ICP-MS and LA‐ICP‐MS-based immunoassays. ► In a personal outlook, we discuss future improvements realistically attainable.

  5. Domestic cat microsphere immunoassays: detection of antibodies during feline immunodeficiency virus infection.

    Science.gov (United States)

    Wood, Britta A; Carver, Scott; Troyer, Ryan M; Elder, John H; VandeWoude, Sue

    2013-10-31

    Microsphere immunoassays (MIAs) allow rapid and accurate evaluation of multiple analytes simultaneously within a biological sample. Here we describe the development and validation of domestic cat-specific MIAs for a) the quantification of total IgG and IgA levels in plasma, and b) the detection of IgG and IgA antibodies to feline immunodeficiency virus (FIV) capsid (CA) and surface (SU) proteins, and feline CD134 in plasma. These assays were used to examine the temporal antibody response of domestic cats infected with apathogenic and pathogenic FIVs, and domestic cats infected with parental and chimeric FIVs of varying pathogenicity. The results from these studies demonstrated that a) total IgG antibodies increase over time after infection; b) α-CA and α-SU IgG antibodies are detectable between 9 and 28 days post-infection and increase over time, and these antibodies combined represent a fraction (1.8 to 21.8%) of the total IgG increase due to infection; c) measurable α-CD134 IgG antibody levels vary among individuals and over time, and are not strongly correlated with viral load; d) circulating IgA antibodies, in general, do not increase during the early stage of infection; and e) total IgG, and α-CA and α-SU IgG antibody kinetics and levels vary with FIV viral strain/pathogenicity. The MIAs described here could be used to screen domestic cats for FIV infection, and to evaluate the FIV-specific or total antibody response elicited by various FIV strains/other diseases. © 2013.

  6. Sensing of RNA viruses

    DEFF Research Database (Denmark)

    Jensen, Søren; Thomsen, Allan Randrup

    2012-01-01

    pathogen-associated molecular patterns have emerged in great detail. This review presents an overview of our current knowledge regarding the receptors used to detect RNA virus invasion, the molecular structures these receptors sense, and the involved downstream signaling pathways.......Our knowledge regarding the contribution of the innate immune system in recognizing and subsequently initiating a host response to an invasion of RNA virus has been rapidly growing over the last decade. Descriptions of the receptors involved and the molecular mechanisms they employ to sense viral...

  7. Development of immunoassays for detecting clothianidin residue in agricultural products.

    Science.gov (United States)

    Li, Ming; Sheng, Enze; Cong, Lujing; Wang, Minghua

    2013-04-17

    Two enzyme-linked immunosorbent assays (ELISAs) based on polyclonal antibodies (PcAbs) for clothianidin are described: colorimetric detection format (ELISA) and pattern of chemiluminescent assay (CLEIA). Clothianidin hapten was synthesized and conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to produce immunogen and coating antigen. Anticlothianidin PcAbs were obtained from immunized New Zealand white rabbits. Under optimal conditions, the half-maximal inhibition concentration (IC₅₀) and the limit of detection (LOD, IC₂₀) of clothianidin were 0.046 and 0.0028 mg/L for the ELISA and 0.015 and 0.0014 mg/L for the CLEIA, respectively. There were no obvious cross-reactivities of the antibodies with its analogues except for dinotefuran. Recoveries of 76.4-116.4% for the immunoassays were achieved from spiked samples. The results of immunoassays for the spiked and authentic samples were largely consistent with gas chromatography. Therefore, the proposed immunoassays would be convenient and satisfactory analytical methods for the monitoring of clothianidin in agricultural products.

  8. Current status and future developments in radiolabelled immunoassays

    International Nuclear Information System (INIS)

    Edwards, R.

    1998-01-01

    Radioisotopes are used extensively in medical practice and their use in RIA or IRMA usually represent a small proportion of the total. Radiolabelled immunoassays based on 125 I constitute a simple didactic, cost effective and robust technology which is still regarded as the reference method in many clinical applications. The IAEA has implemented many successful programmes using the ''bulk reagent'' approach, involving 68 countries in all the different regions. The main achievements have been in technology transfer with self sufficiency in production for some countries; training of large numbers of staff; quality control and quality assurance schemes; devolution of screening programmes for neonatal congenital hypothryoidism. Alternatives to the use of radioisotopic tracers are constrained by many factors and are often only available in restricted commercial packages. They are often not suitable for technology transfer programmes and often lack any didactic component in addition to a relative high cost. The production of radiolabels using 125 I is both simple and adaptable. In addition expertise in their preparation and purification is widespread even in developing countries. Together with the ease of producing antibodies, the facts have made 125 I-radiolabelled immunoassays ideal for investigative procedures for many research activities (30,31) particularly in the medical context where radioisotopes are commonly used. In conclusion, even a superficial examination of public health statistics for various countries throughout the continents indicates a need for a simple, inexpensive and robust analytical tool. In this light, there is a predicted continuing role for radiolabelled immunoassays. (author)

  9. Simple patterned nanofiber scaffolds and its enhanced performance in immunoassay.

    Directory of Open Access Journals (Sweden)

    Jing Wang

    Full Text Available Cancer has become the leading cause of death worldwide; early diagnosis and treatment of cancers is critical for the survival of the patients. The concentration of cancer markers in easy-to-access biological fluids can provide great assistance in screening for occult primary cancers, distinguishing malignant from benign findings, determining prognosis and prediction for cancer patients. The multiplex detection technology of a panel of cancer markers can greatly increase the accuracy of disease diagnosis. Herein, we briefly fabricate a high-throughput micro-immunoassay based on the electrospun polystyrene (PS substrates to improve detection sensitivity. The immunoassay was evaluated by analyzing three different cancer biomarkers (AFP, CEA, VEGF. For AFP, CEA, VEGF immunofluorescence assay, the LOD of assay conducted on electrospun PS substrates before or after plasma and the conventional PS substrates were 0.42, 0.10, 1.12 ng/mL, 0.57, 0.09, 1.24 ng/mL, and 159.75, 26.19, 385.59 pg/mL, respectively (P < 0.05. Due to the high porosity and large surface area-to-volume ratio which is the foremost merit of nanostructures, and the plasma treatment which make the hydrophobic PS nanofibers hydropholic, the nanofibers substrates showed sufficient retention of immunoassay functionality and high potential for capture molecules immobilization. Consequently, the immunofluorescence assay conducted on electrospun PS substrates could significantly enhance the sensitivity and limits of detection.

  10. Real-time pathogen monitoring during enrichment: a novel nanotechnology-based approach to food safety testing.

    Science.gov (United States)

    Weidemaier, Kristin; Carruthers, Erin; Curry, Adam; Kuroda, Melody; Fallows, Eric; Thomas, Joseph; Sherman, Douglas; Muldoon, Mark

    2015-04-02

    We describe a new approach for the real-time detection and identification of pathogens in food and environmental samples undergoing culture. Surface Enhanced Raman Scattering (SERS) nanoparticles are combined with a novel homogeneous immunoassay to allow sensitive detection of pathogens in complex samples such as stomached food without the need for wash steps or extensive sample preparation. SERS-labeled immunoassay reagents are present in the cultural enrichment vessel, and the signal is monitored real-time through the wall of the vessel while culture is ongoing. This continuous monitoring of pathogen load throughout the enrichment process enables rapid, hands-free detection of food pathogens. Furthermore, the integration of the food pathogen immunoassay directly into the enrichment vessel enables fully biocontained food safety testing, thereby significantly reducing the risk of contaminating the surrounding environment with enriched pathogens. Here, we present experimental results showing the detection of E. coli, Salmonella, or Listeria in several matrices (raw ground beef, raw ground poultry, chocolate milk, tuna salad, spinach, brie cheese, hot dogs, deli turkey, orange juice, cola, and swabs and sponges used to sample a stainless steel surface) using the SERS system and demonstrate the accuracy of the approach compared to plating results. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Micromotor-based lab-on-chip immunoassays

    Science.gov (United States)

    García, Miguel; Orozco, Jahir; Guix, Maria; Gao, Wei; Sattayasamitsathit, Sirilak; Escarpa, Alberto; Merkoçi, Arben; Wang, Joseph

    2013-01-01

    Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic polymer/Ni/Pt microtube engine, containing carboxy moieties on its mixed poly(3,4-ethylenedioxythiophene) (PEDOT)/COOH-PEDOT polymeric outermost layer, is further functionalized with the antibody receptor to selectively recognize and capture the target protein. The new motor-based microchip immunoassay operations are carried out without any bulk fluid flow, replacing the common washing steps in antibody-based protein bioassays with the active transport of the captured protein throughout the different reservoirs, where each step of the immunoassay takes place. A first microchip format involving an `on-the-fly' double-antibody sandwich assay (DASA) is used for demonstrating the selective capture of the target protein, in the presence of excess of non-target proteins. A secondary antibody tagged with a polymeric-sphere tracer allows the direct visualization of the binding events. In a second approach the immuno-nanomotor captures and transports the microsphere-tagged antigen through a microchannel network. An anti-protein-A modified microengine is finally used to demonstrate the selective capture, transport and convenient label-free optical detection of a Staphylococcus aureus target bacteria (containing proteinA in its cell wall) in the presence of a large excess of non-target (Saccharomyces cerevisiae) cells. The resulting nanomotor-based microchip immunoassay offers considerable potential for diverse applications in clinical diagnostics, environmental and security monitoring fields.Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic

  12. Lab-on-a-Chip Pathogen Sensors for Food Safety

    Directory of Open Access Journals (Sweden)

    Bumsang Kim

    2012-08-01

    Full Text Available There have been a number of cases of foodborne illness among humans that are caused by pathogens such as Escherichia coli O157:H7, Salmonella typhimurium, etc. The current practices to detect such pathogenic agents are cell culturing, immunoassays, or polymerase chain reactions (PCRs. These methods are essentially laboratory-based methods that are not at all real-time and thus unavailable for early-monitoring of such pathogens. They are also very difficult to implement in the field. Lab-on-a-chip biosensors, however, have a strong potential to be used in the field since they can be miniaturized and automated; they are also potentially fast and very sensitive. These lab-on-a-chip biosensors can detect pathogens in farms, packaging/processing facilities, delivery/distribution systems, and at the consumer level. There are still several issues to be resolved before applying these lab-on-a-chip sensors to field applications, including the pre-treatment of a sample, proper storage of reagents, full integration into a battery-powered system, and demonstration of very high sensitivity, which are addressed in this review article. Several different types of lab-on-a-chip biosensors, including immunoassay- and PCR-based, have been developed and tested for detecting foodborne pathogens. Their assay performance, including detection limit and assay time, are also summarized. Finally, the use of optical fibers or optical waveguide is discussed as a means to improve the portability and sensitivity of lab-on-a-chip pathogen sensors.

  13. Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

    Science.gov (United States)

    Hyytiä, Heidi; Heikkilä, Taina; Brockmann, Eeva-Christine; Kekki, Henna; Hedberg, Pirjo; Puolakanaho, Tarja; Lövgren, Timo; Pettersson, Kim

    2015-03-01

    To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to immunoassay for the detection of cTnI and decreased matrix related interferences, thus resulting in a lower number of falsely elevated cTnI-values. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  14. Tumor specific lung cancer diagnostics with multiplexed FRET immunoassays

    Science.gov (United States)

    Geißler, D.; Hill, D.; Löhmannsröben, H.-G.; Thomas, E.; Lavigne, A.; Darbouret, B.; Bois, E.; Charbonnière, L. J.; Ziessel, R. F.; Hildebrandt, N.

    2010-02-01

    An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5- fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput screening in a broad range of in-vitro diagnostic applications.

  15. Linkage of biomolecules to solid phases for immunoassay

    International Nuclear Information System (INIS)

    Chapman, R.S.

    1998-01-01

    Topics covered by this lecture include a brief review of the principal methods of linkage of biomolecules to solid phase matrices. Copies of the key self explanatory slides are presented as figures together with reprints of two publications by the author dealing with a preferred chemistry for the covalent linkage of antibodies to hydroxyl and amino functional groups and the effects of changes in solid phase matrix and antibody coupling chemistry on the performance of a typical excess reagent immunoassay for thyroid stimulating hormone

  16. On-Chip Immunoassay for Determination of Urinary Albumin

    OpenAIRE

    Laiwattanapaisal, Wanida; Songjaroen, Temsiri; Maturos, Thitima; Lomas, Tanom; Sappat, Assawapong; Tuantranont, Adisorn

    2009-01-01

    An immunoassay performed on a portable microfluidic device was evaluated for the determination of urinary albumin. An increase in absorbance at 500 nm resulting from immunoagglutination was monitored directly on the poly(dimethylsiloxane) (PDMS) microchip using a portable miniature fibre-optic spectrometer. A calibration curve was linear up to 10 mg L–1 (r2 = 0.993), with a detection limit of 0.81 mg L–1 (S/N = 3). The proposed system showed good precision, with relative standard deviations (...

  17. Analytical evaluation of the novel Lumipulse G BRAHMS procalcitonin immunoassay

    OpenAIRE

    Ruzzenente, Orazio; Salvagno, Gian Luca; Gelati, Matteo; Lippi, Giuseppe

    2016-01-01

    Objectives: This study was designed to evaluate the analytical performance of the novel Lumipulse G1200 BRAHMS procalcitonin (PCT) immunoassay. Design and methods: This analytical evaluation encompassed the calculation of the limit of blank (LOB), limit of detection (LOD), functional sensitivity, intra- and inter-assay imprecision, confirmation of linearity and a comparison with the Vidas BRAHMS PCT assay. Results: The LOB, LOD and functional sensitivity were 0.0010 ng/mL, 0.0016 ng/mL and ...

  18. Emerging Pathogens Initiative (EPI)

    Data.gov (United States)

    Department of Veterans Affairs — The Emerging Pathogens Initiative (EPI) database contains emerging pathogens information from the local Veterans Affairs Medical Centers (VAMCs). The EPI software...

  19. An accurate, specific, sensitive, high-throughput method based on a microsphere immunoassay for multiplex detection of three viruses and bacterial fruit blotch bacterium in cucurbits.

    Science.gov (United States)

    Charlermroj, Ratthaphol; Makornwattana, Manlika; Himananto, Orawan; Seepiban, Channarong; Phuengwas, Sudtida; Warin, Nuchnard; Gajanandana, Oraprapai; Karoonuthaisiri, Nitsara

    2017-09-01

    To employ a microsphere immunoassay (MIA) to simultaneously detect multiple plant pathogens (potyviruses, Watermelon silver mottle virus, Melon yellow spot virus, and Acidovorax avenae subsp. citrulli) in actual plant samples, several factors need to be optimized and rigorously validated. Here, a simple extraction method using a single extraction buffer was successfully selected to detect the four pathogens in various cucurbit samples (cucumber, cantaloupe, melon, and watermelon). The extraction method and assay performance were validated with inoculated and field cucurbit samples. The MIA showed 98-99% relative accuracy, 97-100% relative specificity and 92-100% relative sensitivity when compared to commercial ELISA kits and reverse transcription PCR. In addition, the MIA was also able to accurately detect multiple-infected field samples. The results demonstrate that one common extraction method for all tested cucurbit samples could be applied to detect multiple pathogens; avoiding the need for multiple protocols to be employed. This multiplex method can therefore be instrumental for high-throughput screening of multiple plant pathogens with many advantages such as a shorter assay time (2.5h) with single assay format, a lower cost of detection ($5 vs $19.7 for 4 pathogens/sample) and less labor requirement. Its multiplex capacity can also be expanded to detect up to 50 different pathogens upon the availability of specific antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Rui [College of Life Science, Nanchang University, Nanchang, 330031 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Xiong, Yonghua, E-mail: yhxiongchen@163.com [College of Life Science, Nanchang University, Nanchang, 330031 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China)

    2016-12-01

    Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H{sub 2}O{sub 2}) and H{sub 2}O{sub 2}-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H{sub 2}O{sub 2} and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H{sub 2}O{sub 2}, as well as the incubation time between H{sub 2}O{sub 2} and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10{sup 2} CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10{sup 3} CFU/mL to 1.18 × 10{sup 6} CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. - Highlights: • A novel fluorescence immunoassay was developed for the ultrasensitive detection of E. coli O157:H7. • This detection was achieved through the combination of the high bioactivity of CAT and H{sub 2}O{sub 2}-sensitive QDs. • The activity of CAT to H{sub 2}O{sub 2} is 1000 folds higher than that of the HRP

  1. A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

    International Nuclear Information System (INIS)

    Chen, Rui; Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua; Xiong, Yonghua

    2016-01-01

    Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H_2O_2) and H_2O_2-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H_2O_2 and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H_2O_2, as well as the incubation time between H_2O_2 and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10"2 CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10"3 CFU/mL to 1.18 × 10"6 CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. - Highlights: • A novel fluorescence immunoassay was developed for the ultrasensitive detection of E. coli O157:H7. • This detection was achieved through the combination of the high bioactivity of CAT and H_2O_2-sensitive QDs. • The activity of CAT to H_2O_2 is 1000 folds higher than that of the HRP to tetramethylbenzidine. • The limit of detection of the proposed method could

  2. Comparative determination of phenytoin by spectrophotometry, gas chromatography, liquid chromatography, enzyme immunoassay, and radioimmunoassay

    International Nuclear Information System (INIS)

    Castro, A.; Ibanez, J.; DiCesare, J.L.; Adams, R.F.; Malkus, H.

    1978-01-01

    Sera from patients being treated with phenytoin were analyzed for the drug by spectrophotometry, gas chromatography, radioimmunoasay, enzyme immunoassay, and liquid chromatography. The assay values obtained were intercompared statistically. Enzyme immunoassay and liquid chromatography appear to be attractive alternatives to the more traditional methods of spectrophotometry and gas chromatography. Our radioimmunoassay data correlated poorly with results by the four other methods

  3. Determining total thyroxine with the aid of the homogeneous enzyme immunoassay

    International Nuclear Information System (INIS)

    Vogt, W.

    1978-01-01

    Total thyrozine values obtained with the aid of the homogeneous enzyme immunoassay are compared with those delivered by the RIA and the efficiency of the EMIT technique is evaluated. Some results obtained via Enzymun-T4, a heterologous enzyme immunoassay, are also given. (VJ) 891 VJ [de

  4. Evaluation of six immunoassays for detection of dengue virus-specific immunoglobulin M and G antibodies

    NARCIS (Netherlands)

    J. Groen (Jan); P. Koraka (Penelope); J. Velzing (Jans); C. Copra (Cederick); A.D.M.E. Osterhaus (Albert)

    2000-01-01

    textabstractThe performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid

  5. Evaluation of envelope glycoprotein E(rns) of an atypical bovine pestivirus as antigen in a microsphere immunoassay for the detection of antibodies against bovine viral diarrhea virus 1 and atypical bovine pestivirus.

    Science.gov (United States)

    Vijayaraghavan, Balaje; Xia, Hongyan; Harimoorthy, Rajiv; Liu, Lihong; Belák, Sándor

    2012-11-01

    Atypical bovine pestiviruses are related antigenically and phylogenetically to bovine viral diarrhea viruses (BVDV-1 and BVDV-2), and may cause the same clinical manifestations in animals. Glycoprotein E(rns) of an atypical bovine pestivirus Th/04_KhonKaen was produced in a baculovirus expression system and was purified by affinity chromatography. The recombinant E(rns) protein was used as an antigen in a microsphere immunoassay for the detection of antibodies against BVDV-1 and atypical bovine pestivirus. The diagnostic performance of the new method was evaluated by testing a total of 596 serum samples, and the assay was compared with enzyme-linked immunosorbent assay (ELISA). Based on the negative/positive cut-off median fluorescence intensity (MFI) value of 2800, the microsphere immunoassay had a sensitivity of 100% and specificity of 100% compared to ELISA. The immunoassay was able to detect antibodies against both BVDV-1 and the atypical pestivirus. This novel microsphere immunoassay has the potential to be multiplexed for simultaneous detection of antibodies against different bovine pathogens in a high-throughput and economical way. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Aequorin fusion proteins as bioluminescent tracers for competitive immunoassays

    Science.gov (United States)

    Mirasoli, Mara; Michelini, Elisa; Deo, Sapna K.; Dikici, Emre; Roda, Aldo; Daunert, Sylvia

    2004-06-01

    The use of bio- and chemiluminescence for the development of quantitative binding assays offers undoubted advantages over other detection systems, such as spectrophotometry, fluorescence, or radioactivity. Indeed, bio- and chemiluminescence detection provides similar, or even better, sensitivity and detectability than radioisotopes, while avoiding the problems of health hazards, waste disposal, and instability associated with the use of radioisotopes. Among bioluminescent labels, the calcium-activated photoprotein aequorin, originally isolated from Aequorea victoria and today available as a recombinant product, is characterized by very high detectability, down to attomole levels. It has been used as a bioluminescent label for developing a variety of highly sensitive immunoassays, using various analyte-aequorin conjugation strategies. When the analyte is a protein or a peptide, genetic engineering techniques can be used to produce protein fusions where the analyte is in-frame fused with aequorin, thus producing homogeneous one-to-one conjugation products, available in virtually unlimited amount. Various assays were developed using this strategy: a short review of the most interesting applications is presented, as well as the cloning, purification and initial characterization of an endothelin-1-aequorin conjugate suitable for developing a competitive immunoassay for endothelin-1, a potent vasoconstrictor peptide, involved in hypertension.

  7. Determination of phospholipid transfer proteins in rat tissues by immunoassays

    International Nuclear Information System (INIS)

    Teerlink, T.

    1983-01-01

    Several quantitative immunoassays have been developed for two phospholipid transfer proteins from rat liver, i.e. the phosphatidylcholine transfer protein and the non-specific lipid transfer protein. The development of a double-antibody radioimmunoassay for the phosphatidylcholine transfer protein is described. The transfer protein was labelled with iodine-125 by the mild glucose oxidase-lactoperoxidase method. Although less than one tyrosine residue per molecule of transfer protein was labelled, only 20% of the labelled transfer protein was immunoprecipitable. This value could be increased to 80% by purifying the labelled protein by affinity chromatography on a column of anti-phosphatidylcholine transfer protein-IgG coupled to Sepharose 4B. The radioimmunoassay was used to determine the levels of phosphatidylcholine transfer protein in homogenates and 105 000 xg supernatants from various rat tissues as well as several Morris hepatomas. An enzyme immunoassay for the non-specific lipid transfer protein is also described. The antiserum that was raised especially by the author was cross-reactive with the non-specific lipid transfer protein present in 105 000 xg supernatants from human, mouse and bovine liver. The non-specific lipid transfer protein lost its immunoreactivity upon labelling with iodine-125 using different labelling techniques. Therefore, a regular radioimmunoassay could not be developed. The results of these different assays were compared. (Auth.)

  8. Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

    Science.gov (United States)

    Numata, Satoshi; Katakami, Hideki; Inoue, Shinobu; Sawada, Hirotake; Hashida, Seiichi

    2016-07-01

    We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis. © The Author(s) 2016.

  9. Remote Sensing

    CERN Document Server

    Khorram, Siamak; Koch, Frank H; van der Wiele, Cynthia F

    2012-01-01

    Remote Sensing provides information on how remote sensing relates to the natural resources inventory, management, and monitoring, as well as environmental concerns. It explains the role of this new technology in current global challenges. "Remote Sensing" will discuss remotely sensed data application payloads and platforms, along with the methodologies involving image processing techniques as applied to remotely sensed data. This title provides information on image classification techniques and image registration, data integration, and data fusion techniques. How this technology applies to natural resources and environmental concerns will also be discussed.

  10. AN ENVIRONMENTAL TECHNOLOGY VERIFICATION (ETV) TESTING OF THREE IMMUNOASSAY TEST KITS FOR ANTHRAX, BOTULINUM TOXIN AND RICIN

    Science.gov (United States)

    Immunoassay test kits are based on immunoassay methods, where specific antibodies are used to detect and measure the contaminants of interest. Immunoassay test kits rely on the reaction of a contaminant or antigen with a selective antibody to give a product that can be measures....

  11. Multiplex competitive microbead-based flow cytometric immunoassay using quantum dot fluorescent labels

    International Nuclear Information System (INIS)

    Yu, Hye-Weon; Kim, In S.; Niessner, Reinhard; Knopp, Dietmar

    2012-01-01

    Highlights: ► First time, duplex competitive bead-based flow cytometric immunoassay was developed using ODs. ► Antibody-coated QD detection probes and antigen-immobilized microspheres were synthesized. ► The two model target analytes were low molecular weight compounds of microbial and chemical origin. ► The determination of different water types was possible after simple filtration of samples. - Abstract: In answer to the ever-increasing need to perform the simultaneous analysis of environmental hazards, microcarrier-based multiplex technologies show great promise. Further integration with biofunctionalized quantum dots (QDs) creates new opportunities to extend the capabilities of multicolor flow cytometry with their unique fluorescence properties. Here, we have developed a competitive microbead-based flow cytometric immunoassay using QDs fluorescent labels for simultaneous detection of two analytes, bringing the benefits of sensitive, rapid and easy-of-manipulation analytical tool for environmental contaminants. As model target compounds, the cyanobacterial toxin microcystin-LR and the polycyclic aromatic hydrocarbon compound benzo[a]pyrene were selected. The assay was carried out in two steps: the competitive immunological reaction of multiple targets using their exclusive sensing elements of QD/antibody detection probes and antigen-coated microsphere, and the subsequent flow cytometric analysis. The fluorescence of the QD-encoded microsphere was thus found to be inversely proportional to target analyte concentration. Under optimized conditions, the proposed assay performed well within 30 min for the identification and quantitative analysis of the two environmental contaminants. For microcystin-LR and benzo[a]pyrene, dose–response curves with IC 50 values of 5 μg L −1 and 1.1 μg L −1 and dynamic ranges of 0.52–30 μg L −1 and 0.13–10 μg L −1 were obtained, respectively. Recovery was 92.6–106.5% for 5 types of water samples like bottled

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

    Science.gov (United States)

    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, and

  13. New trends in emerging pathogens.

    Science.gov (United States)

    Skovgaard, Niels

    2007-12-15

    The emergence of pathogens is the result of a number of impact in all parts of the food chain. The emerging technologies in food production explain how new pathogens can establish themselves in the food chain and compromise food safety. The impact of the food technology is analysed for several bacteria, such as Yersinia, Campylobacter, Arcobacter, Helicobacter pullorum, Enterobacter sakazakii, Mycobacterium avium spp. paratuberculosis, prions related to vCJD and others. The importance of the ability of many microbes to form VBNC forms is elaborated on. Research on culture independent methods may address this outstanding issue to the better understanding of emerging pathogens. The "demerging" of pathogens also occur, and examples of this are explained. The reaction of bacteria to stresses and sublethal treatments, and how exposure to one stress factor can confer resistance to other stresses, literally speaking causing contagious resistance, are explained. The implication of this e.g. in modern approaches of food preservation, such as Minimally processed Foods, is considerable. Intestinal colonization of EHEC may be regulated by Quorum sensing, and this ability of microbes plays an important role in the colonization of microbes in food and on food processing equipment, an important factor in the emergence of pathogens. The emergence of Saccharomyces cerevisiae, as an opportunistic human pathogen, used for centuries for food and production of alcoholic beverages, calls for research in molecular tools to distinguish between probiotic and clinical strains. Cyclospora cayetanensis and Norovirus outbreaks can no longer be designated as emerging pathogens, they share however one characteristic in the epidemiology of emerging nature, the importance of the hygiene in the primary production stage, including supply of potable water, and the application of GMP and the HACCP principles in the beginning of the food chain. Hepatitis E virus is a potential emerging food borne

  14. Glucose Sensing

    CERN Document Server

    Geddes, Chris D

    2006-01-01

    Topics in Fluorescence Spectroscopy, Glucose Sensing is the eleventh volume in the popular series Topics in Fluorescence Spectroscopy, edited by Drs. Chris D. Geddes and Joseph R. Lakowicz. This volume incorporates authoritative analytical fluorescence-based glucose sensing reviews specialized enough to be attractive to professional researchers, yet also appealing to the wider audience of scientists in related disciplines of fluorescence. Glucose Sensing is an essential reference for any lab working in the analytical fluorescence glucose sensing field. All academics, bench scientists, and industry professionals wishing to take advantage of the latest and greatest in the continuously emerging field of glucose sensing, and diabetes care & management, will find this volume an invaluable resource. Topics in Fluorescence Spectroscopy Volume 11, Glucose Sensing Chapters include: Implantable Sensors for Interstitial Fluid Smart Tattoo Glucose Sensors Optical Enzyme-based Glucose Biosensors Plasmonic Glucose Sens...

  15. Make Sense?

    DEFF Research Database (Denmark)

    Gyrd-Jones, Richard; Törmälä, Minna

    Purpose: An important part of how we sense a brand is how we make sense of a brand. Sense-making is naturally strongly connected to how we cognize about the brand. But sense-making is concerned with multiple forms of knowledge that arise from our interpretation of the brand-related stimuli......: Declarative, episodic, procedural and sensory. Knowledge is given meaning through mental association (Keller, 1993) and / or symbolic interaction (Blumer, 1969). These meanings are centrally related to individuals’ sense of identity or “identity needs” (Wallpach & Woodside, 2009). The way individuals make...... sense of brands is related to who people think they are in their context and this shapes what they enact and how they interpret the brand (Currie & Brown, 2003; Weick, Sutcliffe, & Obstfeld, 2005; Weick, 1993). Our subject of interest in this paper is how stakeholders interpret and ascribe meaning...

  16. Clinical development of placental malaria vaccines and immunoassays harmonization

    DEFF Research Database (Denmark)

    Chêne, Arnaud; Houard, Sophie; Nielsen, Morten A

    2016-01-01

    Placental malaria caused by Plasmodium falciparum infection constitutes a major health problem manifesting as severe disease and anaemia in the mother, impaired fetal development, low birth weight or spontaneous abortion. Prevention of placental malaria currently relies on two key strategies...... that are losing efficacy due to spread of resistance: long-lasting insecticide-treated nets and intermittent preventive treatment during pregnancy. A placental malaria vaccine would be an attractive, cost-effective complement to the existing control tools. Two placental malaria vaccine candidates are currently...... in Phase Ia/b clinical trials. During two workshops hosted by the European Vaccine Initiative, one in Paris in April 2014 and the other in Brussels in November 2014, the main actors in placental malaria vaccine research discussed the harmonization of clinical development plans and of the immunoassays...

  17. Development of an Ultrasensitive Immunoassay for Detecting Tartrazine

    Directory of Open Access Journals (Sweden)

    Chuanlai Xu

    2013-06-01

    Full Text Available We have developed an ultrasensitive indirect competitive enzyme-linked immunosorbent assay for the determination of tartrazine. Two carboxylated analogues of tartrazine with different spacer lengths, and one derivative from commercial tartrazine after a little chemical modification, were synthesized as haptens in order to produce antibodies specific to tartrazine. The effect of sulfonic acid groups on the hapten structure of tartrazine was also studied carefully for the first time. A most specific monoclonal antibody against tartrazine was created and exhibited an IC50 value of 0.105 ng/mL and a limit of detection of 0.014 ng/mL, with no cross-reactivity to other structurally-related pigments. The established immunoassay was applied to the determination of tartrazine in fortified samples of orange juice and in real positive samples of carbonated beverages.

  18. Integrated immunoassay using tuneable surface acoustic waves and lensfree detection.

    Science.gov (United States)

    Bourquin, Yannyk; Reboud, Julien; Wilson, Rab; Zhang, Yi; Cooper, Jonathan M

    2011-08-21

    The diagnosis of infectious diseases in the Developing World is technologically challenging requiring complex biological assays with a high analytical performance, at minimal cost. By using an opto-acoustic immunoassay technology, integrating components commonly used in mobile phone technologies, including surface acoustic wave (SAW) transducers to provide pressure driven flow and a CMOS camera to enable lensfree detection technique, we demonstrate the potential to produce such an assay. To achieve this, antibody functionalised microparticles were manipulated on a low-cost disposable cartridge using the surface acoustic waves and were then detected optically. Our results show that the biomarker, interferon-γ, used for the diagnosis of diseases such as latent tuberculosis, can be detected at pM concentrations, within a few minutes (giving high sensitivity at a minimal cost). This journal is © The Royal Society of Chemistry 2011

  19. Plasma myelin basic protein assay using Gilford enzyme immunoassay cuvettes.

    Science.gov (United States)

    Groome, N P

    1981-10-01

    The assay of myelin basic protein in body fluids has potential clinical importance as a routine indicator of demyelination. Preliminary details of a competitive enzyme immunoassay for this protein have previously been published by the author (Groome, N. P. (1980) J. Neurochem. 35, 1409-1417). The present paper now describes the adaptation of this assay for use on human plasma and various aspects of routine data processing. A commercially available cuvette system was found to have advantages over microtitre plates but required a permuted arrangement of sample replicates for consistent results. For dose interpolation, the standard curve could be fitted to a three parameter non-linear equation by regression analysis or linearised by the logit/log transformation.

  20. A redox-mediated chromogenic reaction and application in immunoassay.

    Science.gov (United States)

    Yu, Ru-Jia; Ma, Wei; Peng, Mao-Pan; Bai, Zhi-Shan; Long, Yi-Tao

    2016-08-31

    A novel redox-mediated chromogenic reaction was demonstrated based on the reaction between HAuCl4 and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), which generate various color responses from red to green in the resulting solutions. Various redox substance could be used to mediate the reaction and trigger a distinct color response. We established a sensitive hydrogen peroxide colorimetric sensor based on the redox-mediated chromogenic reaction and depicted the application both in detection of enzyme and in an immunoassay. Combining the traditional chromogenic reagent with gold nanoparticles, our assay has the advantage in short response time (within three minutes), high sensitivity (10(-12) g mL(-1) for HBsAg) and stability. Copyright © 2016. Published by Elsevier B.V.

  1. Development of an ultrasensitive immunoassay for detecting tartrazine.

    Science.gov (United States)

    Li, Zhuokun; Song, Shanshan; Xu, Liguang; Kuang, Hua; Guo, Shidong; Xu, Chuanlai

    2013-06-25

    We have developed an ultrasensitive indirect competitive enzyme-linked immunosorbent assay for the determination of tartrazine. Two carboxylated analogues of tartrazine with different spacer lengths, and one derivative from commercial tartrazine after a little chemical modification, were synthesized as haptens in order to produce antibodies specific to tartrazine. The effect of sulfonic acid groups on the hapten structure of tartrazine was also studied carefully for the first time. A most specific monoclonal antibody against tartrazine was created and exhibited an IC50 value of 0.105 ng/mL and a limit of detection of 0.014 ng/mL, with no cross-reactivity to other structurally-related pigments. The established immunoassay was applied to the determination of tartrazine in fortified samples of orange juice and in real positive samples of carbonated beverages.

  2. On-Chip Immunoassay for Determination of Urinary Albumin

    Directory of Open Access Journals (Sweden)

    Adisorn Tuantranont

    2009-12-01

    Full Text Available An immunoassay performed on a portable microfluidic device was evaluated for the determination of urinary albumin. An increase in absorbance at 500 nm resulting from immunoagglutination was monitored directly on the poly(dimethylsiloxane (PDMS microchip using a portable miniature fibre-optic spectrometer. A calibration curve was linear up to 10 mg L–1 (r2 = 0.993, with a detection limit of 0.81 mg L–1 (S/N = 3. The proposed system showed good precision, with relative standard deviations (RSDs of 5.1%, when evaluated with 10 mg L–1 albumin (n = 10. Determination of urinary albumin with the proposed system gave results highly similar to those determined by the conventional spectrophotometric method using immunoturbidimetric detection (r2 = 0.995; n = 15.

  3. Performance of immunoassay kits for site characterization and remediation

    International Nuclear Information System (INIS)

    Waters, L.C.; Palausky, A.; Counts, R.W.; Jenkins, R.A.

    1995-01-01

    The US Department of Energy (DOE) is supporting efforts to identify, validate and implement the use of effective, low-cost alternatives to currently used analytical methods for environmental management. As part of that program, we have evaluated the performances of a number of immunoassay (IA) kits with specificities for environmental contaminants of concern to the DOE. The studies were done in the laboratory using both spiked and field test samples. The analyte specificity and manufacturers of the kits evaluated were the following: mercury, BioNebraska; polychlorinated biphenyls (PCBs), EnSys and Millipore; petroleum fuel hydrocarbons, Millipore and Ohmicron; and polyaromatic hydrocarbons (PAHs), Ohmicron and Millipore. The kits were used in either a semiquantitative or quantitative format according to the preference of the manufacturers

  4. Streptavidin-functionalized capillary immune microreactor for highly efficient chemiluminescent immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Yang Zhanjun [State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093 (China); College of Chemistry and Engineering, Yangzhou University, 88 South University Avenue, Yangzhou 225002 (China); Zong Chen [State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093 (China); Ju Huangxian, E-mail: hxju@nju.edu.cn [State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093 (China); Yan Feng, E-mail: yanfeng2007@sohu.com [Jiangsu Institute of Cancer Prevention and Cure, Nanjing 210009 (China)

    2011-11-07

    Highlights: {yields} A novel capillary immune microreactor was proposed for highly efficient flow-through chemiluminescent immunoassay. {yields} The microreactor was prepared by functionalizing capillary inner wall with streptavidin for capture of biotinylated antibody. {yields} The proposed immunoassay method showed wide dynamic range, good reproducibility, stability and practicality. {yields} The microreactor was low-cost and disposable, and possessed several advantages over the conventional immunoreactors. - Abstract: A streptavidin functionalized capillary immune microreactor was designed for highly efficient flow-through chemiluminescent (CL) immunoassay. The functionalized capillary could be used as both a support for highly efficient immobilization of antibody and a flow cell for flow-through immunoassay. The functionalized inner wall and the capture process were characterized using scanning electron microscopy. Compared to conventional packed tube or thin-layer cell immunoreactor, the proposed microreactor showed remarkable properties such as lower cost, simpler fabrication, better practicality and wider dynamic range for fast CL immunoassay with good reproducibility and stability. Using {alpha}-fetoprotein as model analyte, the highly efficient CL flow-through immunoassay system showed a linear range of 3 orders of magnitude from 0.5 to 200 ng mL{sup -1} and a low detection limit of 0.1 ng mL{sup -1}. The capillary immune microreactor could make up the shortcoming of conventional CL immunoreactors and provided a promising alternative for highly efficient flow-injection immunoassay.

  5. Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

    Science.gov (United States)

    Chen, Jing; He, Qing-hua; Xu, Yang; Fu, Jin-heng; Li, Yan-ping; Tu, Zhui; Wang, Dan; Shu, Mei; Qiu, Yu-lou; Yang, Hong-wei; Liu, Yuan-yuan

    2016-01-15

    Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Modulation of pathogen recognition by autophagy

    Directory of Open Access Journals (Sweden)

    Ji Eun eOh

    2012-03-01

    Full Text Available Autophagy is an ancient biological process for maintaining cellular homeostasis by degradation of long-lived cytosolic proteins and organelles. Recent studies demonstrated that autophagy is availed by immune cells to regulate innate immunity. On the one hand, cells exert direct effector function by degrading intracellular pathogens; on the other hand, autophagy modulates pathogen recognition and downstream signaling for innate immune responses. Pathogen recognition via pattern recognition receptors induces autophagy. The function of phagocytic cells is enhanced by recruitment of autophagy-related proteins. Moreover, autophagy acts as a delivery system for viral replication complexes to migrate to the endosomal compartments where virus sensing occurs. In another case, key molecules of the autophagic pathway have been found to negatively regulate immune signaling, thus preventing aberrant activation of cytokine production and consequent immune responses. In this review, we focus on the recent advances in the role of autophagy in pathogen recognition and modulation of innate immune responses.

  7. Description of a nanobody-based competitive immunoassay to detect tsetse fly exposure.

    Directory of Open Access Journals (Sweden)

    Guy Caljon

    2015-02-01

    Full Text Available Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts.A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11 was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao. Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95% and positive predictive values (> 98% but higher negative predictive values and hence increased sensitivity (> 95% and accuracy (> 95%.We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.

  8. AMPK in Pathogens

    OpenAIRE

    Mesquita, Inês Morais; Moreira, Diana; Marques, Belém Sampaio; Laforge, Mireille; Cordeiro-da-Silva, Anabela; Ludovico, Paula; Estaquier, Jérôme; Silvestre, Ricardo Jorge Leal

    2016-01-01

    During host–pathogen interactions, a complex web of events is crucial for the outcome of infection. Pathogen recognition triggers powerful cellular signaling events that is translated into the induction and maintenance of innate and adaptive host immunity against infection. In opposition, pathogens employ active mechanisms to manipulate host cell regulatory pathways toward their proliferation and survival. Among these, subversion of host cell energy metabolism by pathogens is currently recogn...

  9. Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: Implications for immunoassays

    DEFF Research Database (Denmark)

    Jørgensen, Kaj Anker; Karamperis, Nikolaos; Koefoed-Nielsen, Pernille Bundgaard

    2006-01-01

    by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites...... by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition...

  10. Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: implications for immunoassays

    DEFF Research Database (Denmark)

    Karamperis, N; Koefoed-Nielsen, PB; Brahe, P

    2006-01-01

    by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites...... by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition...

  11. Control of (pre-analytical aspects in immunoassay measurements of metabolic hormones in rodents

    Directory of Open Access Journals (Sweden)

    Maximilian Bielohuby

    2018-04-01

    Full Text Available The measurement of circulating hormones by immunoassay remains a cornerstone in preclinical endocrine research. For scientists conducting and interpreting immunoassay measurements of rodent samples, the paramount aim usually is to obtain reliable and meaningful measurement data in order to draw conclusions on biological processes. However, the biological variability between samples is not the only variable affecting the readout of an immunoassay measurement and a considerable amount of unwanted or unintended variability can be quickly introduced during the pre-analytical and analytical phase. This review aims to increase the awareness for the factors ‘pre-analytical’ and ‘analytical’ variability particularly in the context of immunoassay measurement of circulating metabolic hormones in rodent samples. In addition, guidance is provided how to gain control over these variables and how to avoid common pitfalls associated with sample collection, processing, storage and measurement. Furthermore, recommendations are given on how to perform a basic validation of novel single and multiplex immunoassays for the measurement of metabolic hormones in rodents. Finally, practical examples from immunoassay measurements of plasma insulin in mice address the factors ‘sampling site and inhalation anesthesia’ as frequent sources of introducing an unwanted variability during the pre-analytical phase. The knowledge about the influence of both types of variability on the immunoassay measurement of circulating hormones as well as strategies to control these variables are crucial, on the one hand, for planning and realization of metabolic rodent studies and, on the other hand, for the generation and interpretation of meaningful immunoassay data from rodent samples.

  12. A study using an isotope probe comparing immunoassay with serology in detection of Brucella Abortus antibody

    International Nuclear Information System (INIS)

    Devlin, J.G.; Redington, F.; Stephenson, M.

    1986-01-01

    We report a comparison of radio-immunoassay with conventional serology in the detection of brucella abortus antibody from three laboratories. Overall agreement by Chi squared analysis is 5%. There are significant differences between laboratories and a significant number of sero negative suspect sera (from 20% - 60%) were positive by ratio-immunoassay test. We suspect that conventional serology under-reports the incidence of antibody to brucella abortus. (author)

  13. Analytical evaluation of the novel Lumipulse G BRAHMS procalcitonin immunoassay

    Directory of Open Access Journals (Sweden)

    Orazio Ruzzenente

    2016-12-01

    Full Text Available Objectives: This study was designed to evaluate the analytical performance of the novel Lumipulse G1200 BRAHMS procalcitonin (PCT immunoassay. Design and methods: This analytical evaluation encompassed the calculation of the limit of blank (LOB, limit of detection (LOD, functional sensitivity, intra- and inter-assay imprecision, confirmation of linearity and a comparison with the Vidas BRAHMS PCT assay. Results: The LOB, LOD and functional sensitivity were 0.0010 ng/mL, 0.0016 ng/mL and 0.008 ng/mL, respectively. The total analytical imprecision was found to be 2.1% and the linearity was excellent (r=1.00 in the range of concentrations between 0.006–75.5 ng/mL. The correlation coefficient with Vidas BRAHMS PCT was 0.995 and the equation of the Passing and Bablok regression analysis was [Lumipulse G BRAHMS PCT]=0.76×[Vidas BRAHMS PCT]+0.04. The mean overall bias of Lumipulse G BRAHMS PCT versus Vidas BRAHMS PCT was −3.03 ng/mL (95% confidence interval [CI]: −4.32 to −1.74 ng/mL, whereas the mean bias in samples with PCT concentration between 0–10 ng/mL was −0.49 ng/mL (95% CI: −0.77 to −0.24 ng/mL. The diagnostic agreement was 100% at 0.5 ng/mL, 97% at 2.0 ng/mL and 95% at 10 ng/mL, respectively. Conclusions: These results attest that Lumipulse G BRAHMS PCT exhibits excellent analytical performance, among the best of the methods currently available on the diagnostic market. However, the significant bias compared to the Vidas BRAHMS PCT suggests that the methods cannot be used interchangeably. Keywords: Sepsis, Infection, Procalcitonin, Immunoassay

  14. Immune Evasion Strategies of Pathogens in Macrophages: the Potential for Limiting Pathogen Transmission.

    Science.gov (United States)

    Ren, Yuwei; Khan, Faheem Ahmed; Pandupuspitasari, Nuruliarizki Shinta; Zhang, Shujun

    2017-01-01

    Preventing pathogen transmission to a new host is of major interest to the immunologist and could benefit from a detailed investigation of pathogen immune evasion strategies. The first line of defense against pathogen invasion is provided by macrophages. When they sense pathogens, macrophages initiate signals to inflammatory and pro-inflammatory cytokines through pattern recognition receptors (PRRs) subsequently mediating phagocytosis and inflammation. The macrophage immune machinery classically includes two subsets: the activated M1 and the activated M2 that respond accordingly in diverse immune challenges. The lipid and glycogen metabolic pathways work together with the lysosome to help the mature phagosome to degrade and eliminate intracellular pathogens in macrophages. The viral evasion strategies are even more complex due to the interplay between autophagy and apoptosis. However, pathogens evolve several strategies to camouflage themselves against immune responses in order to ensure their survival, replication and transmission. These strategies include the muting of PRRs initiated inflammatory responses, attenuation of M1 and/or induction of M2 macrophages, suppression of autophago-lysosomal formation, interference with lipid and glycogen metabolism, and viral mediation of autophagy and apoptosis cross-talk to enhance viral replication. This review focuses on pathogen immune evasion methods and on the strategies used by the host against camouflaged pathogens.

  15. Catch and measure-mass spectrometry-based immunoassays in biomarker research.

    Science.gov (United States)

    Weiß, Frederik; van den Berg, Bart H J; Planatscher, Hannes; Pynn, Christopher J; Joos, Thomas O; Poetz, Oliver

    2014-05-01

    Mass spectrometry-based (MS) methods are effective tools for discovering protein biomarker candidates that can differentiate between physiological and pathophysiological states. Promising candidates are validated in studies comprising large patient cohorts. Here, targeted protein analytics are used to increase sample throughput. Methods involving antibodies, such as sandwich immunoassays or Western blots, are commonly applied at this stage. Highly-specific and sensitive mass spectrometry-based immunoassays that have been established in recent years offer a suitable alternative to sandwich immunoassays for quantifying proteins. Mass Spectrometric ImmunoAssays (MSIA) and Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA/iMALDI) are two prominent types of MS-based immunoassays in which the capture is done either at the protein or the peptide level. We present an overview of these emerging types of immunoassays and discuss their suitability for the discovery and validation of protein biomarkers. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. © 2013.

  16. Effective gasoline site assessment using the D TECH trademark BTEX immunoassay

    International Nuclear Information System (INIS)

    Hudak, R.T.; Melby, J.M.; Stave, J.W.

    1994-01-01

    The application of immunoassay to environmental testing can greatly aid in assessing sites for various contaminants including PCB, TNT, RDX, PAH and BTEX. Immunoassay offers several benefits to site assessment: it is accurate, reproducible and relatively inexpensive compared to instrumental analysis. In addition, because of its ease-of-use, this technique is ideal for the field and requires minimal user training. To demonstrate not only the effectiveness of the BTEX immunoassay, but also the reliability of the field results, a gasoline contaminated site was assessed comparing the BTEX immunoassay to gas chromatography. All sampling and site related activities were executed in accordance to the USEPA SW-846 guidelines. Three (3) analyses were performed on each sample. One immunoassay analysis was performed in the field by an individual who received two (2) hours of training prior to the start of the study. A technician familiar with the immunoassay ran the second analysis in a laboratory. Finally, an independent GC laboratory certified for BTEX method 8020 and 602 performed the GC analyses. One hundred one (101) samples were analyzed: thirty-nine (39) samples were water, the other sixty-two (62) were soils ranging from clay to silt. The results and costs of the methods are compared

  17. Nanoparticle-based immunosensors and immunoassays for aflatoxins

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xu; Niessner, Reinhard [Institute of Hydrochemistry and Chair of Analytical Chemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München (Germany); Tang, Dianping [Key Laboratory of Analysis and Detection for Food Safety, MOE & Fujian Province, Department of Chemistry, Fuzhou University, Fuzhou 350108 (China); Knopp, Dietmar, E-mail: dietmar.knopp@ch.tum.de [Institute of Hydrochemistry and Chair of Analytical Chemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München (Germany)

    2016-03-17

    Aflatoxins are naturally existing mycotoxins produced mainly by Aspergillus flavus and Aspergillus parasiticus, present in a wide range of food and feed products. Because of their extremely high toxicity and carcinogenicity, strict control of maximum residue levels of aflatoxins in foodstuff is set by many countries. In daily routine, different chromatographic methods are used almost exclusively. As supplement, in several companies enzyme immunoassay-based sample testing as primary screening is performed. Recently, nanomaterials such as noble metal nanoparticles, magnetic particles, carbon nanomaterials, quantum dots, and silica nanomaterials are increasingly utilized for aflatoxin determination to improve the sensitivity and simplify the detection. They are employed either as supports for the immobilization of biomolecules or as electroactive or optical labels for signal transduction and amplification. Several nanoparticle-based electrochemical, piezoelectric, optical, and immunodipstick assays for aflatoxins have been developed. In this review, we summarize these recent advances and illustrate novel concepts and promising applications in the field of food safety. - Highlights: • Novel concepts and promising applications of nanoparticle-based immunological methods for the determination of aflatoxins. • Inclusion of most important nanomaterials and hybrid nanostructures. • Inclusion of electrochemical, optical and mass-sensitive biosensors as well as optical and immunochromatographic assays.

  18. The microassay on a card: A rugged, portable immunoassay

    Science.gov (United States)

    Kidwell, David

    1991-01-01

    The Microassay on a Card (MAC) is a portable, hand-held, non-instrumental immunoassay that can test for the presence of a wide variety of substances in the environment. The MAC is a simple device to use. A drop of test solution is placed on one side of the card and within five minutes a color is developed on the other side in proportion to the amount of substance in the test solution, with sensitivity approaching 10 ng/ml. The MAC is self-contained and self-timed; no reagents or timing is necessary. The MAC may be configured with multiple wells to provide simultaneous testing for multiple species. As envisioned, the MAC will be employed first as an on-site screen for drugs of abuse in urine or saliva. If the MAC can be used as a screen of saliva for drugs of abuse, it could be applied to driving while intoxicated, use of drugs on the job, or testing of the identity of seized materials. With appropriate modifications, the MAC also could be used to test for environmental toxins or pollutants.

  19. Basic and clinical investigation of T3 immunoassay kit

    International Nuclear Information System (INIS)

    Konishi, Junji; Nakajima, Akiko; Morita, Rikushi; Endo, Keigo; Ikekubo, Katsuji

    1976-01-01

    T 3 immunoassay kit was investigated basically and clinically. A good result was obtained at the prescribed incubation temperature and for 16 hours of incubation time. Moreover, it was thought to be possible that incubation time could be shortened to 1 - 4 hours at 37 0 C. Specificity of antibody was good. Recovery of added T 3 was 100+-5 (S.D.) % on an average and parallel of dilution curve of high T 3 serum was also good. Variation coefficient of accuracy of this kit was 1.5 - 2.1 % and that of reproducibility was 1.3 - 6.6 %. Mild hemolysis did not affect measurement value. Serum T 3 level in normals, untreated patients with Basedow's disease and patients with primary hypothyroidism was 142+-21 ng/100 ml, 452+-156 ng/100 ml and 67+-17 ng/100 ml, respectively. Serum T 3 level in patients with Hashimoto's disease was distributed to a wide extent, but that of patients with goiter and simple goiter ranged within normal range. On the other side, serum T 3 level of normal pregnant woman was high and that of patients with anorexia nervosa showed low level. From the above mentioned results, it was concluded that this kit was simple in method and good in sensitivity, specificity and reproducibility and it was also useful for clinical applications. (M. Tsunoda)

  20. Detection of Aspergillus fumigatus mycotoxins: immunogen synthesis and immunoassay development.

    Science.gov (United States)

    Fox, M; Gray, G; Kavanagh, K; Lewis, C; Doyle, S

    2004-02-01

    Immunological detection of secreted low molecular weight toxins represents a potentially novel means of diagnosing infection by the fungus Aspergillus fumigatus. Two such metabolites, gliotoxin and helvolic acid, were selected and conjugated to thyroglobulin for antisera generation in rabbits. Gliotoxin was initially activated using N-[p-maleimidophenyl] isocyanate (PMPI) and subsequently conjugated to S-acetyl thioglycolic acid N-hydroxysuccinimide-activated thyroglobulin, whereas helvolic acid was activated with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in the presence of thyroglobulin prior to immunisation. To facilitate subsequent antisera evaluation, both toxins were similarly conjugated to bovine serum albumin (BSA). Matrix-Assisted Laser Desorption Ionisation-Time Of Flight (MALDI-TOF) mass spectrometry and SDS-PAGE analysis confirmed covalent attachment of toxins to BSA in the ratios of 15 and 2.4 mol per mol BSA for gliotoxin and helvolic acid, respectively. Resultant high titer antisera were capable of detecting both BSA-conjugated toxins (inhibitory concentration (IC)(50): 4-5 microg/ml). Free toxins were also detectable by competitive immunoassay, whereby 10 microg/ml free gliotoxin (30 microM) and helvolic acid (17 microM), respectively, inhibited antibody binding to cognate toxin-BSA previously immobilised on microwells. This work confirms that sensitive and specific antisera can be raised against fungal toxins and may have an application in diagnosing fungal infection.

  1. Enzyme immunoassay for DDT analysis in Lebanese soils

    International Nuclear Information System (INIS)

    Bashour, I.; Dagher, S.; Shammas, G.; Sukkariyah, B.; Kawar, N.

    2000-01-01

    Full text: The use of enzyme-linked immunosorbent assay (ELISA) technique in estimating pesticide residue in soils is a faster, less expensive and easier method to use than the gas chromatography (GC) analysis technique..In the test, DDT pesticide residues in the simple compete with enzyme (horseradish peroxidase)-labeled DDT for a limited number of antibody binding sites on the inside surfaces of the test wells; the envirologix plate kit was tested for the measurement of total DDT in virgin and fortified (0-1000 ng g exp-1) soil samples of different properties from Lebanon. Extraction of DDT from soil was done by shaking the samples for 16 hours on a mechanical shaker with 90% methanol without any clean-up steps. Then the samples were allowed to stand for 30 minutes and an aliquot was taken from the clear supernatant. The DDT in the extract was measured in triplicate by GC and ELISA. The results indicated that the two techniques were highly correlated (r2 =0.9671-0.9973). Differences in soils physical and chemical properties did not accuracy of the detection limits of ELISA when compared to GC-ECD results. Immunoassay technique is a suitable method for rapid and accurate measurement of DDT residue in mineral Lebanese soils

  2. Sensitive, Fast, and Specific Immunoassays for Methyltestosterone Detection

    Directory of Open Access Journals (Sweden)

    Na Kong

    2015-04-01

    Full Text Available An indirect competitive enzyme-linked immunosorbent assay (icELISA and an immunochromatographic strip assay using a highly specific monoclonal antibody, were developed to detect methyltestosterone (MT residues in animal feed. The optimized icELISA had a half-inhibition concentration value of 0.26 ng/mL and a limit of detection value of 0.045 ng/mL. There was no cross-reactivity with eight analogues, revealing high specificity for MT. Based on icELISA results, the recovery rate of MT in animal feed was 82.4%–100.6%. The results were in accordance with those obtained by gas chromatography-mass spectrometry. The developed immunochromatographic strip assay, as the first report for MT detection, had a visual cut-off value of 1 ng/mL in PBS, 2.5 ng/g in fish feed, and 2.5 ng/g in pig feed. Therefore, these immunoassays are useful and fast tools for MT residue detection in animal feed.

  3. Evidence for carcinoembryonic antigen using radioimmunoassay and enzyme immunoassay

    International Nuclear Information System (INIS)

    Kungda Gao, L.

    1980-01-01

    A commercially available radioimmunoassay for the determination of carcinoembryonic antigen (CEA) was initially compared with an enzyme immunoassay (EIA). Considerable differences were found between the individual value. For three patients suffering from carcinomas of the digestive tract a better indication of the disease was given in the RIA than in the EIA. A further 110 patients with various illnesses were examined for serum CEA-levels using RIA. A method for measuring CEA in feces by RIA was developed. The normal range lies below 300 ng/ml. This assay could be of significance for the early recognition of colo-rectal carcinoma. In part II of this dissertation CEA was isolated from colo-rectal carcinomas using three different gel filtration media. It was only possible to obtain almost pure CEA (24 μg CEA per μg protein) by one of the methods. Six guinea pigs were immunized with the isolated CEA and all developed antibodies. The isolated CEA was labelled with 125 I and an own RIA saturation sequence and double antibody separation was developed. One of the antisera was able to distinguish without overlap 7 healthy patients from 7 suffering from colo-rectal carcinomas in non-extracted serum. (orig./MG) [de

  4. Graphene-based chemiluminescence resonance energy transfer for homogeneous immunoassay.

    Science.gov (United States)

    Lee, Joon Seok; Joung, Hyou-Arm; Kim, Min-Gon; Park, Chan Beum

    2012-04-24

    We report on chemiluminescence resonance energy transfer (CRET) between graphene nanosheets and chemiluminescent donors. In contrast to fluorescence resonance energy transfer, CRET occurs via nonradiative dipole-dipole transfer of energy from a chemiluminescent donor to a suitable acceptor molecule without an external excitation source. We designed a graphene-based CRET platform for homogeneous immunoassay of C-reactive protein (CRP), a key marker for human inflammation and cardiovascular diseases, using a luminol/hydrogen peroxide chemiluminescence (CL) reaction catalyzed by horseradish peroxidase. According to our results, anti-CRP antibody conjugated to graphene nanosheets enabled the capture of CRP at the concentration above 1.6 ng mL(-1). In the CRET platform, graphene played a key role as an energy acceptor, which was more efficient than graphene oxide, while luminol served as a donor to graphene, triggering the CRET phenomenon between luminol and graphene. The graphene-based CRET platform was successfully applied to the detection of CRP in human serum samples in the range observed during acute inflammatory stress.

  5. Significance of isolated reactive treponemal chemiluminescence immunoassay results.

    Science.gov (United States)

    Hunter, Michael G; Robertson, Peter W; Post, Jeffrey J

    2013-05-01

    Isolated reactive serum treponemal chemiluminescence immunoassay (CIA) specimens cause clinical uncertainty. Sera were screened by CIA, and reactive samples underwent reflex testing with rapid plasma reagin (RPR), Treponema pallidum particle agglutination (TPPA), and fluorescent treponemal antibody absorption (FTA Abs) assays. Samples reactive only on the CIA were deemed "isolated" reactive CIA samples. We undertook detailed review of a subset of subjects with isolated reactive CIA specimens. Of 28 261 specimens, 1171 (4.1%) were reactive on CIA, of which 133 (11.3%) had isolated CIA reactivity. Most subjects (66 of 82 [80.5%]) with isolated reactive CIA specimens were from high-prevalence populations. We found evidence of CIA, TPPA, and FTA Abs seroreversion. The median chemiluminescent signal-to-cutoff ratio was similar for isolated reactive CIA sera and sera that were reactive on either FTA Abs or TPPA assays (2.19 vs 2.32; P = .15) but lower than for sera reactive on both FTA Abs and TPPA assays (12.37; P < .001) or for sera reactive on RPR assays (25.53; P < .001). A total of 11 of 20 patients (55%) with an isolated reactive CIA specimen who underwent medical record review had previous or subsequent evidence of syphilis infection. Isolated reactive CIA specimens may represent true T. pallidum infection and may be found after seroreversion of traditional treponemal assays.

  6. Serum gastrin: interests and limitations of radio-immunoassay

    International Nuclear Information System (INIS)

    Rougier, P.; Linhart, N.; Bok, B.

    1980-01-01

    Radio-immunoassay of serum gastrin may now be carried out in all laboratories of radio-immunology. Comparison of two commercial kits A: Schwartz-Mann and B: CEA-SORIN according to criteria of specificity, sensitivity and reproducibility within and between systems, shows that they both permit the detection of pathological hypergastrinemias (Zollinger Ellison and atropic gastritis). Both kits have an identical intrasystem coefficient of variation (10 p. cent and 7 p. cent fort A, 5 p. cent and 8 p. cent for B) on the other hand, the inter-system coefficient of variation is better for kit B (22.4 p. cent and 37 p. cent for kit A, and 11.5 p. cent and 14.2 p. cent for kit B). The normal values for each kit are quite different: 97 . 64 pg.ml -1 for A and 51 . 23 pg.ml -1 for B preventing one from comparing estimations carried out with two different kits [fr

  7. Establishment of carcinoembryonic antigen working standard for immunoassay

    International Nuclear Information System (INIS)

    Xu Ligen; Sun Youxiang; Jiao Yan

    2001-01-01

    The author is to prepare the working standard of carcinoembryonic antigen (CEA) for immunoassay and determine its potency. CEA solution of 320 μg/L was prepared from purified CEA solution of 4.6 mg/L and 1% human albumin solution buffered with 50 mmol/L sodium phosphate, pH7.4. This solution was distributed in an aliquot of 0.5 mL (160 ng per ampoule) and lyophilized. The potency of CEA working standard, in terms of present standard of CEA RIA and IRMA kits made by Chinese manufacturers and in terms of 1st IRP CEA HUMAN 73/601 supplied by WHO, has been determined. Mean immunological potency of the working standard is 163 ng per ampoule with confident limit of 159-168 ng per ampoule at 95% probability level. Test of parallelism of dose-response curve for the working standard to that for 1st IRP CEA HUMAN 73/601 has been passed. CEA working standard is suitable to the kits standard for CEA radioimmunoassay and immunoradiometric assay

  8. Designing novel nano-immunoassays: antibody orientation versus sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Puertas, S; Moros, M; Fernandez-Pacheco, R; Ibarra, M R; Grazu, V; De la Fuente, J M, E-mail: vgrazu@unizar.e, E-mail: jmfuente@unizar.e [Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, Campus RIo Ebro, EdifIcio I-D, Mariano Esquillor, s/n, 50018 Zaragoza (Spain)

    2010-12-01

    There is a growing interest in the use of magnetic nanoparticles (MNPs) for their application in quantitative and highly sensitive biosensors. Their use as labels of biological recognition events and their detection by means of some magnetic method constitute a very promising strategy for quantitative high-sensitive lateral-flow assays. In this paper, we report the importance of nanoparticle functionalization for the improvement of sensitivity for a lateral-flow immunoassay. More precisely, we have found that immobilization of IgG anti-hCG through its polysaccharide moieties on MNPs allows more successful recognition of the hCG hormone. Although we have used the detection of hCG as a model in this work, the strategy of binding antibodies to MNPs through its sugar chains reported here is applicable to other antibodies. It has huge potential as it will be very useful for the development of quantitative and high-sensitive lateral-flow assays for its use on human and veterinary, medicine, food and beverage manufacturing, pharmaceutical, medical biologics and personal care product production, environmental remediation, etc.

  9. Subversion of inflammasome activation and pyroptosis by pathogenic bacteria

    Directory of Open Access Journals (Sweden)

    Larissa D Cunha

    2013-11-01

    Full Text Available Activation of the inflammasome occurs in response to a notably high number of pathogenic microbes and is a broad innate immune response that effectively contributes to restriction of pathogen replication and generation of adaptive immunity. Activation of these platforms leads to caspase-1- and/or caspase-11-dependent secretion of proteins, including cytokines, and induction of a specific form of cell death called pyroptosis, which directly or indirectly contribute for restriction of pathogen replication. Not surprisingly, bona fide intracellular pathogens developed strategies for manipulation of cell death to guarantee intracellular replication. In this sense, the remarkable advances in the knowledge of the inflammasome field have been accompanied by several reports characterizing the inhibition of this platform by several pathogenic bacteria. Herein, we review some processes used by pathogenic bacteria, including Yersinia spp., Pseudomonas aeruginosa, Vibrio parahaemolyticus, Chlamydia trachomatis, Francisella tularensis, Shigella flexneri, Legionella pneumophila and Coxiella burnetii to evade the activation of the inflammasome and the induction of pyroptosis.

  10. Molecularly Imprinted Polymer as an Antibody Substitution in Pseudo-immunoassays for Chemical Contaminants in Food and Environmental Samples.

    Science.gov (United States)

    Chen, Chaochao; Luo, Jiaxun; Li, Chenglong; Ma, Mingfang; Yu, Wenbo; Shen, Jianzhong; Wang, Zhanhui

    2018-03-21

    The chemical contaminants in food and the environment are quite harmful to food safety and human health. Rapid, accurate, and cheap detection can effectively control the potential risks derived from these chemical contaminants. Among all detection methods, the immunoassay based on the specific interaction of antibody-analyte is one of the most widely used techniques in the field. However, biological antibodies employed in the immunoassay usually cannot tolerate extreme conditions, resulting in an unstable state in both physical and chemical profiles. Molecularly imprinted polymers (MIPs) are a class of polymers with specific molecular recognition abilities, which are highly robust, showing excellent operational stability under a wide variety of conditions. Recently, MIPs have been used in biomimetic immunoassays for chemical contaminants as an antibody substitute in food and the environment. Here, we reviewed these applications of MIPs incorporated in different analytical platforms, such as enzyme-linked immunosorbent assay, fluorescent immunoassay, chemiluminescent immunoassay, electrochemical immunoassay, microfluidic paper-based immunoassay, and homogeneous immunoassay, and discussed current challenges and future trends in the use of MIPs in biomimetic immunoassays.

  11. Analytical evaluation of the novel Lumipulse G BRAHMS procalcitonin immunoassay.

    Science.gov (United States)

    Ruzzenente, Orazio; Salvagno, Gian Luca; Gelati, Matteo; Lippi, Giuseppe

    2016-12-01

    This study was designed to evaluate the analytical performance of the novel Lumipulse G1200 BRAHMS procalcitonin (PCT) immunoassay. This analytical evaluation encompassed the calculation of the limit of blank (LOB), limit of detection (LOD), functional sensitivity, intra- and inter-assay imprecision, confirmation of linearity and a comparison with the Vidas BRAHMS PCT assay. The LOB, LOD and functional sensitivity were 0.0010 ng/mL, 0.0016 ng/mL and 0.008 ng/mL, respectively. The total analytical imprecision was found to be 2.1% and the linearity was excellent (r=1.00) in the range of concentrations between 0.006-75.5 ng/mL. The correlation coefficient with Vidas BRAHMS PCT was 0.995 and the equation of the Passing and Bablok regression analysis was [Lumipulse G BRAHMS PCT]=0.76×[Vidas BRAHMS PCT]+0.04. The mean overall bias of Lumipulse G BRAHMS PCT versus Vidas BRAHMS PCT was -3.03 ng/mL (95% confidence interval [CI]: -4.32 to -1.74 ng/mL), whereas the mean bias in samples with PCT concentration between 0-10 ng/mL was -0.49 ng/mL (95% CI: -0.77 to -0.24 ng/mL). The diagnostic agreement was 100% at 0.5 ng/mL, 97% at 2.0 ng/mL and 95% at 10 ng/mL, respectively. These results attest that Lumipulse G BRAHMS PCT exhibits excellent analytical performance, among the best of the methods currently available on the diagnostic market. However, the significant bias compared to the Vidas BRAHMS PCT suggests that the methods cannot be used interchangeably.

  12. Highly sensitive immunoassay based on E. coli with autodisplayed Z-domain

    International Nuclear Information System (INIS)

    Jose, Joachim; Park, Min; Pyun, Jae-Chul

    2010-01-01

    The Z-domain of protein A has been known to bind specifically to the F c region of antibodies (IgGs). In this work, the Z-domain of protein A was expressed on the outer membrane of Escherichia coli by using 'Autodisplay' technology as a fusion protein of autotransport domain. The E. coli with autodisplayed Z-domain was applied to the sandwich-type immunoassay as a solid-support of detection-antibodies against a target analyte. For the feasibility demonstration of the E. coli based immunoassay, C-reactive protein (CRP) assay was carried out by using E. coli with autodisplayed Z-domain. The limit of detection (LOD) and binding capacity of the E. coli based immunoassay were estimated to be far more sensitive than the conventional ELISA. Such a far higher sensitivity of E. coli based immunoassay than conventional ELISA was explained by the orientation control of immobilized antibodies and the mobility of E. coli in assay matrix. From the test results of 45 rheumatoid arthritis (RA) patients' serum and 15 healthy samples, a cut-off value was established to have optimal sensitivity and selectivity values for RA. The CRP test result of each individual sample was compared with ELISA which is the reference method for RA diagnosis. From this work, the E. coli with Z-domain was proved to be feasible for the medical diagnosis based on sandwich-type immunoassay.

  13. Sensitivity-Enhancement of FRET Immunoassays by Multiple-Antibody Conjugation on Quantum Dots.

    Science.gov (United States)

    Annio, Giacomo; Jennings, Travis; Tagit, Oya; Hildebrandt, Niko

    2018-05-23

    Quantum dots (QDs) are not only advantageous for color-tuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (ABs) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many ABs per QD will lead to an efficient AB-QD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the ABs on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate. Here, we investigate the performance of Tb-to-QD FRET immunoassays against total prostate specific antigen (TPSA) by changing the number of ABs per QD while leaving all the other assay components unchanged. We first characterize the AB-QD conjugation by various spectroscopic, microscopic, and chromatographic techniques and then quantify the TPSA immunoassay performance regarding sensitivity, limit of detection, and dynamic range. Our results show that an increasing conjugation ratio leads to significantly enhanced FRET immunoassays. These findings will be highly important for developing QD-based immunoassays in which the concentrations of both ABs and QDs can significantly influence the assay performance.

  14. AMPK in Pathogens.

    Science.gov (United States)

    Mesquita, Inês; Moreira, Diana; Sampaio-Marques, Belém; Laforge, Mireille; Cordeiro-da-Silva, Anabela; Ludovico, Paula; Estaquier, Jérôme; Silvestre, Ricardo

    2016-01-01

    During host-pathogen interactions, a complex web of events is crucial for the outcome of infection. Pathogen recognition triggers powerful cellular signaling events that is translated into the induction and maintenance of innate and adaptive host immunity against infection. In opposition, pathogens employ active mechanisms to manipulate host cell regulatory pathways toward their proliferation and survival. Among these, subversion of host cell energy metabolism by pathogens is currently recognized to play an important role in microbial growth and persistence. Extensive studies have documented the role of AMP-activated protein kinase (AMPK) signaling, a central cellular hub involved in the regulation of energy homeostasis, in host-pathogen interactions. Here, we highlight the most recent advances detailing how pathogens hijack cellular metabolism by suppressing or increasing the activity of the host energy sensor AMPK. We also address the role of lower eukaryote AMPK orthologues in the adaptive process to the host microenvironment and their contribution for pathogen survival, differentiation, and growth. Finally, we review the effects of pharmacological or genetic AMPK modulation on pathogen growth and persistence.

  15. Potatoes, pathogens and pests

    NARCIS (Netherlands)

    Lazebnik, Jenny

    2017-01-01

    Currently, fungicides are necessary to protect potato crops against late blight, Phytophthora infestans, one of the world’s most damaging crop pathogens. The introgression of plant resistance genes from wild potato species targeted specifically to the late blight pathogen into

  16. Food-borne pathogens

    International Nuclear Information System (INIS)

    Niemand, J.G.

    1985-01-01

    The Salmonella scare reinforced the importance of never taking chances when it comes to controlling pathogens. The issue has been resolved by radurisation. The article deals with the various pathogens that can effect food and argues the case for radurisation in dealing with them. It also looks at some of the other food products that can be treated using this process

  17. Self-paired monoclonal antibody lateral flow immunoassay strip for rapid detection of Acidovorax avenae subsp. citrulli.

    Science.gov (United States)

    Zeng, Haijuan; Guo, Wenbo; Liang, Beibei; Li, Jianwu; Zhai, Xuzhao; Song, Chunmei; Zhao, Wenjun; Fan, Enguo; Liu, Qing

    2016-09-01

    We screened a highly specific monoclonal antibody (McAb), named 6D, against Acidovorax avenae subsp. citrulli (Aac). Single McAb 6D was used as both nanogold-labeled antibody and test antibody to develop a single self-paired colloidal gold immunochromatographic test strip (Sa-GICS). The detection limit achieved using the Sa-GICS approach was 10(5) CFU/mL, with a result that can be observed by the naked eye within 10 min. Moreover, Sa-GICS can detect eight strains of Aac and display no cross-reactions with other pathogenic plant microorganisms. Artificial contamination experiments demonstrated that Sa-GICS would not be affected by impurities in the leaves or stems of the plants and were consistent with the PCR results. This is the first report on the development of a colloidal gold immunoassay strip with self-paired single McAb for the rapid detection of Aac. Graphical Abstract Schematic representation of the test strip.

  18. Pathogen inactivation techniques.

    Science.gov (United States)

    Pelletier, J P R; Transue, S; Snyder, E L

    2006-01-01

    The desire to rid the blood supply of pathogens of all types has led to the development of many technologies aimed at the same goal--eradication of the pathogen(s) without harming the blood cells or generating toxic chemical agents. This is a very ambitious goal, and one that has yet to be achieved. One approach is to shun the 'one size fits all' concept and to target pathogen-reduction agents at the Individual component types. This permits the development of technologies that might be compatible with, for example, plasma products but that would be cytocidal and thus incompatible with platelet concentrates or red blood cell units. The technologies to be discussed include solvent detergent and methylene blue treatments--designed to inactivate plasma components and derivatives; psoralens (S-59--amotosalen) designed to pathogen-reduce units of platelets; and two products aimed at red blood cells, S-303 (a Frale--frangible anchor-linker effector compound) and Inactine (a binary ethyleneimine). A final pathogen-reduction material that might actually allow one material to inactivate all three blood components--riboflavin (vitamin B2)--is also under development. The sites of action of the amotosalen (S-59), the S-303 Frale, Inactine, and riboflavin are all localized in the nucleic acid part of the pathogen. Solvent detergent materials act by dissolving the plasma envelope, thus compromising the integrity of the pathogen membrane and rendering it non-infectious. By disrupting the pathogen's ability to replicate or survive, its infectivity is removed. The degree to which bacteria and viruses are affected by a particular pathogen-reducing technology relates to its Gram-positive or Gram-negative status, to the sporulation characteristics for bacteria, and the presence of lipid or protein envelopes for viruses. Concerns related to photoproducts and other breakdown products of these technologies remain, and the toxicology of pathogen-reduction treatments is a major ongoing area

  19. Pervasive sensing

    Science.gov (United States)

    Nagel, David J.

    2000-11-01

    The coordinated exploitation of modern communication, micro- sensor and computer technologies makes it possible to give global reach to our senses. Web-cameras for vision, web- microphones for hearing and web-'noses' for smelling, plus the abilities to sense many factors we cannot ordinarily perceive, are either available or will be soon. Applications include (1) determination of weather and environmental conditions on dense grids or over large areas, (2) monitoring of energy usage in buildings, (3) sensing the condition of hardware in electrical power distribution and information systems, (4) improving process control and other manufacturing, (5) development of intelligent terrestrial, marine, aeronautical and space transportation systems, (6) managing the continuum of routine security monitoring, diverse crises and military actions, and (7) medicine, notably the monitoring of the physiology and living conditions of individuals. Some of the emerging capabilities, such as the ability to measure remotely the conditions inside of people in real time, raise interesting social concerns centered on privacy issues. Methods for sensor data fusion and designs for human-computer interfaces are both crucial for the full realization of the potential of pervasive sensing. Computer-generated virtual reality, augmented with real-time sensor data, should be an effective means for presenting information from distributed sensors.

  20. Development, evaluation, and laboratory validation of immunoassays for the diagnosis of equine infectious anemia (EIA) using recombinant protein produced from a synthetic p26 gene of EIA virus.

    Science.gov (United States)

    Singha, Harisankar; Goyal, Sachin K; Malik, Praveen; Khurana, Sandip K; Singh, Raj K

    2013-12-01

    Equine infectious anemia (EIA)-a retroviral disease caused by equine infectious anemia virus (EIAV)-is a chronic, debilitating disease of horses, mules, and donkeys. EIAV infection has been reported worldwide and is recognized as pathogen of significant economic importance to the horse industry. This disease falls under regulatory control program in many countries including India. Control of EIA is based on identification of inapparent carriers by detection of antibodies to EIAV in serologic tests and "Stamping Out" policy. The current internationally accepted test for diagnosis of EIA is the agar gel immune-diffusion test (AGID), which detects antibodies to the major gag gene (p26) product. The objective of this study was to develop recombinant p26 based in-house immunoassays [enzyme linked immunosorbent assays (ELISA), and AGID] for EIA diagnosis. The synthetic p26 gene of EIAV was expressed in Escherichia coli and diagnostic potential of recombinant p26 protein were evaluated in ELISA and AGID on 7,150 and 1,200 equine serum samples, respectively, and compared with commercial standard AGID kit. The relative sensitivity and specificity of the newly developed ELISA were 100 and 98.6 %, respectively. Whereas, relative sensitivity and specificity of the newly developed AGID were in complete agreement in respect to commercial AGID kit. Here, we have reported the validation of an ELISA and AGID on large number of equine serum samples using recombinant p26 protein produced from synthetic gene which does not require handling of pathogenic EIAV. Since the indigenously developed reagents would be economical than commercial diagnostic kit, the rp26 based-immunoassays could be adopted for the sero-diagnosis and control of EIA in India.

  1. Processes for managing pathogens.

    Science.gov (United States)

    Godfree, Alan; Farrell, Joseph

    2005-01-01

    Wastewater contains human, animal, and plant pathogens capable of causing viral, bacterial, or parasitic infections. There are several routes whereby sewage pathogens may affect human health, including direct contact, contamination of food crops, zoonoses, and vectors. The range and numbers of pathogens in municipal wastewater vary with the level of endemic disease in the community, discharges from commercial activities, and seasonal factors. Regulations to control pathogen risk in the United States and Europe arising from land application of biosolids are based on the concept of multiple barriers to the prevention of transmission. The barriers are (i) treatment to reduce pathogen content and vector attraction, (ii) restrictions on crops grown on land to which biosolids have been applied, and (iii) minimum intervals following application and grazing or harvesting. Wastewater treatment reduces number of pathogens in the wastewater by concentrating them with the solids in the sludge. Although some treatment processes are designed specifically to inactivate pathogens, many are not, and the actual mechanisms of microbial inactivation are not fully understood for all processes. Vector attraction is reduced by stabilization (reduction of readily biodegradable material) and/or incorporation immediately following application. Concerns about health risks have renewed interest in the effects of treatment (on pathogens) and advanced treatment methods, and work performed in the United States suggests that Class A pathogen reduction can be achieved less expensively than previously thought. Effective pathogen risk management requires control to the complete chain of sludge treatment, biosolids handling and application, and post-application activities. This may be achieved by adherence to quality management systems based on hazard analysis critical control point (HACCP) principles.

  2. Nitrocellulose membrane-based enzyme-linked immunoassay for dengue serotype-1 IgM detection

    International Nuclear Information System (INIS)

    Leon, S.; Guevara, C.; Chunga, A.

    1999-01-01

    To evaluate the sensitivity and specifity of a nitrocellulose membrane-based immunoassay for dengue IgM, with respect to capture enzyme immunoassay, for the diagnosis of dengue virus infection. 101 serum samples were processed and divided into 2 groups: 53 from dengue serotype 1 (DEN1) infected patients, and 48 from healthy subjects. Both groups were tested with a nitrocellulose membrane-based IgM capture enzyme immunoassay (NMB-EIA) and also with an ELISA as referential pattern. NMB-EIA testing detected IgM anti-DEN1 in 94,34% of samples from infected patients, and in 14,58% of control samples, whereas ELISA fails to report false positive or false negative results: NMB-EIA appears to be a good alternative for dengue infection diagnosis. (authors)

  3. Distribution of Phytophthora spp. in Field Soils Determined by Immunoassay.

    Science.gov (United States)

    Miller, S A; Madden, L V; Schmitthenner, A F

    1997-01-01

    ABSTRACT Populations of Phytophthora spp. were determined by enzyme-linked immunosorbent assay (ELISA) in field soils used for pepper and soybean production in Ohio. Soybean fields were sampled extensively (64 fields, n = 6 samples per field over 2 years) and intensively (4 fields, n = 64 samples per field in 1 year) to assess heterogeneity of P. sojae populations. Four pepper fields (n = 64), three of which had a history of Phytophthora blight (caused by P. capsici), also were sampled intensively during a 6-month period. Mean (m), variance (v), and measures of aggregation (e.g., variance-to-mean ratio [v/m]) of immunoassay values, translated to Phytophthora antigen units (PAU), were related to the disease history in each of the pepper and soybean fields. Mean PAU values for fields in which Phytophthora root rot (soybean) or blight (pepper) had been moderate to severe were higher than in fields in which disease incidence had been low or not observed. A detection threshold value of 11.3 PAU was calculated with values for 64 samples from one pepper field, all of which tested negative for Phytophthora by bioassay and ELISA. Seven of the eight intensively sampled fields contained at least some detectable Phytophthora propagules, with the percentage of positive samples ranging from 1.6 to 73.4. Mean PAU values ranged from 1 to 84 (extensive soybean field sampling), 6 to 24 (intensive soybean field sampling), and 4 to 30 (intensive pepper field sampling); however, variances ranged from 0 to 7,774 (extensive sampling), 30 to 848 (intensive soybean field sampling), and 5 to 2,401 (intensive pepper field sampling). Heterogeneity of PAU was high in most individual soybean and pepper fields, with values of v/m greater than 1, and log(v) increasing with log(m), with a slope of about 2.0. Spatial autocorrelation coefficients were not significant, indicating there was no relationship of PAU values in neighboring sampling units (i.e., field locations) of the intensively sampled

  4. Negative interference by rheumatoid factor in alpha-fetoprotein chemiluminescent microparticle immunoassay.

    Science.gov (United States)

    Wang, Hui; Bi, Xiaohui; Xu, Lei; Li, Yirong

    2017-01-01

    Background Rheumatoid factor causes positive interference in multiple immunoassays. Recently, negative interference has also been found in immunoassays in the presence of rheumatoid factor. The chemiluminescent microparticle immunoassay is widely used to determine serum alpha-fetoprotein. However, it is not clear whether the presence of rheumatoid factor in the serum causes interference in the chemiluminescent microparticle immunoassay of alpha-fetoprotein. Methods Serum alpha-fetoprotein was determined using the ARCHITECT alpha-fetoprotein assay. The estimation of alpha-fetoprotein recovery was carried out in samples prepared by diluting high-concentration alpha-fetoprotein serum with rheumatoid factor-positive or rheumatoid factor-negative serum. Paramagnetic microparticles coated with hepatitis B surface antigen-anti-HBs complexes were used to remove rheumatoid factor from the serum. Results The average recovery of alpha-fetoprotein was 88.4% and 93.8% in the rheumatoid factor-positive and rheumatoid factor-negative serum samples, respectively. The recovery of alpha-fetoprotein was significantly lower in the rheumatoid factor-positive serum samples than in the rheumatoid factor-negative serum samples. In two of five rheumatoid factor-positive samples, a large difference was found (9.8%) between the average alpha-fetoprotein recoveries in the serially diluted and initial recoveries. Fourteen rheumatoid factor-positive serum samples were pretreated with hepatitis B surface antigen-anti-HBs complex-coated paramagnetic microparticles. The alpha-fetoprotein concentrations measured in the pretreated samples increased significantly. Conclusions It was concluded that the alpha-fetoprotein chemiluminescent microparticle immunoassay is susceptible to interference by rheumatoid factor, leading to significantly lower results. Eliminating the incidence of negative interference from rheumatoid factor should be an important goal for immunoassay providers. In the meantime

  5. Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Hyo-Deok; Lee, Joong-jae [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of); Kim, Yu Jung [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Hantschel, Oliver [School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne (Switzerland); Lee, Seung-Goo [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Kim, Hak-Sung, E-mail: hskim76@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of)

    2017-01-15

    Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL{sup −1} for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays. - Highlights: • A human TNF-α (hTNF-α)-specific repebody was selected using a phage display. • A monomeric alkaline phosphatase (mAP) was genetically fused to the repebody. • mAP-fused repebody enabled detection of hTNF-α with high sensitivity and accuracy. • mAP-fused repebody can be widely used as a new immuno-reagent in immunoassays.

  6. BIOTIN INTERFERENCE WITH ROUTINE CLINICAL IMMUNOASSAYS: UNDERSTAND THE CAUSES AND MITIGATE THE RISKS.

    Science.gov (United States)

    Samarasinghe, Shanika; Meah, Farah; Singh, Vinita; Basit, Arshi; Emanuele, Nicholas; Emanuele, Mary Ann; Mazhari, Alaleh; Holmes, Earle W

    2017-08-01

    The objectives of this report are to review the mechanisms of biotin interference with streptavidin/biotin-based immunoassays, identify automated immunoassay systems vulnerable to biotin interference, describe how to estimate and minimize the risk of biotin interference in vulnerable assays, and review the literature pertaining to biotin interference in endocrine function tests. The data in the manufacturer's "Instructions for Use" for each of the methods utilized by seven immunoassay system were evaluated. We also conducted a systematic search of PubMed/MEDLINE for articles containing terms associated with biotin interference. Available original reports and case series were reviewed. Abstracts from recent scientific meetings were also identified and reviewed. The recent, marked, increase in the use of over-the-counter, high-dose biotin supplements has been accompanied by a steady increase in the number of reports of analytical interference by exogenous biotin in the immunoassays used to evaluate endocrine function. Since immunoassay methods of similar design are also used for the diagnosis and management of anemia, malignancies, autoimmune and infectious diseases, cardiac damage, etc., biotin-related analytical interference is a problem that touches every area of internal medicine. It is important for healthcare personnel to become more aware of immunoassay methods that are vulnerable to biotin interference and to consider biotin supplements as potential sources of falsely increased or decreased test results, especially in cases where a lab result does not correlate with the clinical scenario. FDA = U.S. Food & Drug Administration FT3 = free tri-iodothyronine FT4 = free thyroxine IFUs = instructions for use LH = luteinizing hormone PTH = parathyroid hormone SA/B = streptavidin/biotin TFT = thyroid function test TSH = thyroid-stimulating hormone.

  7. Bead-based competitive fluorescence immunoassay for sensitive and rapid diagnosis of cyanotoxin risk in drinking water.

    Science.gov (United States)

    Yu, Hye-Weon; Jang, Am; Kim, Lan Hee; Kim, Sung-Jo; Kim, In S

    2011-09-15

    Due to the increased occurrence of cyanobacterial blooms and their toxins in drinking water sources, effective management based on a sensitive and rapid analytical method is in high demand for security of safe water sources and environmental human health. Here, a competitive fluorescence immunoassay of microcystin-LR (MCYST-LR) is developed in an attempt to improve the sensitivity, analysis time, and ease-of-manipulation of analysis. To serve this aim, a bead-based suspension assay was introduced based on two major sensing elements: an antibody-conjugated quantum dot (QD) detection probe and an antigen-immobilized magnetic bead (MB) competitor. The assay was composed of three steps: the competitive immunological reaction of QD detection probes against analytes and MB competitors, magnetic separation and washing, and the optical signal generation of QDs. The fluorescence intensity was found to be inversely proportional to the MCYST-LR concentration. Under optimized conditions, the proposed assay performed well for the identification and quantitative analysis of MCYST-LR (within 30 min in the range of 0.42-25 μg/L, with a limit of detection of 0.03 μg/L). It is thus expected that this enhanced assay can contribute both to the sensitive and rapid diagnosis of cyanotoxin risk in drinking water and effective management procedures.

  8. Activity assays and immunoassays for plasma Renin and prorenin: information provided and precautions necessary for accurate measurement

    DEFF Research Database (Denmark)

    Campbell, Duncan J; Nussberger, Juerg; Stowasser, Michael

    2009-01-01

    into focus the differences in information provided by activity assays and immunoassays for renin and prorenin measurement and has drawn attention to the need for precautions to ensure their accurate measurement. CONTENT: Renin activity assays and immunoassays provide related but different information...... provided by these assays and of the precautions necessary to ensure their accuracy....

  9. Study on the determination of human placental lactogen (HPL) using an enzyme-immunoassay. Comparison with a commercial radio-immunoassay in the course of normal pregnancies

    International Nuclear Information System (INIS)

    Schneider, B.

    1982-01-01

    A novel enzyme-immunoassay (EIA) for determining human placental lactogen (HPL) was studied for its practicability and quality. The precision of the system in series was tested by using a serum taken each in the 19th, 29th and 40th pregnancy week. A normal range graph between the 10th and the 40th pregnancy week (10 sera per pregnancy week) was established from 310 sera of normal-course pregnancies. The graph practically agreed with the known RIA-established graphs. When comparing with a radio-immunoassay for HPL of routine application and known quality criteria, r=0.93 indicated a close correlation of the values found. (orig./MG) [de

  10. Conversational sensing

    Science.gov (United States)

    Preece, Alun; Gwilliams, Chris; Parizas, Christos; Pizzocaro, Diego; Bakdash, Jonathan Z.; Braines, Dave

    2014-05-01

    Recent developments in sensing technologies, mobile devices and context-aware user interfaces have made it pos- sible to represent information fusion and situational awareness for Intelligence, Surveillance and Reconnaissance (ISR) activities as a conversational process among actors at or near the tactical edges of a network. Motivated by use cases in the domain of Company Intelligence Support Team (CoIST) tasks, this paper presents an approach to information collection, fusion and sense-making based on the use of natural language (NL) and controlled nat- ural language (CNL) to support richer forms of human-machine interaction. The approach uses a conversational protocol to facilitate a ow of collaborative messages from NL to CNL and back again in support of interactions such as: turning eyewitness reports from human observers into actionable information (from both soldier and civilian sources); fusing information from humans and physical sensors (with associated quality metadata); and assisting human analysts to make the best use of available sensing assets in an area of interest (governed by man- agement and security policies). CNL is used as a common formal knowledge representation for both machine and human agents to support reasoning, semantic information fusion and generation of rationale for inferences, in ways that remain transparent to human users. Examples are provided of various alternative styles for user feedback, including NL, CNL and graphical feedback. A pilot experiment with human subjects shows that a prototype conversational agent is able to gather usable CNL information from untrained human subjects.

  11. Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: an anticipated analytical tool for food safety.

    Science.gov (United States)

    Hervás, Miriam; López, Miguel Angel; Escarpa, Alberto

    2009-10-27

    In this work, electrochemical immunoassay involving magnetic beads to determine zearalenone in selected food samples has been developed. The immunoassay scheme has been based on a direct competitive immunoassay method in which antibody-coated magnetic beads were employed as the immobilisation support and horseradish peroxidase (HRP) was used as enzymatic label. Amperometric detection has been achieved through the addition of hydrogen peroxide substrate and hydroquinone as mediator. Analytical performance of the electrochemical immunoassay has been evaluated by analysis of maize certified reference material (CRM) and selected baby food samples. A detection limit (LOD) of 0.011 microg L(-1) and EC(50) 0.079 microg L(-1) were obtained allowing the assessment of the detection of zearalenone mycotoxin. In addition, an excellent accuracy with a high recovery yield ranging between 95 and 108% has been obtained. The analytical features have shown the proposed electrochemical immunoassay to be a very powerful and timely screening tool for the food safety scene.

  12. Remote RemoteRemoteRemote sensing potential for sensing ...

    African Journals Online (AJOL)

    Remote RemoteRemoteRemote sensing potential for sensing potential for sensing potential for sensing potential for sensing potential for sensing potential for sensing potential for sensing potential for sensing potential for sensing potential for sensing p. A Ngie, F Ahmed, K Abutaleb ...

  13. Fluorescent Silica Nanoparticles in the Detection and Control of the Growth of Pathogen

    International Nuclear Information System (INIS)

    Chitra, K.; Annadurai, G.

    2013-01-01

    In this present study the bio conjugated fluorescent silica nanoparticles give an efficient fluorescent-based immunoassay for the detection of pathogen. The synthesized silica nanoparticles were poly dispersed and the size of the silica nanoparticles was in the range of 114-164 nm. The energy dispersive X-ray spectrophotometer showed the presence of silica at 1.8 keV and the selected area diffractometer showed amorphous nature of silica nanoparticles. The FTIR spectrum confirmed the attachment of dye and carboxyl group onto the silica nanoparticles surface. The fluorescent silica nanoparticles showed highly efficient fluorescence and the fluorescent emission of silica nanoparticles occurred at 536 nm. The SEM image showed the aggregation of nanoparticles and bacteria. The growth of the pathogenic E. coli was controlled using silica nanoparticles; therefore silica nanoparticles could be used in food packaging material, biomedical material, and so forth. This work provides a rapid, simple, and accurate method for the detection of pathogen using fluorescent-based immunoassay.

  14. Extracts against Various Pathogens

    Directory of Open Access Journals (Sweden)

    Ritika Chauhan

    2013-07-01

    The present study shows that tested lichen Parmotrema sp. extracts demonstrated a strong antimicrobial effect. That suggests the active components from methanol extracts of the investigated lichen Parmotrema sp. can be used as natural antimicrobial agent against pathogens.

  15. Evolution of microbial pathogens

    National Research Council Canada - National Science Library

    DiRita, Victor J; Seifert, H. Steven

    2006-01-01

    ... A. Hogan vvi ■ CONTENTS 8. Evolution of Pathogens in Soil Rachel Muir and Man-Wah Tan / 131 9. Experimental Models of Symbiotic Host-Microbial Relationships: Understanding the Underpinnings of ...

  16. Indicators for waterborne pathogens

    National Research Council Canada - National Science Library

    Committee on Indicators for Waterborne Pathogens, National Research Council

    2004-01-01

    ... not practical or feasible to monitor for the complete spectrum of microorganisms that may occur in water, and many known pathogens are difficult to detect directly and reliably in water samples.Â...

  17. Host–Pathogen Interactions

    NARCIS (Netherlands)

    Smits, M.A.; Schokker, D.J.

    2011-01-01

    The outcome of an infection is determined by numerous interactions between hosts and pathogens occurring at many different biological levels, ranging from molecule to population. To develop new control strategies for infectious diseases in livestock species, appropriate methodologies are needed

  18. Comparison of enzyme immunoassays for the diagnosis of bovine brucellosis

    International Nuclear Information System (INIS)

    Nielsen, K.; Kelly, L.; Gall, D.; Balsevicius, S.; Bosse, J.; Kelly, W.; Nicoletti, P.

    1998-01-01

    The indirect enzyme immunoassay for measurement of bovine antibody to Brucella abortus was tested on 15,716 Canadian sera to assess the specificity. These sera were also tested by the buffered plate antigen test. Two ELISA formats were used for assessment of data: the targeting procedure using a positive control serum allowed to develop to an optical density of 1.0 and the use of a positive control serum to determine relative positivity at a set time. Two different cut-off values were also assessed for each assay. A total of 763 sera gave reactions above established cut-off values in the ELISA while 216 were positive in the buffered plate antigen test (BPAT). A modification of the indirect ELISA employed divalent cation chelating agents (EDTA/EGTA) incorporated into the serum incubation stage to eliminate some non-specific reactions. This method was applied only to the 763 indirect ELISA reactor sera and it eliminated all but 93 or 37, depending on the cut-off selected, of the reactions. Sensitivity was assessed by testing 424 sera from Brucella abortus culture positive cattle. The indirect ELISA classified all 424 sera as positive by either method of data handling and with or without addition of EDTA/EGTA for a specificity estimate of 100%. In the BPAT, 412 sera gave a positive agglutination reaction. Ten percent of the 15,716 sera were randomly selected and tested by two different competitive ELISAs and by the complement fixation test (CFT). One competitive ELISA used Brucella abortus O-polysaccharide as the antigen and an enzyme conjugated monoclonal antibody to the O-polysaccharide for competition and detection. Of the sera tested, 34 gave false positive reactions. On a retest, the false positive reactions were reduced to 2. The second competitive ELISA used lipopolysaccharide as the antigen, a different monoclonal antibody but also specific for the O-polysaccharide for competition and commercially available goat anti-mouse IgG enzyme conjugate for detection

  19. HORIZON SENSING

    Energy Technology Data Exchange (ETDEWEB)

    Larry G. Stolarczyk

    2003-03-18

    With the aid of a DOE grant (No. DE-FC26-01NT41050), Stolar Research Corporation (Stolar) developed the Horizon Sensor (HS) to distinguish between the different layers of a coal seam. Mounted on mining machine cutter drums, HS units can detect or sense the horizon between the coal seam and the roof and floor rock, providing the opportunity to accurately mine the section of the seam most desired. HS also enables accurate cutting of minimum height if that is the operator's objective. Often when cutting is done out-of-seam, the head-positioning function facilitates a fixed mining height to minimize dilution. With this technology, miners can still be at a remote location, yet cut only the clean coal, resulting in a much more efficient overall process. The objectives of this project were to demonstrate the feasibility of horizon sensing on mining machines and demonstrate that Horizon Sensing can allow coal to be cut cleaner and more efficiently. Stolar's primary goal was to develop the Horizon Sensor (HS) into an enabling technology for full or partial automation or ''agile mining''. This technical innovation (R&D 100 Award Winner) is quickly demonstrating improvements in productivity and miner safety at several prominent coal mines in the United States. In addition, the HS system can enable the cutting of cleaner coal. Stolar has driven the HS program on the philosophy that cutting cleaner coal means burning cleaner coal. The sensor, located inches from the cutting bits, is based upon the physics principles of a Resonant Microstrip Patch Antenna (RMPA). When it is in proximity of the rock-coal interface, the RMPA impedance varies depending on the thickness of uncut coal. The impedance is measured by the computer-controlled electronics and then sent by radio waves to the mining machine. The worker at the machine can read the data via a Graphical User Interface, displaying a color-coded image of the coal being cut, and direct the machine

  20. Radio-immunoassays for glucagon-like peptides 1 and 2 (GLP-1 and GLP-2)

    DEFF Research Database (Denmark)

    Orskov, C; Holst, J J

    1987-01-01

    Gene-sequencing studies have shown that the glucagon precursor contains two additional glucagon-like sequences, the so-called glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). We developed radio-immunoassays against synthetic peptides corresponding to these sequences. Antisera were raised in rabb...

  1. Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder

    NARCIS (Netherlands)

    Haasnoot, W.; Olieman, K.; Cazemier, G.; Verheijen, R.

    2001-01-01

    The low prices of some nonmilk proteins make them attractive as potential adulterants in dairy products. An optical biosensor (BIACORE 3000) was used to develop a direct and combined biosensor immunoassay (BIA) for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powders.

  2. Single biosensor immunoassay for the detection of five aminoglycosides in reconstituted skimmed milk

    NARCIS (Netherlands)

    Haasnoot, W.; Cazemier, G.; Koets, M.; Amerongen, van A.

    2003-01-01

    The application of an optical biosensor (Biacore 3000), with four flow channels (Fcs), in combination with a mixture of four specific antibodies resulted in a competitive inhibition biosensor immunoassay (BIA) for the simultaneous detection of the five relevant aminoglycosides in reconstituted

  3. Multiplex immunoassays for quantification of cytokines, growth factors, and other proteins in stem cell communication

    Czech Academy of Sciences Publication Activity Database

    Valeková, Ivona; Kupcová Skalníková, Helena; Jarkovská, Karla; Motlík, Jan; Kovářová, Hana

    -, č. 1212 (2015), s. 39-63 ISSN 1940 -6029 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : multiplex immunoassays * antibody microarray * luminex xMAP Subject RIV: EB - Genetics ; Molecular Biology

  4. Serum sample containing endogenous antibodies interfering with multiple hormone immunoassays. Laboratory strategies to detect interference

    Directory of Open Access Journals (Sweden)

    Elena García-González

    2016-04-01

    Full Text Available Objectives: Endogenous antibodies (EA may interfere with immunoassays, causing erroneous results for hormone analyses. As (in most cases this interference arises from the assay format and most immunoassays, even from different manufacturers, are constructed in a similar way, it is possible for a single type of EA to interfere with different immunoassays. Here we describe the case of a patient whose serum sample contains EA that interfere several hormones tests. We also discuss the strategies deployed to detect interference. Subjects and methods: Over a period of four years, a 30-year-old man was subjected to a plethora of laboratory and imaging diagnostic procedures as a consequence of elevated hormone results, mainly of pituitary origin, which did not correlate with the overall clinical picture. Results: Once analytical interference was suspected, the best laboratory approaches to investigate it were sample reanalysis on an alternative platform and sample incubation with antibody blocking tubes. Construction of an in-house ‘nonsense’ sandwich assay was also a valuable strategy to confirm interference. In contrast, serial sample dilutions were of no value in our case, while polyethylene glycol (PEG precipitation gave inconclusive results, probably due to the use of inappropriate PEG concentrations for several of the tests assayed. Conclusions: Clinicians and laboratorians must be aware of the drawbacks of immunometric assays, and alert to the possibility of EA interference when results do not fit the clinical pattern. Keywords: Endogenous antibodies, Immunoassay, Interference, Pituitary hormones, Case report

  5. An enzyme immunoassay to quantify neurofilament light chain in cerebrospinal fluid.

    NARCIS (Netherlands)

    Geel, W.J.A. van; Rosengren, L.E.; Verbeek, M.M.

    2005-01-01

    Neurofilament light chain is a component of the axonal cytoskeleton. The concentration of the neurofilament light chain in cerebrospinal fluid may reflect axonal damage or the extent of white matter damage. In this study we describe a sensitive immunoassay for the detection of neurofilament light

  6. Automation on an Open-Access Platform of Alzheimer's Disease Biomarker Immunoassays.

    Science.gov (United States)

    Gille, Benjamin; Dedeene, Lieselot; Stoops, Erik; Demeyer, Leentje; Francois, Cindy; Lefever, Stefanie; De Schaepdryver, Maxim; Brix, Britta; Vandenberghe, Rik; Tournoy, Jos; Vanderstichele, Hugo; Poesen, Koen

    2018-04-01

    The lack of (inter-)laboratory standardization has hampered the application of universal cutoff values for Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarkers and their transfer to general clinical practice. The automation of the AD biomarker immunoassays is suggested to generate more robust results than using manual testing. Open-access platforms will facilitate the integration of automation for novel biomarkers, allowing the introduction of the protein profiling concept. A feasibility study was performed on an automated open-access platform of the commercial immunoassays for the 42-amino-acid isoform of amyloid-β (Aβ 1-42 ), Aβ 1-40 , and total tau in CSF. Automated Aβ 1-42 , Aβ 1-40 , and tau immunoassays were performed within predefined acceptance criteria for bias and imprecision. Similar accuracy was obtained for ready-to-use calibrators as for reconstituted lyophilized kit calibrators. When compared with the addition of a standard curve in each test run, the use of a master calibrator curve, determined before and applied to each batch analysis as the standard curve, yielded an acceptable overall bias of -2.6% and -0.9% for Aβ 1-42 and Aβ 1-40 , respectively, with an imprecision profile of 6.2% and 8.4%, respectively. Our findings show that transfer of commercial manual immunoassays to fully automated open-access platforms is feasible, as it performs according to universal acceptance criteria.

  7. IMMUNOASSAY METHOD FOR THE DETERMINATION OF PENTACHLOROPHENOL IN SOIL AND SEDIMENT

    Science.gov (United States)

    The journal article describes the use of a prototype immunoassay method for the determination of pentacholorphenol (PCP) in soil and sediment. PCP was used as a pesticide and wood preservative and is not currently available to the general public. The paper stresses the importan...

  8. Monoclonal antibody-based broad-specificity immunoassay for monitoring organophosphorus pesticides in environmental water samples

    Science.gov (United States)

    The extensive use of organophosphorus pesticides (OPs) in agriculture and domestic settings can result in widespread water contamination. The development of easy-to-use and rapid-screening immunoassay methods in a class-selective manner is a topic of considerable environmental interest. In this wo...

  9. Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides

    International Nuclear Information System (INIS)

    Krogstad, D.J.; Granich, G.G.; Murray, P.R.; Pfaller, M.A.; Valdes, R.

    1981-01-01

    Heparin interferes with measurement of aminoglycosides in serum by biological, radioenzymatic, and homogeneous enzyme immunoassay techniques, but not with radioimmunoassay. At concentrations greater than or equal to 10 5 and greater than or equal to 3 X 10 6 USP units/L, respectively, it interferes with the radioenzymatic assay by inhibiting the gentamicin 3-acetyltransferase and kanamycin 6'-acetyltransferase enzymes used in the assay. It interferes with the homogeneous enzyme immunoassays for gentamicin and tobramycin (at concentrations greater than or equal to 10 5 and greater than or equal to10 4 USP units/L, respectively), but not with the commercially available homogeneous enzyme immunoassays for other drugs. Heparin interference with the homogeneous enzyme immunoassay for aminoglycosides requires both the heparin polyanion and glucose-6-phosphate dehydrogenase bound to a cationic aminoglycoside. This interference can be reproduced with dextran sulfate (but not dextran), and does not occur with free enzyme (glucose-6-phosphate dehydrogenase) alone. Heparin interference with these two assays and at concentrations that may be present in intravenous infusions or in seriously underfilled blood-collection tubes is described

  10. An ultrasensitive chemiluminescence immunoassay of chloramphenicol based on gold nanoparticles and magnetic beads.

    Science.gov (United States)

    Tao, Xiaoqi; Jiang, Haiyang; Yu, Xuezhi; Zhu, Jinghui; Wang, Xia; Wang, Zhanhui; Niu, Lanlan; Wu, Xiaoping; Shen, Jianzhong

    2013-05-01

    A competitive, direct, chemiluminescent immunoassay based on a magnetic beads (MBs) separation and gold nanoparticles (AuNPs) labelling technique to detect chloramphenicol (CAP) has been developed. Horseradish peroxidase (HRP)-labelled anti-CAP monoclonal antibody conjugated with AuNPs and antigen-immobilized MBs were prepared. After optimization parameters of immunocomplex MBs, the IC50 values of chemiluminescence magnetic nanoparticles immunoassay (CL-MBs-nano-immunoassay) were 0.017 µg L(-1) for extract method I and 0.17 µg L(-1) for extract method II. The immunoassay with two extract methods was applied to detect CAP in milk. Comparison of these two extract methods showed that extract method I was advantageous in better sensitivity, in which the sensitivity was 10 times compared to that of extract method II, while extract method II was superior in simple operation, suitable for high throughout screen. The recoveries were 86.7-98.0% (extract method I) and 80.0-103.0% (extract method II), and the coefficients of variation (CVs) were all recovery with both extract methods and high correlation with traditional ELISA kit in milk system confirmed that the immunomagnetic assay based on AuNPs exhibited promising potential in rapid field screening for trace CAP analysis. Copyright © 2013 John Wiley & Sons, Ltd.

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

    Directory of Open Access Journals (Sweden)

    Jacqueline M. Barnett

    2014-07-01

    Full Text Available 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.

  12. Immunoassay: Principles, development and potential applications in the applied plant sciences

    Energy Technology Data Exchange (ETDEWEB)

    Hofman, P J

    1986-02-01

    The article briefly discusses the general principles of, and the methods involved in, immunoassay, and their development. Emplasis is placed on radioimmunoassay (RIA) and to a lesser extent, enzyme-linked immunosorbent assay (ELISA). The practical applications, with special reference to the citrus and subtropical fruit industries are discussed.

  13. Thermophilic Campylobacter spp. in turkey samples: evaluation of two automated enzyme immunoassays and conventional microbiological techniques

    DEFF Research Database (Denmark)

    Borck, Birgitte; Stryhn, H.; Ersboll, A.K.

    2002-01-01

    Aims: To determine the sensitivity and specificity of two automated enzyme immunoassays (EIA), EiaFoss and Minividas, and a conventional microbiological culture technique for detecting thermophilic Campylobacter spp. in turkey samples. Methods and Results: A total of 286 samples (faecal, meat...

  14. Ervaringen met een solid phase enzyme immunoassay voor het aantonen van gonorroe bij promiscue vrouwen

    NARCIS (Netherlands)

    Ulsen; J.van*; Michel; M.F.*; Strik; R.van*; Joost; T.H.van*; Stolz; E.*; Eijk; R.V.W.van

    1985-01-01

    De Gonozyme test (Abbott Laboratories), een nieuwe enzyme immunoassay (EIA) voor het aantonen van Neisseria gonorrhoeae werd geevalueerd in een grote groep promiscue vrouwen. Als de EIA werd uitgevoerd met materiaal afkomstig van de cervix, bedroeg de prevalentie van gonorroe 8,2%. Vergeleken

  15. Sensitive and rapid immunoassay for parathyroid hormone using magnetic particle labels and magnetic actuation

    NARCIS (Netherlands)

    Dittmer, W.U.; Kievit, de P.; Prins, M.W.J.; Vissers, J.L.M.; Mersch, M.E.C.; Martens, M.F.W.C.

    2008-01-01

    A rapid method for the sensitive detection of proteins using actuated magnetic particle labels, which are measured with a giant magneto-resistive (GMR) biosensor, is described. The technique involves a 1-step sandwich immunoassay with no fluid replacement steps. The various assay binding reactions

  16. A review of promising new immunoassay technology for monitoring forest herbicides

    Science.gov (United States)

    Charles K. McMahon

    1993-01-01

    Rising costs of classical instrumental methods of chemical analysis coupled with an increasing need for environmental monitoring has lead to the development of highly sensitive, low-cost immunochemical methods of analysis for the detection of environmental contaminants. These methods known simply as immunoassays are chemical assays which use antibodies as reagents. A...

  17. False-negative syphilis treponemal enzyme immunoassay results in an HIV-infected case-patient.

    Science.gov (United States)

    Katz, Alan R; Komeya, Alan Y; Tomas, Juval E

    2017-06-01

    We present a case report of a false-negative syphilis treponemal enzyme immunoassay test result in an HIV-infected male. While treponemal tests are widely considered to be more sensitive and specific than non-treponemal tests, our findings point to potential challenges using the reverse sequence syphilis screening algorithm.

  18. Optimized Lateral Flow Immunoassay Reader for the Detection of Infectious Diseases in Developing Countries.

    Science.gov (United States)

    Pilavaki, Evdokia; Demosthenous, Andreas

    2017-11-20

    Detection and control of infectious diseases is a major problem, especially in developing countries. Lateral flow immunoassays can be used with great success for the detection of infectious diseases. However, for the quantification of their results an electronic reader is required. This paper presents an optimized handheld electronic reader for developing countries. It features a potentially low-cost, low-power, battery-operated device with no added optical accessories. The operation of this proof of concept device is based on measuring the reflected light from the lateral flow immunoassay and translating it into the concentration of the specific analyte of interest. Characterization of the surface of the lateral flow immunoassay has been performed in order to accurately model its response to the incident light. Ray trace simulations have been performed to optimize the system and achieve maximum sensitivity by placing all the components in optimum positions. A microcontroller enables all the signal processing to be performed on the device and a Bluetooth module allows transmission of the results wirelessly to a mobile phone app. Its performance has been validated using lateral flow immunoassays with influenza A nucleoprotein in the concentration range of 0.5 ng/mL to 200 ng/mL.

  19. Optimized Lateral Flow Immunoassay Reader for the Detection of Infectious Diseases in Developing Countries

    Directory of Open Access Journals (Sweden)

    Evdokia Pilavaki

    2017-11-01

    Full Text Available Detection and control of infectious diseases is a major problem, especially in developing countries. Lateral flow immunoassays can be used with great success for the detection of infectious diseases. However, for the quantification of their results an electronic reader is required. This paper presents an optimized handheld electronic reader for developing countries. It features a potentially low-cost, low-power, battery-operated device with no added optical accessories. The operation of this proof of concept device is based on measuring the reflected light from the lateral flow immunoassay and translating it into the concentration of the specific analyte of interest. Characterization of the surface of the lateral flow immunoassay has been performed in order to accurately model its response to the incident light. Ray trace simulations have been performed to optimize the system and achieve maximum sensitivity by placing all the components in optimum positions. A microcontroller enables all the signal processing to be performed on the device and a Bluetooth module allows transmission of the results wirelessly to a mobile phone app. Its performance has been validated using lateral flow immunoassays with influenza A nucleoprotein in the concentration range of 0.5 ng/mL to 200 ng/mL.

  20. Electrochemical Methodologies for the Detection of Pathogens.

    Science.gov (United States)

    Amiri, Mandana; Bezaatpour, Abolfazl; Jafari, Hamed; Boukherroub, Rabah; Szunerits, Sabine

    2018-05-25

    electrochemical-based microorganism recognition approaches and putting them into context of other sensing devices for pathogens such as culturing the microorganism on agar plates and the polymer chain reaction (PCR) method, able to identify the DNA of the microorganism. Recent breakthroughs will be highlighted, including the utilization of microfluidic devices and immunomagnetic separation for multiple pathogen analysis in a single device. We will conclude with some perspectives and outlooks to better understand shortcomings. Indeed, there is currently no adequate solution that allows the selective and sensitive binding to a specific microorganism, that is fast in detection and screening, cheap to implement, and able to be conceptualized for a wide range of biologically relevant targets.

  1. Post-translational Control of Intracellular Pathogen Sensing Pathways.

    Science.gov (United States)

    Chiang, Cindy; Gack, Michaela U

    2017-01-01

    Mammalian cells recognize virus-derived nucleic acids using a defined set of intracellular sensors including the DNA sensors cyclic GMP-AMP (cGAMP) synthase (cGAS) and interferon gamma (IFNγ)-inducible protein 16 (IFI16) as well as viral RNA receptors of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family. Following innate immune recognition, these sensors launch an immune response that is characterized by the transcriptional upregulation of many antiviral molecules, including proinflammatory cytokines, chemokines, and IFN-stimulated genes. Recent studies have demonstrated that the signal transduction initiated by these sensors is sophisticatedly regulated by post-translational modifications (PTMs) resulting in a robust yet 'tunable' cytokine response to maintain immune homeostasis. Here we summarize recent advances in our understanding of how PTMs and regulatory enzymes control the signaling activity of RLRs, cGAS, and IFI16 as well as their proximal adaptor proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora

    OpenAIRE

    Sjöblom, Solveig

    2009-01-01

    Erwinia carotovora subsp. carotovora (Ecc) is a Gram-negative enterobacterium that causes soft-rot in potato and other crops. The main virulence determinants, the extracellular plant cell wall -degrading enzymes (PCWDEs), lead to plant tissue maceration. In order to establish a successful infection the production of PCWDEs are controlled by a complex regulatory network, including both specific and global activators and repressors. One of the most important virulence regulation systems in Ecc ...

  3. Probiotic Modulation of Innate Cell Pathogen Sensing and Signaling Events

    Directory of Open Access Journals (Sweden)

    Amy Llewellyn

    2017-10-01

    Full Text Available There is a growing body of evidence documenting probiotic bacteria to have a beneficial effect to the host through their ability to modulate the mucosal immune system. Many probiotic bacteria can be considered to act as either immune activators or immune suppressors, which have appreciable influence on homeostasis, inflammatory- and suppressive-immunopathology. What is becoming apparent is the ability of these probiotics to modulate innate immune responses via direct or indirect effects on the signaling pathways that drive these activatory or suppressive/tolerogenic mechanisms. This review will focus on the immunomodulatory role of probiotics on signaling pathways in innate immune cells: from positive to negative regulation associated with innate immune cells driving gut mucosal functionality. Research investigations have shown probiotics to modulate innate functionality in many ways including, receptor antagonism, receptor expression, binding to and expression of adaptor proteins, expression of negative regulatory signal molecules, induction of micro-RNAs, endotoxin tolerisation and finally, the secretion of immunomodulatory proteins, lipids and metabolites. The detailed understanding of the immunomodulatory signaling effects of probiotic strains will facilitate strain-specific selective manipulation of innate cell signal mechanisms in the modulation of mucosal adjuvanticity, immune deviation and tolerisation in both healthy subjects and patients with inflammatory and suppressive pathology.

  4. Probiotic Modulation of Innate Cell Pathogen Sensing and Signaling Events

    Science.gov (United States)

    Llewellyn, Amy; Foey, Andrew

    2017-01-01

    There is a growing body of evidence documenting probiotic bacteria to have a beneficial effect to the host through their ability to modulate the mucosal immune system. Many probiotic bacteria can be considered to act as either immune activators or immune suppressors, which have appreciable influence on homeostasis, inflammatory- and suppressive-immunopathology. What is becoming apparent is the ability of these probiotics to modulate innate immune responses via direct or indirect effects on the signaling pathways that drive these activatory or suppressive/tolerogenic mechanisms. This review will focus on the immunomodulatory role of probiotics on signaling pathways in innate immune cells: from positive to negative regulation associated with innate immune cells driving gut mucosal functionality. Research investigations have shown probiotics to modulate innate functionality in many ways including, receptor antagonism, receptor expression, binding to and expression of adaptor proteins, expression of negative regulatory signal molecules, induction of micro-RNAs, endotoxin tolerisation and finally, the secretion of immunomodulatory proteins, lipids and metabolites. The detailed understanding of the immunomodulatory signaling effects of probiotic strains will facilitate strain-specific selective manipulation of innate cell signal mechanisms in the modulation of mucosal adjuvanticity, immune deviation and tolerisation in both healthy subjects and patients with inflammatory and suppressive pathology. PMID:29065562

  5. Viral pathogen discovery

    Science.gov (United States)

    Chiu, Charles Y

    2015-01-01

    Viral pathogen discovery is of critical importance to clinical microbiology, infectious diseases, and public health. Genomic approaches for pathogen discovery, including consensus polymerase chain reaction (PCR), microarrays, and unbiased next-generation sequencing (NGS), have the capacity to comprehensively identify novel microbes present in clinical samples. Although numerous challenges remain to be addressed, including the bioinformatics analysis and interpretation of large datasets, these technologies have been successful in rapidly identifying emerging outbreak threats, screening vaccines and other biological products for microbial contamination, and discovering novel viruses associated with both acute and chronic illnesses. Downstream studies such as genome assembly, epidemiologic screening, and a culture system or animal model of infection are necessary to establish an association of a candidate pathogen with disease. PMID:23725672

  6. Intelligent Chiral Sensing Based on Supramolecular and Interfacial Concepts

    Directory of Open Access Journals (Sweden)

    Hironori Izawa

    2010-07-01

    Full Text Available Of the known intelligently-operating systems, the majority can undoubtedly be classed as being of biological origin. One of the notable differences between biological and artificial systems is the important fact that biological materials consist mostly of chiral molecules. While most biochemical processes routinely discriminate chiral molecules, differentiation between chiral molecules in artificial systems is currently one of the challenging subjects in the field of molecular recognition. Therefore, one of the important challenges for intelligent man-made sensors is to prepare a sensing system that can discriminate chiral molecules. Because intermolecular interactions and detection at surfaces are respectively parts of supramolecular chemistry and interfacial science, chiral sensing based on supramolecular and interfacial concepts is a significant topic. In this review, we briefly summarize recent advances in these fields, including supramolecular hosts for color detection on chiral sensing, indicator-displacement assays, kinetic resolution in supramolecular reactions with analyses by mass spectrometry, use of chiral shape-defined polymers, such as dynamic helical polymers, molecular imprinting, thin films on surfaces of devices such as QCM, functional electrodes, FET, and SPR, the combined technique of magnetic resonance imaging and immunoassay, and chiral detection using scanning tunneling microscopy and cantilever technology. In addition, we will discuss novel concepts in recent research including the use of achiral reagents for chiral sensing with NMR, and mechanical control of chiral sensing. The importance of integration of chiral sensing systems with rapidly developing nanotechnology and nanomaterials is also emphasized.

  7. Highly pathogenic avian influenza.

    Science.gov (United States)

    Swayne, D E; Suarez, D L

    2000-08-01

    Highly pathogenic (HP) avian influenza (AI) (HPAI) is an extremely contagious, multi-organ systemic disease of poultry leading to high mortality, and caused by some H5 and H7 subtypes of type A influenza virus, family Orthomyxoviridae. However, most AI virus strains are mildly pathogenic (MP) and produce either subclinical infections or respiratory and/or reproductive diseases in a variety of domestic and wild bird species. Highly pathogenic avian influenza is a List A disease of the Office International des Epizooties, while MPAI is neither a List A nor List B disease. Eighteen outbreaks of HPAI have been documented since the identification of AI virus as the cause of fowl plague in 1955. Mildly pathogenic avian influenza viruses are maintained in wild aquatic bird reservoirs, occasionally crossing over to domestic poultry and causing outbreaks of mild disease. Highly pathogenic avian influenza viruses do not have a recognised wild bird reservoir, but can occasionally be isolated from wild birds during outbreaks in domestic poultry. Highly pathogenic avian influenza viruses have been documented to arise from MPAI viruses through mutations in the haemagglutinin surface protein. Prevention of exposure to the virus and eradication are the accepted methods for dealing with HPAI. Control programmes, which imply allowing a low incidence of infection, are not an acceptable method for managing HPAI, but have been used during some outbreaks of MPAI. The components of a strategy to deal with MPAI or HPAI include surveillance and diagnosis, biosecurity, education, quarantine and depopulation. Vaccination has been used in some control and eradication programmes for AI.

  8. Plasmonic sensing

    DEFF Research Database (Denmark)

    Mogensen, Klaus Bo

    2015-01-01

    Plasmonic sensors typically rely on detection of changes in the refractive index of the surrounding medium. Here, an alternative approach is reported based on electrical surface screening and controlled dissolution of ultrasmall silver nanoparticles (NPs; R ... in the plasmon band. This is demonstrated by using the strong nucleophiles, cyanide and cysteamine, as ligands. The “dissolution paths” in terms of peak wavelength and amplitude shifts differ significantly between different types of analytes, which are suggested as a means to obtain selectivity of the detection...... that cannot be obtained by traditional refractive index sensing, without the use of bioprobes. A simple modified Drude model is used to account for shifts in the plasmon band position due to electrical charging. Here, a screening parameter is introduced in the expression for the free electron density...

  9. False biochemical diagnosis of hyperthyroidism in streptavidin-biotin-based immunoassays: the problem of biotin intake and related interferences.

    Science.gov (United States)

    Piketty, Marie-Liesse; Polak, Michel; Flechtner, Isabelle; Gonzales-Briceño, Laura; Souberbielle, Jean-Claude

    2017-05-01

    Immunoassays are now commonly used for hormone measurement, in high throughput analytical platforms. Immunoassays are generally robust to interference. However, endogenous analytical error may occur in some patients; this may be encountered in biotin supplementation or in the presence of anti-streptavidin antibody, in immunoassays involving streptavidin-biotin interaction. In these cases, the interference may induce both false positive and false negative results, and simulate a seemingly coherent hormonal profile. It is to be feared that this type of errors will be more frequently observed. This review underlines the importance of keeping close interactions between biologists and clinicians to be able to correlate the hormonal assay results with the clinical picture.

  10. Determination of avian influenza A (H9N2) virions by inductively coupled plasma mass spectrometry based magnetic immunoassay with gold nanoparticles labeling

    Science.gov (United States)

    Xiao, Guangyang; Chen, Beibei; He, Man; Shi, Kaiwen; Zhang, Xing; Li, Xiaoting; Wu, Qiumei; Pang, Daiwen; Hu, Bin

    2017-12-01

    Avian influenza viruses are the pathogens of global poultry epidemics, and may even cause the human infections. Here, we proposed a novel inductively coupled plasma mass spectrometry (ICP-MS) based immunoassay with gold nanoparticles (Au NPs) labeling for the determination of H9N2 virions. Magnetic-beads modified with anti-influenza A H9N2 hemagglutinin mono-antibody (mAb-HA) were utilized for the capture of H9N2 virions in complex matrix; and Au NPs conjugated with mAb-HA were employed for the specific labeling of H9N2 virions for subsequent ICP-MS detection. With a sandwich immunoassay strategy, this method exhibited a high specificity for H9N2 among other influenza A virions such as H1N1 and H3N2. Under the optimized conditions, this method could detect as low as 0.63 ng mL- 1 H9N2 virions with the linear range of 2-400 ng mL- 1, the relative standard deviation for seven replicate detections of H9N2 virions was 7.2% (c = 10 ng mL- 1). The developed method was applied for the detection of H9N2 virions in real-world chicken dung samples, and the recovery for the spiking samples was 91.4-116.9%. This method is simple, rapid, sensitive, selective, reliable and has a good application potential for virions detection in real-world samples.

  11. Pemanfaatan Antibodi Monoklonal dalam Immunoassay untuk Deteksi Baculovirus oryctes

    Directory of Open Access Journals (Sweden)

    Susamto Somowiyarjo

    2000-12-01

    Full Text Available The application of non-precoated Indirect Enzyme-Linked Immunosorbent Assay (ELISA and Dot Immunobinding Assay (DIBA employing monoclonal antibodies (MCA against Yogyakarta isolate of Baculovirus oryctes Huger. was described. The MCA-Bv-4 having subclass of IgG2a and titer in vitro of 10^4 - 10^5 proved to be useful antibody for virus detection. The great potential of I-ELISA using MCA-Bv-4 has been it's specificity being able to discriminate between healthy an virus-infected coconut beetle (Oryctes rhinoceros L.. The assay could also differentiate between B. oryctes and Monodon baculovirus, the pathogen of shrimp disease. The best tissue for preparing virus antigen from crude extract was found to be the midgut of the beetle. Head, thorax, abdomen and tibia were not suitable for preparing the test antigen. The crude extract of beetle showed high endogenous enzyme activity to the substrate of DIBA, which precluded the detection of B. oryctes using DIBA. The MCA-Bv-4 could be used to improve the monitoring of the virus to support the program of biological control of coconut beetle using B. oryctes.

  12. Simultaneous detection of IgG antibodies associated with viral hemorrhagic fever by a multiplexed Luminex-based immunoassay.

    Science.gov (United States)

    Wu, Wei; Zhang, Shuo; Qu, Jing; Zhang, Quanfu; Li, Chuan; Li, Jiandong; Jin, Cong; Liang, Mifang; Li, Dexin

    2014-07-17

    Viral hemorrhagic fevers (VHFs) are worldwide diseases caused by several kinds of viruses. With the emergence of new viruses, advanced diagnostic methods are urgently needed for identification of VHFs. Based on Luminex xMAP technology, a rapid, sensitive, multi-pathogen and high-throughput method which could simultaneously detect hemorrhagic fever viruses (HFVs) specific IgG antibodies was developed. Recombinant antigens of nine HFVs including Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), Andes virus (ANDV), Sin Nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) and dengue virus (DENV) were produced and purified from a prokaryotic expression system and the influence of the coupling amount was investigated. Cross-reactions among antigens and their rabbit immune sera were evaluated. Serum samples collected from 51 laboratory confirmed hemorrhagic fever with renal syndrome (HFRS) patients, 43 confirmed SFTS patients and 88 healthy donors were analyzed. Results showed that recombinant nucleocapsid protein of the five viruses belonging to the genus Hantavirus, had serological cross-reactivity with their corresponding rabbit immune sera, but not apparent with immune sera of other four viruses. Evaluation of this new method with clinical serum samples showed 98.04% diagnostic sensitivity for HFRS, 90.70% for SFTS detection and the specificity was ranging from 66.67% to 100.00%. The multiplexed Luminex-based immunoassay has firstly been established in our study, which provides a potentially reliable diagnostic tool for IgG antibody detection of VHFs. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Development of improved enzyme-based and lateral flow immunoassays for rapid and accurate serodiagnosis of canine brucellosis.

    Science.gov (United States)

    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.

  14. Human pathogen avoidance adaptations

    NARCIS (Netherlands)

    Tybur, J.M.; Lieberman, D.

    2016-01-01

    Over the past few decades, researchers have become increasingly interested in the adaptations guiding the avoidance of disease-causing organisms. Here we discuss the latest developments in this area, including a recently developed information-processing model of the adaptations underlying pathogen

  15. Optical Properties and Immunoassay Applications of Noble Metal Nanoparticles

    International Nuclear Information System (INIS)

    Zhu, S.; Zhou, W.

    2010-01-01

    Noble metal, especially gold (Au) and silver (Ag) nanoparticles exhibit unique and tunable optical properties on account of their surface plasmon resonance (SPR). In this paper, we mainly discussed the theory background of the enhanced optical properties of noble metal nanoparticles. Mie theory, transfer matrix method, discrete dipole approximation (DDA) method, and finite-difference time domain (FDTD) method applied brute-force computational methods for different nanoparticles optical properties. Some important nanostructure fabrication technologies such as nanosphere lithography (NSL) and focused ion beam (FIB) are also introduced in this paper. Moreover, these fabricated nanostructures are used in the plasmonic sensing fields. The binding signal between the antibody and antigen, amyloid-derived diffusible ligands (ADDLs)-potential Alzheimer's disease (AD) biomarkers, and staphylococcal enterotoxin B (SEB) in nano-Moore per liter (nM) concentration level are detected by our designed nanobiosensor. They have many potential applications in the biosensor, environment protection, food security, and medicine safety for health, and so forth, fields.

  16. Optical Properties and Immunoassay Applications of Noble Metal Nanoparticles

    Directory of Open Access Journals (Sweden)

    Shaoli Zhu

    2010-01-01

    Full Text Available Noble metal, especially gold (Au and silver (Ag nanoparticles exhibit unique and tunable optical properties on account of their surface plasmon resonance (SPR. In this paper, we mainly discussed the theory background of the enhanced optical properties of noble metal nanoparticles. Mie theory, transfer matrix method, discrete dipole approximation (DDA method, and finite-difference time domain (FDTD method applied brute-force computational methods for different nanoparticles optical properties. Some important nanostructure fabrication technologies such as nanosphere lithography (NSL and focused ion beam (FIB are also introduced in this paper. Moreover, these fabricated nanostructures are used in the plasmonic sensing fields. The binding signal between the antibody and antigen, amyloid-derived diffusible ligands (ADDLs-potential Alzheimer's disease (AD biomarkers, and staphylococcal enterotixn B (SEB in nano-Moore per liter (nM concentration level are detected by our designed nanobiosensor. They have many potential applications in the biosensor, environment protection, food security, and medicine safety for health, and so forth, fields.

  17. Autophagy in plant pathogenic fungi.

    Science.gov (United States)

    Liu, Xiao-Hong; Xu, Fei; Snyder, John Hugh; Shi, Huan-Bin; Lu, Jian-Ping; Lin, Fu-Cheng

    2016-09-01

    Autophagy is a conserved cellular process that degrades cytoplasmic constituents in vacuoles. Plant pathogenic fungi develop special infection structures and/or secrete a range of enzymes to invade their plant hosts. It has been demonstrated that monitoring autophagy processes can be extremely useful in visualizing the sequence of events leading to pathogenicity of plant pathogenic fungi. In this review, we introduce the molecular mechanisms involved in autophagy. In addition, we explore the relationship between autophagy and pathogenicity in plant pathogenic fungi. Finally, we discuss the various experimental strategies available for use in the study of autophagy in plant pathogenic fungi. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. In vitro conditions modify immunoassayability of bovine pituitary prolactin and growth hormone: insights into their secretory granule storage forms

    International Nuclear Information System (INIS)

    Lorenson, M.Y.

    1985-01-01

    The amount of immunoassayable intracellular bovine (b) PRL and GH varies depending on treatment conditions. The present studies were designed to characterize the mechanisms involved and to compare immunoassayability of both hormones under similar conditions. Pituitary homogenate and secretory granule hormones displayed both time- and temperature-dependent increases when incubated at pH 10.5 with reduced glutathione. Changes in immunoassayability seem to reflect conversion from poorly immunoactive tissue hormone oligomers to monomeric hormone. The data indicate that oligomeric bPRL is stabilized primarily by intermolecular disulfide bonds, although it is also susceptible to urea, SDS, and EDTA; granule thiols may also influence the conversion to monomer. The storage form of bGH appears to be stabilized differently. Maneuvers demonstrated in these studies to influence immunoassayability correlate very well with their previously established effects on hormone release and secretion, strengthening the likelihood that a functional link exists between assayability and secretion

  19. DEMONSTRATION BULLETIN: PCP IMMUNOASSAY TECHNOLOGIES - PENTA RISC BY ENSYS INC., PENTA RAPID BY OHMICRON CORP., ENVIROGARD BY MILLIPORE

    Science.gov (United States)

    The objectives of this demonstration were to test these field screening technologies for accuracy and precision in detecting Pentachlorophenol (PCP) levels in soil and water by comparing their results with those of a confirmatory laboratory. The three immunoassay technologies ...

  20. IFSA: a microfluidic chip-platform for frit-based immunoassay protocols

    Science.gov (United States)

    Hlawatsch, Nadine; Bangert, Michael; Miethe, Peter; Becker, Holger; Gärtner, Claudia

    2013-03-01

    Point-of-care diagnostics (POC) is one of the key application fields for lab-on-a-chip devices. While in recent years much of the work has concentrated on integrating complex molecular diagnostic assays onto a microfluidic device, there is a need to also put comparatively simple immunoassay-type protocols on a microfluidic platform. In this paper, we present the development of a microfluidic cartridge using an immunofiltration approach. In this method, the sandwich immunoassay takes place in a porous frit on which the antibodies have immobilized. The device is designed to be able to handle three samples in parallel and up to four analytical targets per sample. In order to meet the critical cost targets for the diagnostic market, the microfluidic chip has been designed and manufactured using high-volume manufacturing technologies in mind. Validation experiments show comparable sensitivities in comparison with conventional immunofiltration kits.

  1. A competitive chemiluminescence enzyme immunoassay for rapid and sensitive determination of enrofloxacin

    Science.gov (United States)

    Yu, Fei; Wu, Yongjun; Yu, Songcheng; Zhang, Huili; Zhang, Hongquan; Qu, Lingbo; Harrington, Peter de B.

    With alkaline phosphatase (ALP)-adamantane (AMPPD) system as the chemiluminescence (CL) detection system, a highly sensitive, specific and simple competitive chemiluminescence enzyme immunoassay (CLEIA) was developed for the measurement of enrofloxacin (ENR). The physicochemical parameters, such as the chemiluminescent assay mediums, the dilution buffer of ENR-McAb, the volume of dilution buffer, the monoclonal antibody concentration, the incubation time, and other relevant variables of the immunoassay have been optimized. Under the optimal conditions, the detection linear range of 350-1000 pg/mL and the detection limit of 0.24 ng/mL were provided by the proposed method. The relative standard deviations were less than 15% for both intra and inter-assay precision. This method has been successfully applied to determine ENR in spiked samples with the recovery of 103%-96%. It showed that CLEIA was a good potential method in the analysis of residues of veterinary drugs after treatment of related diseases.

  2. A sandwich immunoassay for human prolyl 4-hydroxylase using monoclonal antibody

    International Nuclear Information System (INIS)

    Yoshida, Shinichi

    1986-01-01

    Monoclonal antibody was used in a sandwich enzyme immunoassay and in a radioimmunoassay for human serum immunoreactive prolyl 4-hydroxylase. The enzyme immunoassay utilized a monoclonal antibody as a solid phase and horseradish peroxidase-labeled rabbit antibody to human prolyl 4-hydroxylase as a conjugate. Sensitivity was 0.1 ng of enzyme per tube. With a conjugate purified by an enzyme-bound affinity column, sensitivity was increased to 0.01 ng per tube, and linearity was obtained between 0.01 to 30 ng per tube. The radioimmunoassay used a 125 I-labeled rabbit antibody (IgG) as the conjugate. Sensitivity of this technique was 0.4 ng of enzyme per tube. (Auth.)

  3. On-chip signal amplification of magnetic bead-based immunoassay by aviating magnetic bead chains.

    Science.gov (United States)

    Jalal, Uddin M; Jin, Gyeong Jun; Eom, Kyu Shik; Kim, Min Ho; Shim, Joon S

    2017-11-06

    In this work, a Lab-on-a-Chip (LOC) platform is used to electromagnetically actuate magnetic bead chains for an enhanced immunoassay. Custom-made electromagnets generate a magnetic field to form, rotate, lift and lower the magnetic bead chains (MBCs). The cost-effective, disposable LOC platform was made with a polymer substrate and an on-chip electrochemical sensor patterned via the screen-printing process. The movement of the MBCs is controlled to improve the electrochemical signal up to 230% when detecting beta-type human chorionic gonadotropin (β-hCG). Thus, the proposed on-chip MBC-based immunoassay is applicable for rapid, qualitative electrochemical point-of-care (POC) analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Comparative studies on the determination of alphafetoprotein by enzyme immunoassay and by radioimmunoassay

    International Nuclear Information System (INIS)

    Haller, G.; Linneke, P.; Voss, P.; Jeske, W.

    1987-01-01

    Alphafetoprotein (AFP) was determined in serum of pregnant women in the tenth till sixteenth week of pregnancy by means of two enzyme immunoassays (Enzymun-Test AFP, Boehringer Mannheim, FRG and AFP EIA 'Dessau' 1000, Research Institute for Vaccine Dessau, GDR) and a radioimmunoassay (Radioimmunoassay Kit, AFP-PR, CIS, France). Parallel determinations in sera of 438 patients, who had come to surveillance for the first consultation were estimated. A comparison between the methods showed a good correlation. (author)

  5. Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals

    International Nuclear Information System (INIS)

    Lattanzio, Veronica M.T.; Nivarlet, Noan; Lippolis, Vincenzo; Gatta, Stefania Della; Huet, Anne-Catherine; Delahaut, Philippe; Granier, Benoit; Visconti, Angelo

    2012-01-01

    Highlights: ► We developed a rapid method based on a multiplex dipstick immunoassay. ► The assay allowed the determination of major Fusarium toxins in wheat, oats, maize. ► We obtained cut off levels close to EU regulatory levels. - Abstract: A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin–BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73–109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 μg kg −1 , respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 μg kg −1 , respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC–MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.

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

    Directory of Open Access Journals (Sweden)

    Raymond L Houghton

    2014-03-01

    Full Text Available 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.

  7. Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals

    Energy Technology Data Exchange (ETDEWEB)

    Lattanzio, Veronica M.T., E-mail: veronica.lattanzio@ispa.cnr.it [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy); Nivarlet, Noan [UNISENSOR S.A., Zoning industriel du Dossay, Rue du Dossay no 3, B-4020 Liege (Belgium); Lippolis, Vincenzo; Gatta, Stefania Della [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy); Huet, Anne-Catherine; Delahaut, Philippe [Centre d' Economie Rurale (CER Groupe), Rue du Point du Jour no 8, B-6900 Marloie (Belgium); Granier, Benoit [UNISENSOR S.A., Zoning industriel du Dossay, Rue du Dossay no 3, B-4020 Liege (Belgium); Visconti, Angelo [National Research Council of Italy, Institute of Sciences of Food Production (ISPA-CNR), Via Amendola 122/O, 70126 Bari (Italy)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer We developed a rapid method based on a multiplex dipstick immunoassay. Black-Right-Pointing-Pointer The assay allowed the determination of major Fusarium toxins in wheat, oats, maize. Black-Right-Pointing-Pointer We obtained cut off levels close to EU regulatory levels. - Abstract: A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin-BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73-109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 {mu}g kg{sup -1}, respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 {mu}g kg{sup -1}, respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC-MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins

  8. In-electrode vs. on-electrode: ultrasensitive Faraday cage-type electrochemiluminescence immunoassay.

    Science.gov (United States)

    Guo, Zhiyong; Sha, Yuhong; Hu, Yufang; Wang, Sui

    2016-03-28

    A new-concept of an "in-electrode" Faraday cage-type electrochemiluminescence immunoassay (ECLIA) method for the ultrasensitive detection of neurotensin (NT) was reported with capture antibody (Ab1)-nanoFe3O4@graphene (GO) and detector antibody (Ab2)&N-(4-aminobutyl)-N-ethylisoluminol (ABEI)@GO, which led to about 1000-fold improvement in sensitivity by extending the Helmholtz plane (OHP) of the proposed electrode assembly effectively.

  9. Competitive enzyme immunoassay for human chorionic somatomammotropin using the avidin-biotin system

    International Nuclear Information System (INIS)

    Rappuoli, R.; Leoncini, P.; Tarli, P.; Neri, P.

    1981-01-01

    Human chorionic somatomammotropin (HCS) is determined by an enzyme immunoassay where HCS competes with biotin-labeled HCS for insolubilized anti-HCS antibodies. Enzyme-labeled avidin is then used to reveal the amount of bound HCS. The system proves to be sensitive (1 ng/ml of HCS can be detected) and results agree with radioimmunoassay determinations (correlation coefficient = 0.979). Kinetics of the avidin-biotin reaction and coating of polystyrene wells are also investigated

  10. Evaluation of automated enzyme immunoassays for five anticonvulsants and theophylline adapted to a centrifugal analyzer.

    Science.gov (United States)

    Urquhart, N; Godolphin, W; Campbell, D J

    1979-05-01

    We report a clinical evaluation of the enzyme immunoassay (EMIT) performed with the GEMSAEC centrifugal analyzer as compared to gas-liquid and liquid chromatography for anticonvulsant drugs and theophylline, respectively. A good correlation was obtained for all drugs, although some difficulties were experienced with one lot of reagent for ethosuximide. The analyzer has an economic advantage if many samples are being analyzed for few drugs in each sample.

  11. Deoxynivalenol-mimic nanobody isolated from a naïve phage display nanobody library and its application in immunoassay.

    Science.gov (United States)

    Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Bhunia, Arun K; Tu, Zhui; Chen, Bo; Liu, Yuan-Yuan

    2015-08-05

    In this study, using mycotoxin deoxynivalenol (DON) as a model hapten, we developed a nanobody-based environmental friendly immunoassay for sensitive detection of DON. Two nanobodies (N-28 and N-31) which bind to anti-DON monoclonal antibody (MAb) were isolated from a naive phage display library. These nanobodies are clonable, thermally stable and mycotoxin-free products and can be served as coating antigen mimetics in heterologous immunoassay. The half inhibition concentration (IC50) of the immunoassay developed with N-28 and N-31 was 8.77 ± 0.41 ng mL(-1) and 19.97 ± 0.84 ng mL(-1), respectively, which were 18- and 8-fold more sensitive than the conventional coating antigen (DON-BSA) based immunoassay. In order to better understand the molecular mechanism of antigen mimicry by nanobody, the 3D structure of "nanobody (N-28) - anti-DON MAb" complex was presented and verified by molecular modeling and alanine-scanning mutagenesis. The results showed that hydrogen bond and hydrophobic interaction formed between Thr 102 - Ser 106 of N-28 and CDR H3 residues of anti-DON antibody may contribute to their binding. This novel concept of enhancing sensitivity of immunoassay for DON based on nanobody may provide potential applications in a general method for immunoassay of various food chemical contaminants. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. A fast universal immobilization of immunoglobulin G at 4 °C for the development of array-based immunoassays.

    Directory of Open Access Journals (Sweden)

    Shu-Lin Guo

    Full Text Available To maintain the antibody activity and enhance performance of array-based immunoassays, protein G was used to allow a shorter duration of immunoglobulin G immobilization at 4 °C, with the antibody placed in the appropriate orientation. The multiplexed detection of six pain-related message molecules (PRMMs was used as examples for the development of array-based immunoassays: substance P, calcitonin gene-related peptide, nerve growth factor, brain-derived neurotrophic factor, tumor necrosis factor-α, and β-endorphin. Protein G- and non-protein G-coated slides were tested. Compared to non-protein G immunoassays, protein G shortened the antibody immobilization time at 4 °C from overnight to 2 hours. Only protein G-facilitated immunoassays succeeded in simultaneously detecting all six PRMMs with high specificity. Dose-response curves showed that the limits of detection of the protein G-multiplexed immunoassays for the PRMMs was approximately 164, 167, 120, 60, 80, and 92 pg/ml, respectively. Thus, protein G effectively shortens the duration of antibody immobilization at 4 °C, allowing the use of sensitive array-based immunoassays for the simultaneous detection of PRMMs.

  13. Evolution of microbial pathogens.

    OpenAIRE

    Morschhäuser, J; Köhler, G; Ziebuhr, W; Blum-Oehler, G; Dobrindt, U; Hacker, J

    2000-01-01

    Various genetic mechanisms including point mutations, genetic rearrangements and lateral gene transfer processes contribute to the evolution of microbes. Long-term processes leading to the development of new species or subspecies are termed macroevolution, and short-term developments, which occur during days or weeks, are considered as microevolution. Both processes, macro- and microevolution need horizontal gene transfer, which is particularly important for the development of pathogenic micr...

  14. Diagnostic effectiveness of immunoassays systems for hepatitis C virus in samples from multi-transfusion patients

    International Nuclear Information System (INIS)

    Rivero Jimenez, Rene A; Merlin Linares, Julio C; Blanco de Armas, Madelin; Navea Leyva, Leonor M

    2009-01-01

    Hepatitis C virus (CHV) blood-transmission is a health problem in Cuba and in the world. Some types of diagnostic immunoassays have been developed for the blood certification and in general have a high diagnostic sensitivity and specificity in healthy donors. However, its behavior in samples from multi-transfusion patients could by less effective. To assess the diagnostic effectiveness of the UMELISA HCV third generation Cuban immunoassay (TecnoSUMA, S.A. La Habana), Cuba) in samples from multi-transfusion patients, in parallel, 335 sera from patients were processed by UBI HCV EIA 4.0 (United Biomedical, EE.UU) and UMELISA HCV third generation, and the samples with incongruous results were verified by PCR COBAS AmpliScreen HCV Test, v2 system (Roche, EE.UU.) Comparing the UMELISA HCV third generation system with the UBI HCV EIA 4.0 it was achieved a Sd of 95,8% CI(95%): 92,5-99,15 and a Ed of 100% CI (95%): 99,7-100, with IY: 0,96 (0,93-0,99) with k: 0,0582 ID (95%): 0,9276-0,9888, p = 0,000. Both immunoassay systems were satisfactory for immunodiagnosis of multi-transfusion patients

  15. Detection of alpha-fetoprotein in magnetic immunoassay of thin channels using biofunctional nanoparticles

    Science.gov (United States)

    Tsai, H. Y.; Gao, B. Z.; Yang, S. F.; Li, C. S.; Fuh, C. Bor

    2014-01-01

    This paper presents the use of fluorescent biofunctional nanoparticles (10-30 nm) to detect alpha-fetoprotein (AFP) in a thin-channel magnetic immunoassay. We used an AFP model biomarker and s-shaped deposition zones to test the proposed detection method. The results show that the detection using fluorescent biofunctional nanoparticle has a higher throughput than that of functional microparticle used in previous experiments on affinity reactions. The proposed method takes about 3 min (versus 150 min of previous method) to detect 100 samples. The proposed method is useful for screening biomarkers in clinical applications, and can reduce the run time for sandwich immunoassays to less than 20 min. The detection limits (0.06 pg/ml) and linear ranges (0.068 pg/ml-0.68 ng/ml) of AFP using fluorescent biofunctional nanoparticles are the same as those of using functional microparticles within experimental errors. This detection limit is substantially lower and the linear range is considerably wider than those of enzyme-linked immunosorbent assay (ELISA) and other methods in sandwich immunoassay methods. The differences between this method and an ELISA in AFP measurements of serum samples were less than 12 %. The proposed method provides simple, fast, and sensitive detection with a high throughput for biomarkers.

  16. Targeted selected reaction monitoring mass spectrometric immunoassay for insulin-like growth factor 1.

    Directory of Open Access Journals (Sweden)

    Eric E Niederkofler

    Full Text Available Insulin-like growth factor 1 (IGF1 is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM mode. The resulting quantitative mass spectrometric immunoassay (MSIA exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories.

  17. Magnetofluorescent nanocomposites and quantum dots used for optimal application in magnetic fluorescence-linked immunoassay.

    Science.gov (United States)

    Tsai, H Y; Li, S Y; Fuh, C Bor

    2018-03-01

    Magnetofluorescent nanocomposites with optimal magnetic and fluorescent properties were prepared and characterized by combining magnetic nanoparticles (iron oxide@polymethyl methacrylate) with fluorescent nanoparticles (rhodamine 6G@mSiO 2 ). Experimental parameters were optimized to produce nanocomposites with high magnetic susceptibility and fluorescence intensity. The detection of a model biomarker (alpha-fetoprotein) was used to demonstrate the feasibility of applying the magnetofluorescent nanocomposites combined with quantum dots and using magnetic fluorescence-linked immunoassay. The magnetofluorescent nanocomposites enable efficient mixing, fast re-concentration, and nanoparticle quantization for optimal reactions. Biofunctional quantum dots were used to confirm the alpha-fetoprotein (AFP) content in sandwich immunoassay after mixing and washing. The analysis time was only one third that required in ELISA. The detection limit was 0.2 pg mL -1 , and the linear range was 0.68 pg mL -1 -6.8 ng mL -1 . This detection limit is lower, and the linear range is wider than those of ELISA and other methods. The measurements made using the proposed method differed by less than 13% from those obtained using ELISA for four AFP concentrations (0.03, 0.15, 0.75, and 3.75 ng mL -1 ). The proposed method has a considerable potential for biomarker detection in various analytical and biomedical applications. Graphical abstract Magnetofluorescent nanocomposites combined with fluorescent quantum dots were used in magnetic fluorescence-linked immunoassay.

  18. Evaluation of field test kits including immunoassays for the detection of contaminants in soil and water

    International Nuclear Information System (INIS)

    Waters, L.C.; Smith, R.R.; Counts, R.W.; Stewart, J.H.; Jenkins, R.A.

    1993-01-01

    Effective field test methods are needed for hazardous waste site characterization and remediation. Useful field methods should be rapid, analyte-specific, cost-effective and accurate in the concentration range at which the analyte is regulated. In this study, field test kits for polychlorinated biphenyls (PCBs), mercury, lead and nitrate were evaluated with reference to these criteria. PCBs and mercury, in soils, were analyzed by immunoassay. Ionic lead and nitrate, in water, were measured chemically using test strips. Except for lead, each analyte was measured in both spiked and actual field samples. Twenty to 40 samples per day can be analyzed with the immunoassays and even more with the strip tests. The sensitivity of the immunoassays is in the 1-3 ppM range. Nitrate was consistently detected at ≥5 ppM; lead ions at ≥20 ppM. Results obtained using these methods compared favorably with those obtained by standard laboratory methods. In addition to being useful field screening methods, these kits can be used in the laboratory to sort out negative samples and/or to define proper dilutions for positive samples requiring further analysis

  19. Comparison of pre-processing methods for multiplex bead-based immunoassays.

    Science.gov (United States)

    Rausch, Tanja K; Schillert, Arne; Ziegler, Andreas; Lüking, Angelika; Zucht, Hans-Dieter; Schulz-Knappe, Peter

    2016-08-11

    High throughput protein expression studies can be performed using bead-based protein immunoassays, such as the Luminex® xMAP® technology. Technical variability is inherent to these experiments and may lead to systematic bias and reduced power. To reduce technical variability, data pre-processing is performed. However, no recommendations exist for the pre-processing of Luminex® xMAP® data. We compared 37 different data pre-processing combinations of transformation and normalization methods in 42 samples on 384 analytes obtained from a multiplex immunoassay based on the Luminex® xMAP® technology. We evaluated the performance of each pre-processing approach with 6 different performance criteria. Three performance criteria were plots. All plots were evaluated by 15 independent and blinded readers. Four different combinations of transformation and normalization methods performed well as pre-processing procedure for this bead-based protein immunoassay. The following combinations of transformation and normalization were suitable for pre-processing Luminex® xMAP® data in this study: weighted Box-Cox followed by quantile or robust spline normalization (rsn), asinh transformation followed by loess normalization and Box-Cox followed by rsn.

  20. Development and analytical performance of a new ARCHITECT automated dipeptidyl peptidase-4 immunoassay

    Directory of Open Access Journals (Sweden)

    Philip M. Hemken

    2017-12-01

    Full Text Available Background: Dipeptidyl peptidase-4 (DPP-4 may be a suitable biomarker to identify people with severe asthma who have greater activation of the interleukin-13 (IL-13 pathway and may therefore benefit from IL-13-targeted treatments. We report the analytical performance of an Investigational Use Only immunoassay and provide data on the biological range of DPP-4 concentrations. Methods: We assessed assay performance, utilising analyses of precision, linearity and sensitivity; interference from common endogenous assay interferents, and from asthma and anti-diabetic medications, were also assessed. The assay was used to measure the range of serum DPP-4 concentrations in healthy volunteers and subjects with diabetes and severe, uncontrolled asthma. Results: The total precision of DPP-4 concentration measurement (determined using percentage coefficient of variation was ≤5% over 20 days. Dilution analysis yielded linear results from 30 to 1305 ng/mL; the limit of quantitation was 19.2 ng/mL. No notable endogenous or drug interferences were observed at the expected therapeutic concentration. Median DPP-4 concentrations in healthy volunteers and subjects with asthma or Type 1 diabetes were assessed, with concentrations remaining similar in subjects with diabetes and asthma across different demographics. Conclusion: These analyses indicate that the ARCHITECT DPP-4 Immunoassay is a reliable and robust method for measuring serum DPP-4 concentration. Keywords: Asthma, Automated immunoassay, Biomarker, Dipeptidyl peptidase-4, IL-13

  1. Which amphetamine-type stimulants can be detected by oral fluid immunoassays?

    Science.gov (United States)

    Souza, Daniele Z; Boehl, Paula O; Comiran, Eloisa; Prusch, Débora S; Zancanaro, Ivomar; Fuentefria, Alexandre M; Pechansky, Flavio; Duarte, Paulina C A V; De Boni, Raquel B; Fröehlich, Pedro E; Limberger, Renata P

    2012-02-01

    The use of oral fluid for monitoring drug consumption on roads has many advantages over conventional biological fluids; therefore, several immunoassays have been developed for this purpose. In this work, the ability of 3 commercial immunoassays to detect amphetamine-type stimulants (ATSs) in oral fluid was assessed. In addition, it was reviewed the main controlled ATSs available worldwide, as well as the oral fluid immunological screening tests that have been used for identifying ATSs in drivers. The analytical specificity of amphetamine direct enzyme-linked immunosorbent assay (ELISA), methamphetamine direct ELISA (Immunalysis Corporation), and Oral-View saliva multidrug of abuse test (Alfa Scientific Designs) was evaluated using ATS-spiked oral fluid. Legislation and published articles that report the use of immunological screening tests to detect ATS consumption in conductors were reviewed, including the kit's technical information, project reports, police and drug databases. Even at high concentrations, the tested assays were not able to detect methylphenidate, fenproporex, or diethylpropion, controlled ATSs legally marketed in many countries. This evidences the need to develop new kits that enable one to control the misuse of prescription ATSs on roads through oral fluid immunoassays.

  2. Factitious Graves' Disease Due to Biotin Immunoassay Interference-A Case and Review of the Literature.

    Science.gov (United States)

    Elston, Marianne S; Sehgal, Shekhar; Du Toit, Stephen; Yarndley, Tania; Conaglen, John V

    2016-09-01

    Biotin (vitamin B7) is an essential co-factor for four carboxylases involved in fatty acid metabolism, leucine degradation, and gluconeogenesis. The recommended daily intake (RDI) of biotin is approximately 30 μg per day. Low-moderate dose biotin is a common component of multivitamin preparations, and high-dose biotin (10 000 times RDI) has been reported to improve clinical outcomes and quality of life in patients with progressive multiple sclerosis. Biotin is also a component of immunoassays, and supplementation may cause interference in both thyroid and non-thyroid immunoassays. To assess whether biotin ingestion caused abnormal thyroid function tests (TFTs) in a patient through assay interference. We report a patient with biotin-associated abnormal TFTs and a systematic review of the literature. A tertiary endocrine service in Hamilton, New Zealand. The patient had markedly abnormal TFTs that did not match the clinical context. After biotin cessation, TFTs normalized far more rapidly than possible given the half-life of T4, consistent with assay interference by biotin. Multiple other analytes also tested abnormal in the presence of biotin. Biotin ingested in moderate to high doses can cause immunoassay interference. Depending on the assay format, biotin interference can result in either falsely high or low values. Interference is not limited to thyroid tests and has the potential to affect a wide range of analytes. It is important for clinicians to be aware of this interaction to prevent misdiagnosis and inappropriate treatment.

  3. Discordant Analytical Results Caused by Biotin Interference on Diagnostic Immunoassays in a Pediatric Hospital.

    Science.gov (United States)

    Ali, Mahesheema; Rajapakshe, Deepthi; Cao, Liyun; Devaraj, Sridevi

    2017-09-01

    Recent studies have reported that biotin interferes with certain immunoassays. In this study, we evaluated the analytical interference of biotin on immunoassays that use streptavidin-biotin in our pediatric hospital. We tested the effect of different concentrations of biotin (1.5-200 ng/ml) on TSH, Prolactin, Ferritin, CK-MB, β-hCG, Troponin I, LH, FSH, Cortisol, Anti-HAV antibody (IgG and IgM), assays on Ortho Clinical Diagnostic Vitros 5600 Analyzer. Biotin (up to 200 ng/mL) did not significantly affect Troponin I and HAV assays. Biotin (up to 12.5 ng/ml) resulted in biotin >6.25 ng/mL significantly affected TSH (>20% bias) assay. Prolactin was significantly affected even at low levels (Biotin 1.5 ng/mL). Thus, we recommend educating physicians about biotin interference in common immunoassays and adding an electronic disclaimer. © 2017 by the Association of Clinical Scientists, Inc.

  4. Chemiluminescence enzyme immunoassay based on magnetic nanoparticles for detection of hepatocellular carcinoma marker glypican-3

    Directory of Open Access Journals (Sweden)

    Qian-Yun Zhang

    2011-08-01

    Full Text Available Glypican-3 (GPC3 is reported as a great promising tumor marker for hepatocellular carcinoma (HCC diagnosis. Highly sensitive and accurate analysis of serum GPC3 (sGPC3, in combination with or instead of traditional HCC marker alpha-fetoprotein (AFP, is essential for early diagnosis of HCC. Biomaterial-functionalized magnetic particles have been utilized as solid supports with good biological compatibility for sensitive immunoassay. Here, the magnetic nanoparticles (MnPs and magnetic microparticles (MmPs with carboxyl groups were further modified with streptavidin, and applied for the development of chemiluminescence enzyme immunoassay (CLEIA. After comparing between MnPs- and MmPs-based CLEIA, MnPs-based CLEIA was proved to be a better method with less assay time, greater sensitivity, better linearity and longer chemiluminescence platform. MnPs-based CLEIA was applied for detection of sGPC3 in normal liver, hepatocirrhosis, secondary liver cancer and HCC serum samples. The results indicated that sGPC3 was effective in diagnosis of HCC with high performance. Keywords: Magnetic nanoparticle, Magnetic microparticle, Chemiluminescence enzyme immunoassay, Glypican-3, Hepatocellular carcinoma

  5. A Novel Colorimetric Immunoassay Utilizing the Peroxidase Mimicking Activity of Magnetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Hyun Gyu Park

    2013-05-01

    Full Text Available A simple colorimetric immunoassay system, based on the peroxidase mimicking activity of Fe3O4 magnetic nanoparticles (MNPs, has been developed to detect clinically important antigenic molecules. MNPs with ca. 10 nm in diameter were synthesized and conjugated with specific antibodies against target molecules, such as rotaviruses and breast cancer cells. Conjugation of the MNPs with antibodies (MNP-Abs enabled specific recognition of the corresponding target antigenic molecules through the generation of color signals arising from the colorimetric reaction between the selected peroxidase substrate, 3,3',5,5'-tetramethylbenzidine (TMB and H2O2. Based on the MNP-promoted colorimetric reaction, the target molecules were detected and quantified by measuring absorbance intensities corresponding to the oxidized form of TMB. Owing to the higher stabilities and economic feasibilities of MNPs as compared to horseradish peroxidase (HRP, the new colorimetric system employing MNP-Abs has the potential of serving as a potent immunoassay that should substitute for conventional HRP-based immunoassays. The strategy employed to develop the new methodology has the potential of being extended to the construction of simple diagnostic systems for a variety of biomolecules related to human cancers and infectious diseases, particularly in the realm of point-of-care applications.

  6. Rapid Methods for the Detection of Foodborne Bacterial Pathogens: Principles, Applications, Advantages and Limitations

    Directory of Open Access Journals (Sweden)

    Law eJodi Woan-Fei

    2015-01-01

    Full Text Available The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR, multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA, loop-mediated isothermal amplification (LAMP and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases.

  7. Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: An anticipated analytical tool for food safety

    International Nuclear Information System (INIS)

    Hervas, Miriam; Lopez, Miguel Angel; Escarpa, Alberto

    2009-01-01

    In this work, electrochemical immunoassay involving magnetic beads to determine zearalenone in selected food samples has been developed. The immunoassay scheme has been based on a direct competitive immunoassay method in which antibody-coated magnetic beads were employed as the immobilisation support and horseradish peroxidase (HRP) was used as enzymatic label. Amperometric detection has been achieved through the addition of hydrogen peroxide substrate and hydroquinone as mediator. Analytical performance of the electrochemical immunoassay has been evaluated by analysis of maize certified reference material (CRM) and selected baby food samples. A detection limit (LOD) of 0.011 μg L -1 and EC 50 0.079 μg L -1 were obtained allowing the assessment of the detection of zearalenone mycotoxin. In addition, an excellent accuracy with a high recovery yield ranging between 95 and 108% has been obtained. The analytical features have shown the proposed electrochemical immunoassay to be a very powerful and timely screening tool for the food safety scene.

  8. Immunoassay for Capsular Antigen of Bacillus anthracis Enables Rapid Diagnosis in a Rabbit Model of Inhalational Anthrax.

    Directory of Open Access Journals (Sweden)

    Marcellene A Gates-Hollingsworth

    Full Text Available Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the study was to evaluate detection of poly-γ-D-glutamic acid (PGA, the capsular antigen of B. anthracis, as a biomarker surrogate for blood culture in a rabbit model of inhalational anthrax. The mean time to a positive blood culture was 26 ± 5.7 h (mean ± standard deviation, whereas the mean time to a positive ELISA was 22 ± 4.2 h; P = 0.005 in comparison with blood culture. A lateral flow immunoassay was constructed for detection of PGA in plasma at concentrations of less than 1 ng PGA/ml. Use of the lateral flow immunoassay for detection of PGA in the rabbit model found that antigen was detected somewhat earlier than the earliest time point at which the blood culture became positive. The low cost, ease of use, and rapid time to result of the lateral flow immunoassay format make an immunoassay for PGA a viable surrogate for blood culture for detection of infection in individuals who have a likelihood of exposure to B. anthracis.

  9. The management of isolated positive syphilis enzyme immunoassay results in HIV-negative patients attending a sexual health clinic.

    Science.gov (United States)

    Thorley, Nicola; Adebayo, Michael; Smit, Erasmus; Radcliffe, Keith

    2016-08-01

    An unconfirmed positive treponemal enzyme immunoassay (enzyme immunoassay positive, Treponema pallidum particle agglutination negative and rapid plasma reagin negative) presents a clinical challenge to distinguish early syphilis infection from false-positive results. These cases are referred for syphilis line assay (INNO-LIA) and recalled for repeat syphilis serology. We performed a retrospective audit to establish the proportion of HIV-negative cases with unconfirmed positive enzyme immunoassay results, the proportion of these cases that received an INNO-LIA test and repeat syphilis serology testing and reviewed the clinical outcomes; 0.35% (80/22687) cases had an unconfirmed positive treponemal enzyme immunoassay result. Repeat syphilis serology was performed in 80% (64/80) cases, but no additional cases of syphilis were identified. Eighty-eight per cent (70/80) received an INNO-LIA test; 14% (5/37) unconfirmed enzyme immunoassay-positive cases with no prior history of syphilis were confirmed on INNO-LIA assay, supporting a diagnosis of latent syphilis. As a confirmatory treponemal test, the INNO-LIA assay may be more useful than repeat syphilis serological testing. © The Author(s) 2016.

  10. Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: An anticipated analytical tool for food safety

    Energy Technology Data Exchange (ETDEWEB)

    Hervas, Miriam; Lopez, Miguel Angel [Departamento Quimica Analitica, Universidad de Alcala, Ctra. Madrid-Barcelona, Km. 33600, E-28871 Alcala de Henares, Madrid (Spain); Escarpa, Alberto, E-mail: alberto.escarpa@uah.es [Departamento Quimica Analitica, Universidad de Alcala, Ctra. Madrid-Barcelona, Km. 33600, E-28871 Alcala de Henares, Madrid (Spain)

    2009-10-27

    In this work, electrochemical immunoassay involving magnetic beads to determine zearalenone in selected food samples has been developed. The immunoassay scheme has been based on a direct competitive immunoassay method in which antibody-coated magnetic beads were employed as the immobilisation support and horseradish peroxidase (HRP) was used as enzymatic label. Amperometric detection has been achieved through the addition of hydrogen peroxide substrate and hydroquinone as mediator. Analytical performance of the electrochemical immunoassay has been evaluated by analysis of maize certified reference material (CRM) and selected baby food samples. A detection limit (LOD) of 0.011 {mu}g L{sup -1} and EC{sub 50} 0.079 {mu}g L{sup -1} were obtained allowing the assessment of the detection of zearalenone mycotoxin. In addition, an excellent accuracy with a high recovery yield ranging between 95 and 108% has been obtained. The analytical features have shown the proposed electrochemical immunoassay to be a very powerful and timely screening tool for the food safety scene.

  11. False-positive ethyl glucuronide immunoassay screening caused by a propyl alcohol-based hand sanitizer.

    Science.gov (United States)

    Arndt, Torsten; Grüner, Joachim; Schröfel, Stefanie; Stemmerich, Karsten

    2012-11-30

    Urine ethyl glucuronide (EtG) is considered as a specific marker of recent ethanol consumption. We describe false-positive DRI(®) EIA EtG enzyme immunoassay results caused by propyl glucuronides in urine after using a propanol-based hand sanitizer. EtG screening was done with the DRI(®) EIA EtG assay (Microgenics), using a cut-off of 0.5 mg/L as recommended by the manufacturer and of 0.1 mg/L as demanded by the German Regulations for Reissuing Drivers Licenses. Confirmatory EtG analysis was done with the ClinMass(®) EtG LC-MS/MS testkit (Recipe), extended by the mass transitions 235.1→75.1, 235.1→85.1, and 235.1→113.1 for the detection of the 1- and 2-propyl glucuronides. Self-experiments were done by staff members of our lab (n=7), using 3 mL Sterillium(®) Classic Pure (30 g/100 g 1-propanol and 45 g/100 g 2-propanol) for hand sanitation every quarter of an hour for 8 h according to DIN EN 1500:2011-05 with and without an exhauster and by passive inhalation of the sanitizer vapor. Spot urine samples were taken immediately before and up to 24 h after the first sanitizer use. False-positive immunoassay results of up to 4 mg/L or 2.3 mg/g creatinine were obtained after normal use of the sanitizer and also after passive inhalation of the sanitizer vapor (up to 0.89 mg/L or 0.61 mg/g). Immunoassay results were positive even after 4-fold use of the sanitizer (up to 0.14 mg/L or 0.38 mg/g) and up to 6 h after the last sanitizer contact (maximum 0.63 mg/L and 0.33 mg/g for sanitizer users and 0.25 mg/g after passive inhalation). Spiking of EtG-free urine with 1-propyl glucuronide (Athena Environmental Sciences) between 0.05 and 10 mg/L clearly demonstrated a cross reaction of the immunoassay of approx. 10% as compared to EtG. LC-MS/MS of urines with a positive immunoassay EtG result did not show EtG signals, but distinct signals of 1-propyl glucuronide (n-propyl glucuronide) and 2-propyl glucuronide (iso-propyl glucuronide). An exhauster effectively prevented

  12. Development of nanobody-based flow injection chemiluminescence immunoassay for sensitive detection of human prealbumin.

    Science.gov (United States)

    Ma, Lei; Sun, Yanyan; Kang, Xuejun; Wan, Yakun

    2014-11-15

    Nanobodies, derived from camelid heavy-chain antibodies, have novel and impactful applications in clinical diagnostics. Our objective is to develop a nanobody-based chemiluminescence immunoassay for sensitive detection of human prealbumin (PA). In this context, a phage display nanobody library is constructed via immunizing dromedary camel with human prealbumin. Three nanobodies have been identified by five successive bio-panning steps. Based on their high expression level and good affinity, two out of three are chosen for further study. Magnetic beads (MBs) were functionalized with PEI by acylamide bond formed between the carboxyl group on the surface of the MB. Then, an anti-PA nanobody (Nb1) can be effectively immobilized onto the surface of the functionalized MB using glutaradehyde as the link. The modified MBs with Nb1 can specifically capture the target PA and reacted with silica nanoparticles with co-immobilized HRP and anti-PA nanobody (Nb2). The concentration of PA was detected by flow injection chemiluminescence. When using MB/PEI as the carrier of anti-PA Nb1, the CL signal significantly increased to 4-fold compared with the signal using MB without PEI modification. The CL signal was further amplified to 5-fold when Si/Nb2 was used as the signal probe. Under optimized conditions, the present immunoassay exhibited a wide quantitative range from 0.05 to 1000 μg L(-1) with a detection limit of 0.01 μg L(-1). The sensitivity of the proposed immunoassay offers great promises in providing a sensitive, specific, time saving, and potential method for detecting PA in clinical settings. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Structural basis for the high specificity of a Trypanosoma congolense immunoassay targeting glycosomal aldolase.

    Directory of Open Access Journals (Sweden)

    Joar Pinto

    2017-09-01

    Full Text Available Animal African trypanosomosis (AAT is a neglected tropical disease which imposes a heavy burden on the livestock industry in Sub-Saharan Africa. Its causative agents are Trypanosoma parasites, with T. congolense and T. vivax being responsible for the majority of the cases. Recently, we identified a Nanobody (Nb474 that was employed to develop a homologous sandwich ELISA targeting T. congolense fructose-1,6-bisphosphate aldolase (TcoALD. Despite the high sequence identity between trypanosomatid aldolases, the Nb474-based immunoassay is highly specific for T. congolense detection. The results presented in this paper yield insights into the molecular principles underlying the assay's high specificity.The structure of the Nb474-TcoALD complex was determined via X-ray crystallography. Together with analytical gel filtration, the structure reveals that a single TcoALD tetramer contains four binding sites for Nb474. Through a comparison with the crystal structures of two other trypanosomatid aldolases, TcoALD residues Ala77 and Leu106 were identified as hot spots for specificity. Via ELISA and surface plasmon resonance (SPR, we demonstrate that mutation of these residues does not abolish TcoALD recognition by Nb474, but does lead to a lack of detection in the Nb474-based homologous sandwich immunoassay.The results show that the high specificity of the Nb474-based immunoassay is not determined by the initial recognition event between Nb474 and TcoALD, but rather by its homologous sandwich design. This (i provides insights into the optimal set-up of the assay, (ii may be of great significance for field applications as it could explain the potential detection escape of certain T. congolense strains, and (iii may be of general interest to those developing similar assays.

  14. Comparison of Immunoassay methods for T3, T4 and TSH

    International Nuclear Information System (INIS)

    Alonso Rodríguez, Celia A.

    2016-01-01

    Measurements of T3, T4 and TSH have been considered very important in the diagnosis and monitoring of thyroid diseases both overt and subclinical. These subclinical diseases are actively seeking for years, both in healthy patients and hospitalized for other illnesses; and in the population over 35 years, especially women, in health checkups. The active search for these diseases requires the use of rapid and reliable techniques; that can be developed massively, with good level of detectability and comparable. The overall objective is to present the evaluation of different immunoassay techniques with respect to the RIA and IRMA: ELISA, chemiluminescence, Amplified Chemiluminescence, electrochemiluminescence Immunofluorescence. Compare including automatic methods and analyze the cost and feasibility of them for laboratory immunoassay. ELISA colorimetric technique for dosing was comparable to RIA T4, not for T3. Chemiluminescence (AMERLITE) compared to dosing RIA and IRMA T4 to TSH proved to be valid for both. Amplified Chemiluminescence (Immulite) compared to IRMA for TSH was no significant difference. Electrochemiluminescence (Elecsys 2010) compared to T3 and T4 RIA and IRMA for TSH, no significant differences for T4 and TSH; but no variation to T3. Immunofluorescence (AIA-600) used to compare with RIA for T3 and T4, and TSH IRMA, no significant differences for the measured analytes. Benchmarking of automatic methods suggests that the most thrifty of trials is Immunofluorescence the AIA-600, regarding calibration and control, programming time, randomization and the ability to save the value of the fluorescence deferred calculations for tests without valid at the time of realizing calibration. Analyzing the cost and feasibility of these methods for laboratory immunoassay, we must consider that their characteristics electrochemiluminescence is the fastest, but its price is prohibitive for our health systems. The AIA-600 appears to be the method of choice for its

  15. A competitive immunoassay for sensitive detection of small molecules chloramphenicol based on luminol functionalized silver nanoprobe.

    Science.gov (United States)

    Yu, Xiuxia; He, Yi; Jiang, Jie; Cui, Hua

    2014-02-17

    Chloramphenicol (CHL) as a broad-spectrum antibiotic has a broad action spectrum against Gram-positive and Gram-negative bacteria, as well as anaerobes. The use of CHL is strictly restricted in poultry because of its toxic effect. However, CHL is still illegally used in animal farming because of its accessibility and low cost. Therefore, sensitive methods are highly desired for the determination of CHL in foodstuffs. The immunoassays based on labeling as an important tool have been reported for the detection of CHL residues in food-producing animals. However, most of the labeling procedures require multi-step reactions and purifications and thus they are complicated and time-consuming. Recently, in our previous work, luminol functionalized silver nanoparticles have been successfully synthesized, which exhibits higher CL efficiency than luminol functionalized gold nanoparticles. In this work, the new luminol functionalized silver nanoparticles have been used for the labeling of small molecules CHL for the first time and a competitive chemiluminescent immunoassay has been developed for the detection of CHL. Owing to the amplification of silver nanoparticles, high sensitivity for CHL could be achieved with a low detection limit of 7.6×10(-9) g mL(-1) and a wide linear dynamic range of 1.0×10(-8)-1.0×10(-6) g mL(-1). This method has also been successfully applied to determine CHL in milk and honey samples with a good recoveries (92% and 102%, 99% and 107% respectively), indicating that the method is feasible for the determination of CHL in real milk and honey samples. The labeling procedure is simple, convenient and fast, superior to previously reported labeling procedures. The immunoassay is also simple, fast, sensitive and selective. It is of application potential for the determination of CHL in foodstuffs. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Accuracy of immunoassay and mass spectrometry urinary free cortisol in the diagnosis of Cushing's syndrome.

    Science.gov (United States)

    Aranda, G; Careaga, M; Hanzu, F A; Patrascioiu, I; Ríos, P; Mora, M; Morales-Romero, B; Jiménez, W; Halperin, I; Casals, G

    2016-10-01

    Urinary free cortisol (UFC) determination by highly specific methods as mass spectrometry instead of commercially available antibody-based immunoassays is increasingly recommended. However, clinical comparisons of both analytical approaches in the screening of Cushing's syndrome (CS) are not available. The aim of this study was to evaluate the diagnostic value of mass spectrometry versus immunoassay measurements of 24 h-UFC in the screening of CS. Cross-sectional study of 33 histologically confirmed CS patients: 25 Cushing's disease, 5 adrenal CS and 3 ectopic CS; 92 non-CS patients; and 35 healthy controls. UFC by immunoassay (UFCxIA) and mass spectrometry (UFCxMS), urinary free cortisone (UFCo) and UFC:UFCo ratio were measured, together with creatinine-corrected values. Sensitivity, specificity, AUC and Landis and Koch concordance index were determined. AUC for UFCxIA and UFCxMS were 0.77 (CI 0.68-0.87) and 0.77 (CI 0.67-0.87) respectively, with a kappa coefficient 0.60 and strong Landis and Koch concordance index. The best calculated cutoff values were 359 nmol/24 h for UFCxIA (78 % sensitivity, 62 % specificity) and 258.1 nmol/24 h for UCFxMS (53 % sensitivity, 86 % specificity). The upper limit of UFCxIA and UCFxMS reference ranges were 344.7 and 169.5 nmol/24 h respectively. Sensitivity and specificity for CS diagnosis at these cutpoints were 84 and 56 % for UFCxIA and 81 and 54 % for UFCxMS. According to our data, both methods present a very similar diagnostic value. However, results suggest that lower cutoff points for mass spectrometry may be necessary in order to improve clinical sensitivity.

  17. Design of novel hybrid organic-inorganic nanostructured biomaterials for immunoassay applications

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, G [Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, PO Box 486, 31270.901, Belo Horizonte, MG (Brazil); Barbosa-Stancioli, E F [Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, PO Box 486, 31270.901, Belo Horizonte, MG (Brazil); Piscitelli Mansur, A A [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil); Vasconcelos, W L [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil); Mansur, H S [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil)

    2006-12-01

    The purpose of this study was to develop novel hybrid organic-inorganic materials based on poly(vinyl alcohol) (PVA) polymer chemically crosslinked network to be tested as solid support on bovine herpesvirus immunoassay. Hybrids were synthesized by reacting PVA with three different alkoxysilanes modifying chemical groups: tetraethoxysilane (TEOS), 3-mercaptopropyltrimethoxysilane (MPTMS) and 3-glycidoxypropyltrimethoxysilane (GPTMS). PVA-derived hybrids were also modified by chemically crosslinking with glutaraldehyde (GA) during the synthesis reaction. In order to investigate the structure in the nanometer-scale, PVA-derived hybrids were characterized by using small-angle x-ray scattering synchrotron radiation (SAXS) and x-ray diffraction (XRD). PVA hybrids' chemical functionalities and their interaction with herpesviruses were also characterized by Fourier transform infrared spectroscopy (FTIR). The bioactivity assays were tested through enzyme linked immunosorbent assay (ELISA). SAXS results have indicated nano-ordered disperse domains for PVA hybrids with different x-ray scattering patterns for PVA polymer and PVA-derived hybrids. FTIR spectra have shown major vibration bands associated with organic-inorganic chemical groups present in the PVA, PVA-derived by silane modifier and PVA chemically crosslinked by GA. The immunoassay results have shown that PVA hybrids with chemically functionalized structures regulated to some extent the specific bioimmobilization of herpesvirus onto solid phase. We think that it is due to the overall balance of forces associated with van der Waals interaction, hydrophilic and hydrophobic forces and steric hindrance acting at the surface. PVA and PVA-derived hybrid materials were successfully produced with GA crosslinking in a nanometer-scale network. Also, such a PVA-based material could be advantageously used in immunoassays with enhanced specificity for diagnosis.

  18. Evaluation of the Lumipulse G TP-N Chemiluminescent Immunoassay as a Syphilis Screening Test.

    Science.gov (United States)

    Ortiz, Daniel A; Loeffelholz, Michael J

    2017-11-01

    A syphilis diagnosis is often aided by the detection of treponemal and nontreponemal antibodies. Automated treponemal antibody detection systems enable high-volume clinical laboratories to perform syphilis screening at a faster pace with lower labor costs. The Lumipulse G TP-N chemiluminescent immunoassay is an automated system that qualitatively detects IgG and IgM antibodies against Treponema pallidum antigens in human serum and plasma. To assess performance characteristics and workflow efficiency, the Lumipulse G TP-N assay was compared to the Bioplex 2200 Syphilis IgG multiplex flow immunoassay. Among the 4,134 routine and HIV samples tested by the two automated assays, the percentage of agreement was excellent at 99.0% (95% confidence interval [CI], 98.6% to 99.2%; κ, 0.89), with the Lumipulse G TP-N having a shorter time to first and subsequent results. All specimens with reactive syphilis screening results were further tested by rapid plasma reagin (RPR) and Treponema pallidum particle agglutination (TP·PA) testing ( n = 231). The results from the RPR-reactive samples ( n = 82) showed complete concordance with the two automated assays, while the TP·PA assay displayed some discrepancies. The positive percent agreement (PPA) and negative percent agreement (NPA) between the TP·PA test and the Lumipulse G TP-N test were 98.9% and 77.3%, respectively. The Bioplex 2200 Syphilis IgG immunoassay displayed a similar PPA (100%) but a substantially lower NPA (15.9%). Patient chart reviews of discrepant results suggested that the Lumipulse G TP-N assay produced 27 fewer falsely reactive results and can reduce the amount of additional confirmatory RPR and TP·PA testing needed. The analogous performance characteristics of the two automated systems indicate that the Lumipulse G TP-N assay is suitable for high-throughput syphilis screening. Copyright © 2017 American Society for Microbiology.

  19. Evaluation of Beckman Coulter DxI 800 immunoassay system using clinically oriented performance goals.

    Science.gov (United States)

    Akbas, Neval; Schryver, Patricia G; Algeciras-Schimnich, Alicia; Baumann, Nikola A; Block, Darci R; Budd, Jeffrey R; Gaston, S J Stephen; Klee, George G

    2014-11-01

    We evaluated the analytical performance of 24 immunoassays using the Beckman Coulter DxI 800 immunoassay systems at Mayo Clinic, Rochester, MN for trueness, precision, detection limits, linearity, and consistency (across instruments and reagent lots). Clinically oriented performance goals were defined using the following methods: trueness-published desirable accuracy limits, precision-published desirable biologic variation; detection limits - 0.1 percentile of patient test values, linearity - 50% of total error, and consistency-percentage test values crossing key decision points. Local data were collected for precision, linearity, and consistency. Data were provided by Beckman Coulter, Inc. for trueness and detection limits. All evaluated assays except total thyroxine were within the proposed goals for trueness. Most of the assays met the proposed goals for precision (86% of intra-assay results and 75% of inter-assay results). Five assays had more than 15% of the test results below the minimum detection limits. Carcinoembryonic antigen, total thyroxine and free triiodothyronine exceeded the proposed goals of ±6.3%, ±5% and ±5.7% for dilution linearity. All evaluated assays were within the proposed goals for instrument consistency. Lot-to-lot consistency results for cortisol, ferritin and total thyroxine exceeded the proposed goals of 3.3%, 11.4% and 7% at one medical decision level, while vitamin B12 exceeded the proposed goals of 5.2% and 3.8% at two decision levels. The Beckman Coulter DxI 800 immunoassay system meets most of these proposed goals, even though these clinically focused performance goals represent relatively stringent limits. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  20. Design of novel hybrid organic-inorganic nanostructured biomaterials for immunoassay applications

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, G [Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, PO Box 486, 31270.901, Belo Horizonte, MG (Brazil); Barbosa-Stancioli, E F [Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, PO Box 486, 31270.901, Belo Horizonte, MG (Brazil); Piscitelli Mansur, A A [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil); Vasconcelos, W L [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil); Mansur, H S [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil)

    2006-12-01

    The purpose of this study was to develop novel hybrid organic-inorganic materials based on poly(vinyl alcohol) (PVA) polymer chemically crosslinked network to be tested as solid support on bovine herpesvirus immunoassay. Hybrids were synthesized by reacting PVA with three different alkoxysilanes modifying chemical groups: tetraethoxysilane (TEOS), 3-mercaptopropyltrimethoxysilane (MPTMS) and 3-glycidoxypropyltrimethoxysilane (GPTMS). PVA-derived hybrids were also modified by chemically crosslinking with glutaraldehyde (GA) during the synthesis reaction. In order to investigate the structure in the nanometer-scale, PVA-derived hybrids were characterized by using small-angle x-ray scattering synchrotron radiation (SAXS) and x-ray diffraction (XRD). PVA hybrids' chemical functionalities and their interaction with herpesviruses were also characterized by Fourier transform infrared spectroscopy (FTIR). The bioactivity assays were tested through enzyme linked immunosorbent assay (ELISA). SAXS results have indicated nano-ordered disperse domains for PVA hybrids with different x-ray scattering patterns for PVA polymer and PVA-derived hybrids. FTIR spectra have shown major vibration bands associated with organic-inorganic chemical groups present in the PVA, PVA-derived by silane modifier and PVA chemically crosslinked by GA. The immunoassay results have shown that PVA hybrids with chemically functionalized structures regulated to some extent the specific bioimmobilization of herpesvirus onto solid phase. We think that it is due to the overall balance of forces associated with van der Waals interaction, hydrophilic and hydrophobic forces and steric hindrance acting at the surface. PVA and PVA-derived hybrid materials were successfully produced with GA crosslinking in a nanometer-scale network. Also, such a PVA-based material could be advantageously used in immunoassays with enhanced specificity for diagnosis.

  1. Effector-triggered immunity: from pathogen perception to robust defense.

    Science.gov (United States)

    Cui, Haitao; Tsuda, Kenichi; Parker, Jane E

    2015-01-01

    In plant innate immunity, individual cells have the capacity to sense and respond to pathogen attack. Intracellular recognition mechanisms have evolved to intercept perturbations by pathogen virulence factors (effectors) early in host infection and convert it to rapid defense. One key to resistance success is a polymorphic family of intracellular nucleotide-binding/leucine-rich-repeat (NLR) receptors that detect effector interference in different parts of the cell. Effector-activated NLRs connect, in various ways, to a conserved basal resistance network in order to transcriptionally boost defense programs. Effector-triggered immunity displays remarkable robustness against pathogen disturbance, in part by employing compensatory mechanisms within the defense network. Also, the mobility of some NLRs and coordination of resistance pathways across cell compartments provides flexibility to fine-tune immune outputs. Furthermore, a number of NLRs function close to the nuclear chromatin by balancing actions of defense-repressing and defense-activating transcription factors to program cells dynamically for effective disease resistance.

  2. Pathogenic mycoflora on carrot seeds

    Directory of Open Access Journals (Sweden)

    Bogdan Nowicki

    2013-12-01

    Full Text Available Altogether 300 seed samples were collected during 9 years in 8 regions of Poland and the fungi Were isolated and their pathogenicity to carrot seedlings was examined. Alternaria rudicina provcd to be the most important pathogen although. A. alternata was more common. The other important pathogens were Fusarium spp., Phoma spp. and Botrytis cinerea. The infection of carrot seeds by A. radicina should be used as an important criterium in seed quality evaluation.

  3. Good performance of an immunoassay based method for nevirapine measurements in human breast milk

    DEFF Research Database (Denmark)

    Salado-Rasmussen, Kirsten; Theilgaard, Zahra Persson; Chiduo, Mercy

    2011-01-01

    from HIV-uninfected women. Clinical samples from HIV-infected women receiving a single-dose of nevirapine were analyzed. Results: Precision and accuracy were evaluated with two concentrations of quality control materials analyzed in three replicates on four different days and was......, requires complicated extraction techniques. The ARK method employs an immunoassay technology and requires a small sample volume (40 μL) and no pre-treatment of the samples. Methods: Commercial enzyme and antibody were used and calibration standards and quality controls were prepared from pooled breast milk...

  4. The Use of Recombinant Hemagglutinine Protein of Rinderpest Virus in Enzyme Immunoassay

    OpenAIRE

    BULUT, Hakan; BOLAT, Yusuf

    2003-01-01

    In this study, Rinderpest virus (RPV) recombinant hemagglutinine protein (rH) fused with protein A region of Staphylococcus aureus was expressed in Escherichia coli and purified by IgG affinity chromatography. rH protein was also used to establish enzyme immunoassay. Therefore, to prevent IgG binding to the protein A the wells coated with the rH proteins were blocked by human serum. Afterwards, RPV antigens were added to the wells to evaluate this assay. To this end, serum from mice immunized...

  5. Solid-Phase Immunoassay of Polystyrene-Encapsulated Semiconductor Coreshells for Cardiac Marker Detection

    Directory of Open Access Journals (Sweden)

    Sanghee Kim

    2012-01-01

    Full Text Available A solid-phase immunoassay of polystyrene-encapsulated semiconductor nanoparticles was demonstrated for cardiac troponin I (cTnI detection. CdSe/ZnS coreshells were encapsulated with a carboxyl-functionalized polystyrene nanoparticle to capture the target antibody through a covalent bonding and to eliminate the photoblinking and toxicity of semiconductor luminescent immunosensor. The polystyrene-encapsulated CdSe/ZnS fluorophores on surface-modified glass chip identified cTnI antigens at the level of ~ng/mL. It was an initial demonstration of diagnostic chip for monitoring a cardiovascular disease.

  6. Upconverting nanophosphors as reporters in a highly sensitive heterogeneous immunoassay for cardiac troponin I

    Energy Technology Data Exchange (ETDEWEB)

    Sirkka, Nina, E-mail: nkelon@utu.fi; Lyytikäinen, Annika; Savukoski, Tanja; Soukka, Tero

    2016-06-21

    Photon upconverting nanophosphors (UCNPs) have a unique capability to produce anti-Stokes emission at visible wavelengths via sequential multiphoton absorption upon infrared excitation. Since the anti-Stokes emission can be easily spectrally resolved from the Stokes' shifted autofluorescence, the upconversion luminescence (UCL) is a highly attractive reporter technology for optical biosensors and biomolecular binding assays – potentially enabling unprecedented sensitivity in separation-based solid-phase immunoassays. UCL technology has not previously been applied in sensitive detection of cardiac troponin I (cTnI), which requires highly sensitive detection to enable accurate and timely diagnosis of myocardial infarction. We have developed an UCL-based immunoassay for cTnI using NaYF{sub 4}: Yb{sup 3+}, Er{sup 3+} UCNPs as reporters. Biotinylated anti-cTnI monoclonal antibody (Mab) and Fab fragment immobilized to streptavidin-coated wells were used to capture cTnI. Captured cTnI was detected from dry well surface after a 15 min incubation with poly(acrylic acid) coated UCNPs conjugated to second anti-cTnI Mab. UCL was measured with a dedicated UCL microplate reader. The UCL-based immunoassay allowed sensitive detection of cTnI. The limit of detection was 3.14 ng L{sup −1}. The calibration curve was linear up to cTnI concentration 50,000 ng L{sup −1}. Plasma recoveries of added cTnI were 92–117%. Obtained cTnI concentrations from five normal plasma samples were 4.13–10.7 ng L{sup −1} (median 5.06 ng L{sup −1}). There is yet significant potential for even further improved limit of detection by reducing non-specifically bound fraction of the Mab-conjugated UCNPs. The assay background with zero calibrator was over 40-fold compared to the background obtained from wells where the reporter conjugate had been excluded. - Highlights: • Detection of attomole analyte quantity in microwell using upconversion luminescence. • Upconverting

  7. Results of the HPL determination using a new enzymatic immunoassay - comparison with a commercial radioimmunoassay

    International Nuclear Information System (INIS)

    Leis, D.; Schneider, B.; Keller, A.; Braun, S.

    1982-01-01

    An enzymatic immunoassay recently developed by the firm Organon, Oss, Netherlands, to determine HPL in the serum of pregnant women was compared to a widely used radioimmunoassay marketed by the firm Amersham-Buchler, Braunschweig. The values determined showed a good correlation. The accuracy of measurement was within the generally accepted range for immunological assays of this kind. The assay procedures are comparable with regard to the expenditure of time and work. A great advantage of the EIA is due to the absence of radioactive substances, which permits this test to be performed in any laboratory equipped with a centrifuge and photometer. (orig.) [de

  8. Dot immunoassay for the simultaneous determination of postvaccination immunity against pertussis, diphtheria, and tetanus.

    Science.gov (United States)

    Khramtsov, Pavel; Bochkova, Maria; Timganova, Valeria; Zamorina, Svetlana; Rayev, Mikhail

    2017-06-01

    A dot immunoassay for simultaneous semiquantitative detection of IgG against tetanus toxoid (Ttx) and diphtheria toxoid (Dtx) and qualitative detection of anti-Bordetella pertussis IgGs in human blood serum using carbon nanoparticles functionalized with streptococcal protein G was developed. Inactivated B. pertussis cells in suspension form were used as an antigen in the immunoassay. Pertussis, tetanus, and diphtheria antigens were separately spotted onto nitrocellulose strips, and then the immunostrips were successively incubated with blood sera and a suspension of carbon nanoparticles. The immunostrips were then scanned with a flatbed scanner, and the images obtained were processed with ImageJ. One hundred fifty-five venous blood serum samples from children vaccinated with diphtheria, tetanus, and whole-cell pertussis (DTwP) vaccine were tested in comparison with a conventional ELISA and agglutination test. The total time required for analysis of 32 serum samples was less than 3 h. Comparison between the results of the dot immunoassay and the corresponding ELISA/agglutination test revealed a high level of agreement (Cohen's kappa between 0.765 and 0.813). The lower limit of quantification was 0.06 IU/ml for anti-Ttx and anti-Dtx. The intra-assay coefficients of variation were less than 15% for anti-Ttx and anti-Dtx and less than 10% for anti-pertussis. The diagnostic sensitivity of detection of the antibody protection level was 93.5% for anti-Ttx [95% confidence interval (CI) 83.5-97.9%], 92.4% for anti-Dtx (95% CI 80.9297.5%), and 90.2% for anti-pertussis (95% CI 75.9-96.8%). The diagnostic specificity was 90.9% for anti-Ttx (95% CI 57.1-99.5%), 85% for anti-Dtx (95% CI 61.1-96.0%), and 89.3% for anti-pertussis (95%CI 80.8-94.5%). The dot immunoassay developed does not require expensive reading equipment, and allows detection of antibodies against three antigens in a single analysis. The immunostrips can be stored for a long time without changes in the

  9. A competitive enzyme immunoassay for the quantitative detection of cocaine from banknotes and latent fingermarks.

    Science.gov (United States)

    van der Heide, Susan; Garcia Calavia, Paula; Hardwick, Sheila; Hudson, Simon; Wolff, Kim; Russell, David A

    2015-05-01

    A sensitive and versatile competitive enzyme immunoassay (cEIA) has been developed for the quantitative detection of cocaine in complex forensic samples. Polyclonal anti-cocaine antibody was purified from serum and deposited onto microtiter plates. The concentration of the cocaine antibody adsorbed onto the plates, and the dilution of the cocaine-HRP hapten were both studied to achieve an optimised immunoassay. The method was successfully used to quantify cocaine in extracts taken from both paper currency and latent fingermarks. The limit of detection (LOD) of 0.162ngmL(-1) achieved with the assay compares favourably to that of conventional chromatography-mass spectroscopy techniques, with an appropriate sensitivity for the quantification of cocaine at the low concentrations present in some forensic samples. The cEIA was directly compared to LC-MS for the analysis of ten UK banknote samples. The results obtained from both techniques were statistically similar, suggesting that the immunoassay was unaffected by cross-reactivity with potentially interfering compounds. The cEIA was used also for the detection of cocaine in extracts from latent fingermarks. The results obtained were compared to the cocaine concentrations detected in oral fluid sampled from the same individual. Using the cEIA, we have shown, for the first time, that endogeneously excreted cocaine can be detected and quantified from a single latent fingermark. Additionally, it has been shown that the presence of cocaine, at similar concentrations, in more than one latent fingermark from the same individual can be linked with those concentrations found in oral fluid. These results show that detection of drugs in latent fingermarks could directly indicate whether an individual has consumed the drug. The specificity and feasibility of measuring low concentrations of cocaine in complex forensic samples demonstrate the effectiveness and robustness of the assay. The immunoassay presents a simple and cost

  10. Radio-immunoassay of plasma arginine vasopressin in man. Comparison of two methods of extraction

    International Nuclear Information System (INIS)

    Wolf, J.P.; Dumoulin, G.; Henriet, M.T.; Baulay, A.; Berthelay, S.

    1987-01-01

    Two methods of extraction, prior to radio-immunoassay (RIA) of human plasma arginine vasopressin (AVP) were tested: ethanol extraction (ETH), chromatography with ODS-Silice (ODS-Sil). Recovery of 125 I AVP was higher when using ODS-Sil than when using ETH. Recovery of standard AVP was found to be more efficient and reproducible for ODS-Sil. However, the RIA used, performed after chromatography with ODS-Sil, is not enough sensitive to detect low concentrations but is able to detect high concentrations and physiological variations of plasma AVP [fr

  11. A direct radio-immunoassay for plasma aldosterone: significance of endogenous cortisol

    International Nuclear Information System (INIS)

    Man, A.J.M. de; Hofman, J.A.; Hendriks, Th.; Rosmalen, F.M.A.; Ross, H.A.; Benraad, Th.J.

    1980-01-01

    A direct radio-immunoassay for plasma aldosterone was developed, using a highly specific antiserum raised in sheep. An excellent correlation was observed between its results and the levels measured after extraction and chromatography. The necessity of including a blocking agent to inhibit the binding of aldosterone to plasma proteins was investigated. It was found that in low-cortisol ( 10 μg/100 ml) the final assay result was independent of the presence of ANS. The effect of cortisol was interpreted in terms of its influence on aldosterone binding to plasma proteins in the absence of a blocking agent. (Auth.)

  12. Synthesis of polycyclic aromatic hydrocarbon-protein conjugates for preparation and immunoassay of antibodies.

    Science.gov (United States)

    Glushkov, Andrey N; Kostyanko, Mikhail V; Cherno, Sergey V; Vasilchenko, Ilya L

    2002-04-01

    The method is described dealing with the synthesis of conjugates protein-polycyclic aromatic hydrocarbons (PAHs), highly soluble in water, stable without special stabilizers and containing the minimum quantity of cross-linked products. The reaction of protein with PAH containing an aldehyde group, has been carried out in an alkaline solution, and stabilization of the conjugate has been achieved by reduction with sodium borohydride in the presence of a compound blocking the formation of an insoluble polymeric fraction. The efficiency of synthesized conjugates for the induction and immunoassay of Abs to PAH for benzo[a]pyrene is shown.

  13. Enzyme immunoassay of oestrogen receptors in needle biopsies from human liver

    DEFF Research Database (Denmark)

    Becker, U; Andersen, J; Poulsen, H S

    1991-01-01

    For quantitative assessments of sex hormone receptors in liver tissue, ligand binding assays are inconvenient, as they require large biopsies (0.5-1.0 g). The present study shows that it is possible to measure oestrogen receptors (ER) quantitatively in needle biopsy specimens as small as 10 mg...... by modifications of a commercial enzyme immunoassay employing monoclonal antibodies. Sucrose gradient centrifugation and the dextran charcoal method served as reference methods. A consecutive series of needle biopsies from patients suspected of liver disease were investigated. The biopsies (n = 37) had a median...

  14. Mobile Sensing Systems

    Science.gov (United States)

    Macias, Elsa; Suarez, Alvaro; Lloret, Jaime

    2013-01-01

    Rich-sensor smart phones have made possible the recent birth of the mobile sensing research area as part of ubiquitous sensing which integrates other areas such as wireless sensor networks and web sensing. There are several types of mobile sensing: individual, participatory, opportunistic, crowd, social, etc. The object of sensing can be people-centered or environment-centered. The sensing domain can be home, urban, vehicular… Currently there are barriers that limit the social acceptance of mobile sensing systems. Examples of social barriers are privacy concerns, restrictive laws in some countries and the absence of economic incentives that might encourage people to participate in a sensing campaign. Several technical barriers are phone energy savings and the variety of sensors and software for their management. Some existing surveys partially tackle the topic of mobile sensing systems. Published papers theoretically or partially solve the above barriers. We complete the above surveys with new works, review the barriers of mobile sensing systems and propose some ideas for efficiently implementing sensing, fusion, learning, security, privacy and energy saving for any type of mobile sensing system, and propose several realistic research challenges. The main objective is to reduce the learning curve in mobile sensing systems where the complexity is very high. PMID:24351637

  15. Mobile sensing systems.

    Science.gov (United States)

    Macias, Elsa; Suarez, Alvaro; Lloret, Jaime

    2013-12-16

    Rich-sensor smart phones have made possible the recent birth of the mobile sensing research area as part of ubiquitous sensing which integrates other areas such as wireless sensor networks and web sensing. There are several types of mobile sensing: individual, participatory, opportunistic, crowd, social, etc. The object of sensing can be people-centered or environment-centered. The sensing domain can be home, urban, vehicular… Currently there are barriers that limit the social acceptance of mobile sensing systems. Examples of social barriers are privacy concerns, restrictive laws in some countries and the absence of economic incentives that might encourage people to participate in a sensing campaign. Several technical barriers are phone energy savings and the variety of sensors and software for their management. Some existing surveys partially tackle the topic of mobile sensing systems. Published papers theoretically or partially solve the above barriers. We complete the above surveys with new works, review the barriers of mobile sensing systems and propose some ideas for efficiently implementing sensing, fusion, learning, security, privacy and energy saving for any type of mobile sensing system, and propose several realistic research challenges. The main objective is to reduce the learning curve in mobile sensing systems where the complexity is very high.

  16. Mobile Sensing Systems

    Directory of Open Access Journals (Sweden)

    Elsa Macias

    2013-12-01

    Full Text Available Rich-sensor smart phones have made possible the recent birth of the mobile sensing research area as part of ubiquitous sensing which integrates other areas such as wireless sensor networks and web sensing. There are several types of mobile sensing: individual, participatory, opportunistic, crowd, social, etc. The object of sensing can be people-centered or environment-centered. The sensing domain can be home, urban, vehicular… Currently there are barriers that limit the social acceptance of mobile sensing systems. Examples of social barriers are privacy concerns, restrictive laws in some countries and the absence of economic incentives that might encourage people to participate in a sensing campaign. Several technical barriers are phone energy savings and the variety of sensors and software for their management. Some existing surveys partially tackle the topic of mobile sensing systems. Published papers theoretically or partially solve the above barriers. We complete the above surveys with new works, review the barriers of mobile sensing systems and propose some ideas for efficiently implementing sensing, fusion, learning, security, privacy and energy saving for any type of mobile sensing system, and propose several realistic research challenges. The main objective is to reduce the learning curve in mobile sensing systems where the complexity is very high.

  17. 340 nm pulsed UV LED system for europium-based time-resolved fluorescence detection of immunoassays

    DEFF Research Database (Denmark)

    Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter

    2016-01-01

    We report on the design, development and investigation of an optical system based on UV light emitting diode (LED) excitation at 340 nm for time-resolved fluorescence detection of immunoassays. The system was tested to measure cardiac marker Troponin I with a concentration of 200 ng....../L in immunoassay. The signal-to-noise ratio was comparable to state-of-the-art Xenon flash lamp based unit with equal excitation energy and without overdriving the LED. We performed a comparative study of the flash lamp and the LED based system and discussed temporal, spatial, and spectral features of the LED...... excitation for time-resolved fluorimetry. Optimization of the suggested key parameters of the LED promises significant increase of the signal-to-noise ratio and hence of the sensitivity of immunoassay systems....

  18. 340 nm pulsed UV LED system for europium-based time-resolved fluorescence detection of immunoassays.

    Science.gov (United States)

    Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter; Petersen, Paul Michael; Pedersen, Christian

    2016-09-19

    We report on the design, development and investigation of an optical system based on UV light emitting diode (LED) excitation at 340 nm for time-resolved fluorescence detection of immunoassays. The system was tested to measure cardiac marker Troponin I with a concentration of 200 ng/L in immunoassay. The signal-to-noise ratio was comparable to state-of-the-art Xenon flash lamp based unit with equal excitation energy and without overdriving the LED. We performed a comparative study of the flash lamp and the LED based system and discussed temporal, spatial, and spectral features of the LED excitation for time-resolved fluorimetry. Optimization of the suggested key parameters of the LED promises significant increase of the signal-to-noise ratio and hence of the sensitivity of immunoassay systems.

  19. The fabrication of magnetic particle-based chemiluminescence immunoassay for human epididymis protein-4 detection in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Xiaoling Fu

    2018-03-01

    Full Text Available The magnetic particles have a significant influence on the immunoassay detection and cancer therapy. Herein, the chemiluminescence immunoassay combined with the magnetic particles (MPCLIA was presented for the clinical determination and analysis of human epididymis protein 4 (HE4 in the human serum. Under the optimized experiment conditions, the secure MPCLIA method can detect HE4 in the broader range of 0–1000 pmol/L, with a lower detection limit of 1.35 pmol/L. The satisfactory recovery rate of the method in the serum ranged from 83.62% to 105.10%, which was well within the requirement of clinical analysis. Moreover, the results showed the good correlation with enzyme-linked immunosorbent assay (ELISA, with the correlation coefficient of 0.9589. This proposed method has been successfully applied to the clinical determination of HE4 in the human serum. Keywords: Chemiluminescence immunoassay, Magnetic particles, Human epididymis protein 4

  20. Nano-bio-sensing

    CERN Document Server

    Carrara, Sandro

    2011-01-01

    This book examines state-of-the-art applications of nano-bio-sensing. It brings together researchers from nano-electronics and bio-technology, providing multidisciplinary content from nano-structures fabrication to bio-sensing applications.

  1. Pathogenic agents in freshwater resources

    Science.gov (United States)

    Geldreich, Edwin E.

    1996-02-01

    Numerous pathogenic agents have been found in freshwaters used as sources for water supplies, recreational bathing and irrigation. These agents include bacterial pathogens, enteric viruses, several protozoans and parasitic worms more common to tropical waters. Although infected humans are a major source of pathogens, farm animals (cattle, sheep, pigs), animal pets (dogs, cats) and wildlife serve as significant reservoirs and should not be ignored. The range of infected individuals within a given warm-blooded animal group (humans included) may range from 1 to 25%. Survival times for pathogens in the water environment may range from a few days to as much as a year (Ascaris, Taenia eggs), with infective dose levels varying from one viable cell for several primary pathogenic agents to many thousands of cells for a given opportunistic pathogen.As pathogen detection in water is complex and not readily incorporated into routine monitoring, a surrogate is necessary. In general, indicators of faecal contamination provide a positive correlation with intestinal pathogen occurrences only when appropriate sample volumes are examined by sensitive methodology.Pathways by which pathogens reach susceptible water users include ingestion of contaminated water, body contact with polluted recreational waters and consumption of salad crops irrigated by polluted freshwaters. Major contributors to the spread of various water-borne pathogens are sewage, polluted surface waters and stormwater runoff. All of these contributions are intensified during periods of major floods. Several water-borne case histories are cited as examples of breakdowns in public health protection related to water supply, recreational waters and the consumption of contaminated salad crops. In the long term, water resource management must focus on pollution prevention from point sources of waste discharges and the spread of pathogens in watershed stormwater runoff.

  2. Introduction to remote sensing

    CERN Document Server

    Cracknell, Arthur P

    2007-01-01

    Addressing the need for updated information in remote sensing, Introduction to Remote Sensing, Second Edition provides a full and authoritative introduction for scientists who need to know the scope, potential, and limitations in the field. The authors discuss the physical principles of common remote sensing systems and examine the processing, interpretation, and applications of data. This new edition features updated and expanded material, including greater coverage of applications from across earth, environmental, atmospheric, and oceanographic sciences. Illustrated with remotely sensed colo

  3. Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae

    Directory of Open Access Journals (Sweden)

    Klein Cátia S

    2009-12-01

    Full Text Available Abstract Background Mycoplasma hyopneumoniae is a highly infectious swine pathogen and is the causative agent of enzootic pneumonia (EP. Following the previous report of a proteomic survey of the pathogenic 7448 strain of swine pathogen, Mycoplasma hyopneumoniae, we performed comparative protein profiling of three M. hyopneumoniae strains, namely the non-pathogenic J strain and the two pathogenic strains 7448 and 7422. Results In 2DE comparisons, we were able to identify differences in expression levels for 67 proteins, including the overexpression of some cytoadherence-related proteins only in the pathogenic strains. 2DE immunoblot analyses allowed the identification of differential proteolytic cleavage patterns of the P97 adhesin in the three strains. For more comprehensive protein profiling, an LC-MS/MS strategy was used. Overall, 35% of the M. hyopneumoniae genome coding capacity was covered. Partially overlapping profiles of identified proteins were observed in the strains with 81 proteins identified only in one strain and 54 proteins identified in two strains. Abundance analysis of proteins detected in more than one strain demonstrates the relative overexpression of 64 proteins, including the P97 adhesin in the pathogenic strains. Conclusions Our results indicate the physiological differences between the non-pathogenic strain, with its non-infective proliferate lifestyle, and the pathogenic strains, with its constitutive expression of adhesins, which would render the bacterium competent for adhesion and infection prior to host contact.

  4. Burkholderia glumae: next major pathogen of rice?

    Science.gov (United States)

    Ham, Jong Hyun; Melanson, Rebecca A; Rush, Milton C

    2011-05-01

    Burkholderia glumae causes bacterial panicle blight of rice, which is an increasingly important disease problem in global rice production. Toxoflavin and lipase are known to be major virulence factors of this pathogen, and their production is dependent on the TofI/TofR quorum-sensing system, which is mediated by N-octanoyl homoserine lactone. Flagellar biogenesis and a type III secretion system are also required for full virulence of B. glumae. Bacterial panicle blight is thought to be caused by seed-borne B. glumae; however, its disease cycle is not fully understood. In spite of its economic importance, neither effective control measures for bacterial panicle blight nor rice varieties showing complete resistance to the disease are currently available. A better understanding of the molecular mechanisms underlying B. glumae virulence and of the rice defence mechanisms against the pathogen would lead to the development of better methods of disease control for bacterial panicle blight. Bacteria; Proteobacteria; Betaproteobacteria; Burkholderiales; Burkholderiaceae; Burkholderia. Gram-negative, capsulated, motile, lophotrichous flagella, pectolytic. Aborted seed, empty grains as a result of failure of grain filling, brown spots on panicles, seedling rot. Seed sterilization, planting partially resistant lines (no completely resistant line is available). KNOWN VIRULENCE FACTORS: Toxoflavin, lipase, type III effectors. © 2010 LSU AGCENTER. MOLECULAR PLANT PATHOLOGY © 2010 BSPP AND BLACKWELL PUBLISHING LTD.

  5. Sense of moving

    DEFF Research Database (Denmark)

    Christensen, Mark Schram; Grünbaum, Thor

    2017-01-01

    In this chapter, we assume the existence of a sense of “movement activity” that arises when a person actively moves a body part. This sense is usually supposed to be part of sense of agency (SoA). The purpose of the chapter is to determine whether the already existing experimental paradigms can...

  6. 340 nm pulsed UV LED system for europium-based time-resolved fluorescence detection of immunoassays

    OpenAIRE

    Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter; Petersen, Paul Michael; Pedersen, Christian

    2016-01-01

    We report on the design, development and investigation of an optical system based on UV light emitting diode (LED) excitation at 340 nm for time-resolved fluorescence detection of immunoassays. The system was tested to measure cardiac marker Troponin I with a concentration of 200 ng/L in immunoassay. The signal-to-noise ratio was comparable to state-of-the-art Xenon flash lamp based unit with equal excitation energy and without overdriving the LED. We performed a comparative study of the flas...

  7. Detection of Avian Influenza Virus by Fluorescent DNA Barcode-based Immunoassay with Sensitivity Comparable to PCR

    DEFF Research Database (Denmark)

    Cao, Cuong; Dhumpa, Raghuram; Bang, Dang Duong

    2010-01-01

    involves the sandwiching of the target AIV between magnetic immunoprobes and barcode-carrying immunoprobes. Because each barcode-carrying immunoprobe is functionalized with a multitude of fluorophore-DNA barcode strands, many DNA barcodes are released for each positive binding event resulting......In this paper, a coupling of fluorophore-DNA barcode and bead-based immunoassay for detecting avian influenza virus (AIV) with PCR-like sensitivity is reported. The assay is based on the use of sandwich immunoassay and fluorophore-tagged oligonucleotides as representative barcodes. The detection...

  8. Rapid screening of aflatoxin B1 in beer by fluorescence polarization immunoassay.

    Science.gov (United States)

    Beloglazova, N V; Eremin, S A

    2015-09-01

    This manuscript describes the development of a sensitive, fast and easily-performed fluorescence polarization immunoassay (FPIA) for the mycotoxin aflatoxin B1 (AFB1) in various beer samples, both lager and dark. The highest sensitivity was determined for six poly- and monoclonal antibodies selective towards aflatoxins. The sample pretreatment design was emphasized since beer samples are characterized by extremely diverse matrices. Herein, the choice of sorbent for effective removal of matrix interferences prior to analysis was crucial. The samples were diluted with a borate buffer solution containing 1% PEG 6000 and passed through the clean-up column packed with NH2-derivated silica. This sample pretreatment technique was perfectly suitable for the FPIA of lager beer samples, but for dark beer and ale it did not suffice. An artificial matrix was constructed to plot a calibration curve and quantify the results of the latter samples. The developed immunoassay was characterized by a limit of detection of 1 ng mL(-1). Apparent recovery values of 89-114% for lager and 80-125% for dark beer were established. The FPIA data for AFB1 was characterized by elevated linear regression coefficients, 0.9953 for spiked lager and 0.9895 for dark beer samples respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. A harmonized immunoassay with liquid chromatography-mass spectrometry analysis in egg allergen determination.

    Science.gov (United States)

    Nimata, Masaomi; Okada, Hideki; Kurihara, Kei; Sugimoto, Tsukasa; Honjoh, Tsutomu; Kuroda, Kazuhiko; Yano, Takeo; Tachibana, Hirofumi; Shoji, Masahiro

    2018-01-01

    Food allergy is a serious health issue worldwide. Implementing allergen labeling regulations is extremely challenging for regulators, food manufacturers, and analytical kit manufacturers. Here we have developed an "amino acid sequence immunoassay" approach to ELISA. The new ELISA comprises of a monoclonal antibody generated via an analyte specific peptide antigen and sodium lauryl sulfate/sulfite solution. This combination enables the antibody to access the epitope site in unfolded analyte protein. The newly developed ELISA recovered 87.1%-106.4% ovalbumin from ovalbumin-incurred model processed foods, thereby demonstrating its applicability as practical egg allergen determination. Furthermore, the comparison of LC-MS/MS and the new ELISA, which targets the amino acid sequence conforming to the LC-MS/MS detection peptide, showed a good agreement. Consequently the harmonization of two methods was demonstrated. The complementary use of the new ELISA and LC-MS analysis can offer a wide range of practical benefits in terms of easiness, cost, accuracy, and efficiency in food allergen analysis. In addition, the new assay is attractive in respect to its easy antigen preparation and predetermined specificity. Graphical abstract The ELISA composing of the monoclonal antibody targeting the amino acid sequence conformed to LC-MS detection peptide, and the protein conformation unfolding reagent was developed. In ovalbumin determination, the developed ELISA showed a good agreement with LC-MS analysis. Consequently the harmonization of immunoassay with LC-MS analysis by using common target amino acid sequence was demonstrated.

  10. Plasma exchange to remove HIT antibodies: dissociation between enzyme-immunoassay and platelet activation test reactivities.

    Science.gov (United States)

    Warkentin, Theodore E; Sheppard, Jo-Ann I; Chu, F Victor; Kapoor, Anil; Crowther, Mark A; Gangji, Azim

    2015-01-01

    Repeated therapeutic plasma exchange (TPE) has been advocated to remove heparin-induced thrombocytopenia (HIT) IgG antibodies before cardiac/vascular surgery in patients who have serologically-confirmed acute or subacute HIT; for this situation, a negative platelet activation assay (eg, platelet serotonin-release assay [SRA]) has been recommended as the target serological end point to permit safe surgery. We compared reactivities in the SRA and an anti-PF4/heparin IgG-specific enzyme immunoassay (EIA), testing serial serum samples in a patient with recent (subacute) HIT who underwent serial TPE precardiac surgery, as well as for 15 other serially-diluted HIT sera. We observed that post-TPE/diluted HIT sera-when first testing SRA-negative-continue to test strongly positive by EIA-IgG. This dissociation between the platelet activation assay and a PF4-dependent immunoassay for HIT antibodies indicates that patients with subacute HIT undergoing repeated TPE before heparin reexposure should be tested by serial platelet activation assays even when their EIAs remain strongly positive. © 2015 by The American Society of Hematology.

  11. Highly sensitive immunoassay of protein molecules based on single nanoparticle fluorescence detection in a nanowell

    Science.gov (United States)

    Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

    2011-03-01

    A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

  12. Characterization of hapten-protein conjugates: antibody generation and immunoassay development for chlorophenoxyacetic acid pesticides.

    Science.gov (United States)

    Boro, Robin C; Singh, K Vikas; Suri, C Raman

    2009-01-01

    The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In this study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density for two different chlorophenoxyacetic acid pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenoxybutyric acid (2,4-DB), coupled to carrier protein. Highly sensitive anti-2,4-D and anti-2,4-DB antibodies were obtained using these well-characterized hapten-protein conjugates. The generated antibodies were used in an immunoassay format demonstrating inhibitory concentration (IC50) values equal to 30 and 7 ng/mL for 2,4-D and 2,4-DB, respectively. Linearity was observed in the concentration range between 0.1-500 nglmL with LODs around 4 and 3 ng/mL for 2,4-D and 2,4-DB, respectively, in standard water samples. The proposed method was successfully applied for the determination of the extent of hapten-protein conjugation to produce specific antibodies for immunoassay development against pesticides.

  13. Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance.

    Science.gov (United States)

    Tu, Bailin; Tieman, Bryan; Moore, Jeffrey; Pan, You; Muerhoff, A Scott

    2017-06-01

    Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.

  14. New antibody and immunoassay pretreatment strategy to screen polychlorinated biphenyls in Korean transformer oil.

    Science.gov (United States)

    Terakado, Shingo; Ohmura, Naoya; Park, Seok-Un; Lee, Seung-Min; Glass, Thomas R

    2013-01-01

    Development and modifications are described that expand the application of an immunoassay from the detection of Kanechlors (Japanese technical PCBs mixtures) to the detection of Aroclors (U. S. technical PCB mixtures, used in Korea) in contaminated Korean transformer oil. The first necessary modification was the development of a new antibody with a reactivity profile favorable for Aroclors. The second modification was the addition of a second column to the solid-phase extraction method to reduce assay interference caused by the Korean oil matrix. The matrix interference is suspected to be caused by the presence of synthetic oils (or similar materials) present as contaminants. The modified assay was validated by comparison to high-resolution gas chromatography/high-resolution mass spectrometry analysis, and was shown to be tolerant of up to 10% of several common synthetic insulating oils. Finally the screening performance of the modified assay was evaluated using 500 used transformer oil samples of Korean origin, and was shown to have good performance in terms of false positive and false negative rates. This report provides evidence for the first establishment of immunoassay screening for Aroclor based PCB contamination in Korean transformer oil.

  15. A summary report on the 23rd quality control survey for immunoassays in Japan, 2001

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2002-10-01

    The purpose of the survey is to improve the quality of in vitro tests and this report is its summary of immunoassays (the old name: RI in vitro tests) conducted in 2001. The survey was performed in 143 facilities out of 1,655 in Japan, which involved 20 national and public university hospitals, 16 private university hospitals, 21 national and public hospitals, 26 private hospitals, 41 hygiene test institutes and 19 reagent manufacturers. Tests examined were on 6 substances related to functions of pituitary, 5 of thyroid, 1 of parathyroid, 4 of gastro-intestine and pancreas, 5 of gonad and placenta, 4 of adrenal, 1 of renal-blood pressure regulation, on IgE, on digoxin and on 12 tumor-related substances. Tests were done on 2-3 samples supplied from the Committee and the mean, standard deviation and coefficient of variation were calculated for one way analysis of variance of within- and between-kit. Methods included those (65.4% vs 62.7% in 2000) with non-radioisotope like enzyme immunoassay (non-RI) and with radioisotopes like radioimmunoassay (RI). Dissociation between manufacturers of non-RI values without a common standard substance and between non-RI and RI values even with the same antibody was noted: the Committee considered these problems as their future task. (K.H.)

  16. Photoelectrochemical detection of enzymatically generated CdS nanoparticles: Application to development of immunoassay.

    Science.gov (United States)

    Barroso, Javier; Saa, Laura; Grinyte, Ruta; Pavlov, Valeri

    2016-03-15

    We report an innovative photoelectrochemical process (PEC) based on graphite electrode modified with electroactive polyvinylpyridine bearing osmium complex (Os-PVP). The system relies on the in situ enzymatic generation of CdS quantum dots (QDs). Alkaline phosphatase (ALP) catalyzes the hydrolisis of sodium thiophosphate (TP) to hydrogen sulfide (H2S) which in the presence Cd(2+) ions yields CdS semiconductor nanoparticles (SNPs). Irradiation of SNPs with the standard laboratory UV-illuminator (wavelength of 365 nm) results in photooxidation of 1-thioglycerol (TG) mediated by Os-PVP complex on the surface of graphite electrode at applied potential of 0.31 V vs. Ag/AgCl. A novel immunoassay based on specific enzyme linked immunosorbent assay (ELISA) combined with the PEC methodology was developed. Having selected the affinity interaction between bovine serum albumine (BSA) with anti-BSA antibody (AB) as a model system, we built the PEC immunoassay for AB. The new assay displays a linear range up to 20 ngmL(-1) and a detection limit (DL) of 2 ngmL(-1) (S/N=3) which is lower 5 times that of the traditional chromogenic ELISA test employing p-nitro-phenyl phosphate (pNPP). Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Development of a simple extraction procedure for chlorpyrifos determination in food samples by immunoassay.

    Science.gov (United States)

    Gabaldón, J A; Maquieira, A; Puchades, R

    2007-02-28

    The suitability of immunoassay methodology for rapid and accurate determination of chlorpyrifos in vegetables was tested. The optimised ELISA detection limit was 0.32ng/ml, with a working range from 0.69 to 6.21ng/ml and an immunoassay test-mid point (IC(50)) of 2.08ng/ml. A rapid sample preparation procedure considering different parameters such as the amount of sample, volume of extractant, extraction time and dilution factor was optimised. The developed direct extraction (DE) and multiresidue (ME) standard procedures were performed in different fortified fresh and processed vegetable samples (tomato, bonnet pepper, bean, pea, asparagus, broccoli, watermelon, melon, lettuce, cucumber, celery and red pepper). Recoveries were in all cases in the whole range 85.2-108.9% for both DE and ME extracts. Also, the comparison of the results obtained by both immunochemical and chromatographic methods for spiked fruits and vegetables were good with a correlation coefficient (r) of 0.97.

  18. Dog cloning with in vivo matured oocytes obtained using electric chemiluminescence immunoassay-predicted ovulation method.

    Science.gov (United States)

    Lee, Seunghoon; Zhao, Minghui; No, Jingu; Nam, Yoonseok; Im, Gi-Sun; Hur, Tai-Young

    2017-01-01

    Radioactive immunoassay (RIA) is a traditional serum hormone assay method, but the application of the method in reproductive studies is limited by the associated radioactivity. The aim of present study was to evaluate the reliability of RIA and to compare its canine serum progesterone concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). In vivo matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum progesterone levels were assessed to accurately determine ovulation and oocyte maturation. Canine serum progesterone concentrations during both proestrus and estrus were analyzed by RIA and ECLI to determine the ovulation day. Although both methods detected similar progesterone levels before ovulation, the mean progesterone concentration determined using ECLI was significantly higher than of RIA three days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of progesterone were used for determination of ovulation. A high percentage of mature oocytes was observed using ECLI when 6-15 ng/mL of progesterone was used for ovulation determination. To determine whether ECLI could be used for canine cloning, six canines were selected as oocyte donors, and two puppies were obtained after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning.

  19. Screening for cocaine on Euro banknotes by a highly sensitive enzyme immunoassay.

    Science.gov (United States)

    Abdelshafi, Nahla A; Panne, Ulrich; Schneider, Rudolf J

    2017-04-01

    This study focused on quantitative detection of cocaine on Euro banknotes in Germany. A sensitive direct competitive immunoassay was developed and optimized with a limit of detection (LOD) of 5.6ng/L. Exhaustive cocaine extraction by solvent was tested using different methanol concentrations and buffered solutions. Cross-reactivity studies were performed to determine the degree of interference of cocaine metabolites with the immunoassay. Sixty-five Euro banknotes obtained from different districts in Berlin were evaluated. A 100% contamination frequency with cocaine was detected. A comparison between the amount of cocaine extracted by cotton swabbing of one square centimeter of the banknote showed a good correlation for lower contamination levels. This assay showed high sensitivity of detecting pg of cocaine per 1cm 2 of one banknote by swabbing 1cm 2 : 0, 14, and 21pg/cm 2 . Moreover, three notes of different denominations revealed high cocaine concentration; 1.1mg/note, and twice 55µg/note. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Evaluation and comparison of radio-, fluorescence, and enzyme-linked immunoassays for serum thyroxine

    International Nuclear Information System (INIS)

    Kaplan, L.A.; Gau, N.; Fearn, J.; Steain, E.A.; Chen, I.W.; Maxon, H.; Volle, C.

    1981-01-01

    We have compared three analytical systems for the measurement of serum thyroxine: enzyme-linked immunoassay (EIA), fluorescent immunoassay (FIA) and a radioimmunoassay (RIA). These were evaluated with respect to their precision, accuracy, analytical sensitivity and sample throughput. The RIA is more sensitive than the EIA (10 μg/L vs. 35 μg/L). Both systems have excellent precision (X=86 μg/L, C.V.sub(RIA)=C.V.sub(EIA)=4.6 percent). Both the EIA and RIA demonstrate good accuracy with recovery of between 97-98 percent of added thyroxine. The FIA has an apparent sensitiviity between that of the RIA and EIA (25 μg/L), but a precision consistently lower than the other two systems (C.V. =7.4 percent, X=86 μg/L). Patients' results by RIA compared well with those from EIA (r=0.91,P 0.05). Although not fully automated, the EIA performed on the Abbott ABA-100 analyzer has a sample throughput equal to the automated RIA system (Micromedic, Concept 4)

  1. Alkaline phosphatase labeled SERS active sandwich immunoassay for detection of Escherichia coli

    Science.gov (United States)

    Bozkurt, Akif Goktug; Buyukgoz, Guluzar Gorkem; Soforoglu, Mehmet; Tamer, Ugur; Suludere, Zekiye; Boyaci, Ismail Hakki

    2018-04-01

    In this study, a sandwich immunoassay method utilizing enzymatic activity of alkaline phosphatase (ALP) on 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for Escherichia coli (E. coli) detection was developed using surface enhanced Raman spectroscopy (SERS). For this purpose, spherical magnetic gold coated core-shell nanoparticles (MNPs-Au) and rod shape gold nanoparticles (Au-NRs) were synthesized and modified for immunomagnetic separation (IMS) of E. coli from the solution. In order to specify the developed method to ALP activity, Au-NRs were labeled with this enzyme. After successful construction of the immunoassay, BCIP substrate was added to produce the SERS-active product; 5-bromo-4-chloro-3-indole (BCI). A good linearity (R2 = 0.992) was established between the specific SERS intensity of BCI at 600 cm- 1 and logarithmic E. coli concentration in the range of 1.7 × 101-1.7 × 106 cfu mL- 1. LOD and LOQ values were also calculated and found to be 10 cfu mL- 1 and 30 cfu mL- 1, respectively.

  2. Comparison of immunoassays for differentiation of herpes simplex virus type 1 and 2 antibodies

    International Nuclear Information System (INIS)

    Klapper, Paul E.; Valley, Pam J.; Cleator, Gerham M.; Mandall, D.; Qutub, Mohammed O.

    2006-01-01

    To asses the commercial available enzyme-linked immunosorbent assays (ELISA) for differentiation of herpes simplex virus type 1 (Hs-1) and type 2 (HSV-2) antibodies. The study was performed between January 1997 to November 2002 in the Division ofVirology,Department of Pathological Sciences, Central Manchester Healthcare Trust and University of Manchester, Manchester, United Kingdom. Assays based upon type-specific glycoprotein G-1 (gG-1) for HSV-1, and glycoprotein G-2 (gG-2) from HSV-2 were evaluated to differentiate between HSV-1 and HSV-2 antibodies. Using 5 different ELISA tests, 2 panels of serum samples were tested. Panel one consisted of 88 sera, selected from the serum bank of the Clinical Virology Laboratory, Manchester Royal Infirmary; panel 2 comprised of 90 sera selected from samples collected from Bangladeshi female commercial workers.The data of this study showed that a high rate of gG-1 based immunoassays ranged from 87.9-100% for sensitivity and 51.5-100% specificity. Although there are several immunoassays were claimed to differentiate between HSV-1 and HSV-2 antibodies, selection of these assays should be carefully interpreted with the overall clinical framework provided by detailed sexual history and genital examination. (author)

  3. [Membrane-filtration immunoassay: reagents, methods and the diagnostic and technical means for detection].

    Science.gov (United States)

    Khramov, E N; Osin, N S; Pomelova, V G; Vikha, I V; Bychenkova, T A; Smirnova, V G; Grakina, G I; Kas'ianova, T A

    1999-01-01

    The comprehensive development of dot-EIA made at the State Research Institute of Biological Instrument-Making Industry has provided devices KIMF-02 and KIMF-03), a base of chemical reagents, immunoassays, test systems for detection of a wide range of causative agents of viral and bacterial infections, that of serodiagnosis of their related diseases. The KIMF-02 kit has undergone engineering and medical tests and recommended for the Ministry of Health of the Russian Federation to produce them in stock. The kit includes all required for analysis even in an ill-equipped laboratory, a set of attached agents ensures a valid visual recording of results. The developed procedures and test systems allow the immunoassay to be as sensitive as TIFA; however, they are laborious and much simpler in design. The simple and rapid procedures of dot-EIA are recommended for incorporation into the a package of laboratory methods for verification of the accumulation of virus-specific antigens in various biological substrata, environmental samples, for control of the activity of antigens and antibodies used in serological tests, for detection of specific antigens in the clinical samples, and for serodiagnosis of infections.

  4. Development of a mass spectrometry immunoassay for unambiguous detection of egg allergen traces in wines.

    Science.gov (United States)

    Pilolli, Rosa; Chaudhari, Ravindra; Palmisano, Francesco; Monaci, Linda

    2017-02-01

    A mass spectrometry immunoassay (MSIA) specifically designed for the detection of egg allergens in wines is described. MSIA is based on an immunoaffinity enrichment procedure combined with targeted MS/MS detection of selected egg peptide markers. Polyclonal antibodies raised against native ovalbumin, chosen as the target protein tracing for egg powder, were immobilized onto low backpressure monolithic MSIA customized disposable tips. Ovalbumin-free wine samples were fortified with standard protein at different concentrations in the low microgram-per-milliliter range. A simple protocol was devised consisting of a 1:4 dilution of the wine sample with a basic solution for pH adjustment, followed by a semi-automated purification/enrichment step on MSIA customized disposable tips fitted on a multichannel electronic pipette. Among the main figures of merit, LOD and LOQ values as low as 0.01 and 0.03 μg/mL, respectively, and within-day precision of 18% should be noticed. Noteworthy, the developed assay outperformed current MS-based methods for the detection of allergenic protein in wine matrices, thanks to the immunoaffinity enrichment. In addition, compared to other immunoassays, the present approach boasts the unquestionable advantage of providing an unambiguous identification of the target protein by simultaneous detection of three unique peptide markers each giving three specific MS/MS transitions.

  5. Enzyme immunoassay

    DEFF Research Database (Denmark)

    Feldt-Rasmussen, B; Dinesen, B; Deckert, M

    1985-01-01

    variation of the 24 h urine albumin excretion of different days was high in patients with incipient diabetic nephropathy (51.5%) and was only slightly reduced by taking the variation of creatinine excretion into account (39.5%). No correlation was found between albumin excretion, and HbA1c or urine glucose...

  6. Immunoassay technique

    International Nuclear Information System (INIS)

    Goodearl, P.D.

    1981-01-01

    For quantitatively assaying antigen in a fluid, a mixture thereof with further antigen, labelled for example by substitution or addition of a radioactive molecule is caused to flow along a paper wick or other carrier on which antibodies to the antigen have been immobilised, e.g. by activating the paper by application of cyanogen bromide, then bringing it into contact with a solution of the antibodies. The paper wick or other carrier is developed by causing a glycerol/water solution to rise through the wick by capillary action, and there is measured the distance travelled along the wick by the labelled antigen. (author)

  7. Correlation of antinuclear antibody immunofluorescence patterns with immune profile using line immunoassay in the Indian scenario

    Directory of Open Access Journals (Sweden)

    Sebastian Wendy

    2010-07-01

    Full Text Available Background: Immunity status, individual response to disease and types of antibodies produced are well known to vary from person to person, place to place and probably from population to population. A broad spectrum of specific auto antibodies that have so far been associated with specific rheumatic diseases, as noted in Western literature, has been well taken as a reference standard all over the world. There is neither research work nor any data correlating the auto antibodies and their antinuclear antibody (ANA patterns with the immunoprofile in the Indian population to date. Aims: To understand a definite association between ANA patterns and specific antibodies in the serum in the Indian study population and to document similarities / differences with the West. Settings and Design: This prospective and retrospective double blind study was undertaken on the South Indian population referred for ANA testing by Indirect Immunofluorescence method and by immunoline methods. Materials and Methods: Serum samples of patients from a random South Indian population who sought medical help for rheumatic disease were subjected for ANA testing by indirect immunofluorescence (IIF method and line immunoassay during the study period of 27 months. Serum samples were processed in dilution of 1:100 using HEp - 2010 / liver biochip (Monkey (EUROIMMUN AG. The serum samples which were further processed for line immunoassay were treated in 1:100 dilution on nylon strips coated with recombinant and purified antigens as discrete lines with plastic backing (EUROIMMUN AG coated with antigens nRNP / Sm, Sm, SSA, Ro-52, SSB, Scl-70, PM-Scl, PCNA, Jo-1, CENP-B, dsDNA, nucleosomes, histones, ribosomal protein-P, anti-mitochondrial antibodies (AMA-M2 along with a control band. The analysis was done by comparing the intensity of the reaction with positive control line by image analysis. Results: The antinuclear antibody indirect immunofluorescence (ANA - IIF patterns obtained

  8. Qualitative and Quantitative Determination of Quorum Sensing Inhibition In Vitro

    DEFF Research Database (Denmark)

    Jakobsen, Tim Holm; Alhede, Maria; Hultqvist, Louise Dahl

    2018-01-01

    The formation of biofilms in conjunction with quorum sensing (QS) regulated expression of virulence by opportunistic pathogens contributes significantly to immune evasion and tolerance to a variety of antimicrobial treatments. The present protocol describes methods to determine the in vitro effic...

  9. A broad range quorum sensing inhibitor working through sRNA inhibition

    DEFF Research Database (Denmark)

    Jakobsen, Tim H.; Warming, Anders N.; Vejborg, Rebecca M.

    2017-01-01

    that the repressing effect of ajoene on quorum sensing occurs by inhibition of small regulatory RNAs (sRNA) in P. aeruginosa as well as in Staphylococcus aureus, another important human pathogen that employs quorum sensing to control virulence gene expression. Using various reporter constructs, we found that ajoene......-spectrum compounds transcending the Gram negative-positive borderline....

  10. The Quantitative Basis of the Arabidopsis Innate Immune System to Endemic Pathogens Depends on Pathogen Genetics.

    Directory of Open Access Journals (Sweden)

    Jason A Corwin

    2016-02-01

    Full Text Available The most established model of the eukaryotic innate immune system is derived from examples of large effect monogenic quantitative resistance to pathogens. However, many host-pathogen interactions involve many genes of small to medium effect and exhibit quantitative resistance. We used the Arabidopsis-Botrytis pathosystem to explore the quantitative genetic architecture underlying host innate immune system in a population of Arabidopsis thaliana. By infecting a diverse panel of Arabidopsis accessions with four phenotypically and genotypically distinct isolates of the fungal necrotroph B. cinerea, we identified a total of 2,982 genes associated with quantitative resistance using lesion area and 3,354 genes associated with camalexin production as measures of the interaction. Most genes were associated with resistance to a specific Botrytis isolate, which demonstrates the influence of pathogen genetic variation in analyzing host quantitative resistance. While known resistance genes, such as receptor-like kinases (RLKs and nucleotide-binding site leucine-rich repeat proteins (NLRs, were found to be enriched among associated genes, they only account for a small fraction of the total genes associated with quantitative resistance. Using publically available co-expression data, we condensed the quantitative resistance associated genes into co-expressed gene networks. GO analysis of these networks implicated several biological processes commonly connected to disease resistance, including defense hormone signaling and ROS production, as well as novel processes, such as leaf development. Validation of single gene T-DNA knockouts in a Col-0 background demonstrate a high success rate (60% when accounting for differences in environmental and Botrytis genetic variation. This study shows that the genetic architecture underlying host innate immune system is extremely complex and is likely able to sense and respond to differential virulence among pathogen

  11. Microfluidic devices for sample preparation and rapid detection of foodborne pathogens.

    Science.gov (United States)

    Kant, Krishna; Shahbazi, Mohammad-Ali; Dave, Vivek Priy; Ngo, Tien Anh; Chidambara, Vinayaka Aaydha; Than, Linh Quyen; Bang, Dang Duong; Wolff, Anders

    2018-03-10

    Rapid detection of foodborne pathogens at an early stage is imperative for preventing the outbreak of foodborne diseases, known as serious threats to human health. Conventional bacterial culturing methods for foodborne pathogen detection are time consuming, laborious, and with poor pathogen diagnosis competences. This has prompted researchers to call the current status of detection approaches into question and leverage new technologies for superior pathogen sensing outcomes. Novel strategies mainly rely on incorporating all the steps from sample preparation to detection in miniaturized devices for online monitoring of pathogens with high accuracy and sensitivity in a time-saving and cost effective manner. Lab on chip is a blooming area in diagnosis, which exploits different mechanical and biological techniques to detect very low concentrations of pathogens in food samples. This is achieved through streamlining the sample handling and concentrating procedures, which will subsequently reduce human errors and enhance the accuracy of the sensing methods. Integration of sample preparation techniques into these devices can effectively minimize the impact of complex food matrix on pathogen diagnosis and improve the limit of detections. Integration of pathogen capturing bio-receptors on microfluidic devices is a crucial step, which can facilitate recognition abilities in harsh chemical and physical conditions, offering a great commercial benefit to the food-manufacturing sector. This article reviews recent advances in current state-of-the-art of sample preparation and concentration from food matrices with focus on bacterial capturing methods and sensing technologies, along with their advantages and limitations when integrated into microfluidic devices for online rapid detection of pathogens in foods and food production line. Copyright © 2018. Published by Elsevier Inc.

  12. Help in the Choice of Automated or Semiautomated Immunoassays for Serological Diagnosis of Toxoplasmosis: Evaluation of Nine Immunoassays by the French National Reference Center for Toxoplasmosis.

    Science.gov (United States)

    Villard, O; Cimon, B; L'Ollivier, C; Fricker-Hidalgo, H; Godineau, N; Houze, S; Paris, L; Pelloux, H; Villena, I; Candolfi, E

    2016-12-01

    Toxoplasmosis, a benign infection, is asymptomatic or paucisymptomatic in over 80% of cases, except in immunocompetent patients suffering from ocular toxoplasmosis or in immunocompromised patients with opportunistic or congenital toxoplasmosis. Diagnosis is based mainly on serology testing. Thus, we compared the performance of the nine most commonly used commercial automated or semiautomated immunoassays for IgG and IgM Toxoplasma gondii antibody detection, that is, the Advia Centaur, Architect, AxSYM, Elecsys, Enzygnost, Liaison, Platelia, VIDAS, and VIDIA assays. The assays were conducted on four panels of serum samples derived during routine testing from patients with an interfering disease and who exhibited a low IgG antibody level in one of two clinical settings, namely, acute or chronic toxoplasmosis. As a result, IgG sensitivities ranged from 97.1% to 100%, and IgG specificities ranged from 99.5% to 100%. For IgG quantification, strong differences in IgG titers (expressed in IU/ml) were noted depending on the assay used. IgM sensitivities ranged from 65% to 97.9%, and IgM specificities ranged from 92.6% to 100%. For defining the best serological strategies to be implemented, it appears crucial to compare the diagnostic performance of the different tests with respect to their specificity and sensitivity in detecting the presence of IgG and IgM antibodies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. Transient virulence of emerging pathogens.

    Science.gov (United States)

    Bolker, Benjamin M; Nanda, Arjun; Shah, Dharmini

    2010-05-06

    Should emerging pathogens be unusually virulent? If so, why? Existing theories of virulence evolution based on a tradeoff between high transmission rates and long infectious periods imply that epidemic growth conditions will select for higher virulence, possibly leading to a transient peak in virulence near the beginning of an epidemic. This transient selection could lead to high virulence in emerging pathogens. Using a simple model of the epidemiological and evolutionary dynamics of emerging pathogens, along with rough estimates of parameters for pathogens such as severe acute respiratory syndrome, West Nile virus and myxomatosis, we estimated the potential magnitude and timing of such transient virulence peaks. Pathogens that are moderately evolvable, highly transmissible, and highly virulent at equilibrium could briefly double their virulence during an epidemic; thus, epidemic-phase selection could contribute significantly to the virulence of emerging pathogens. In order to further assess the potential significance of this mechanism, we bring together data from the literature for the shapes of tradeoff curves for several pathogens (myxomatosis, HIV, and a parasite of Daphnia) and the level of genetic variation for virulence for one (myxomatosis). We discuss the need for better data on tradeoff curves and genetic variance in order to evaluate the plausibility of various scenarios of virulence evolution.

  14. Ecological niche of plant pathogens

    Directory of Open Access Journals (Sweden)

    Ecaterina Fodor

    2011-06-01

    Full Text Available Disease ecology is a new approach to the understanding of the spread and dynamics of pathogens in natural and man-made environments. Defining and describing the ecological niche of the pathogens is one of the major tasks for ecological theory, as well as for practitioners preoccupied with the control and forecasting of established and emerging diseases. Niche theory has been periodically revised, not including in an explicit way the pathogens. However, many progresses have been achieved in niche modeling of disease spread, but few attempts were made to construct a theoretical frame for the ecological niche of pathogens. The paper is a review of the knowledge accumulated during last decades in the niche theory of pathogens and proposes an ecological approach in research. It quest for new control methods in what concerns forest plant pathogens, with a special emphasis on fungi like organisms of the genus Phytophthora. Species of Phytophthora are the most successful plant pathogens of the moment, affecting forest and agricultural systems worldwide, many of them being invasive alien organisms in many ecosystems. The hyperspace of their ecological niche is defined by hosts, environment and human interference, as main axes. To select most important variables within the hyperspace, is important the understanding of the complex role of pathogens in the ecosystems as well as for control programs. Biotic relationships within ecosystem of host-pathogen couple are depicted by ecological network and specific metrics attached to this. The star shaped network is characterized by few high degree nodes, by short path lengths and relatively low connectivity, premises for a rapid disturbance spread. 

  15. Ecological niche of plant pathogens

    Directory of Open Access Journals (Sweden)

    Ecaterina Fodor

    2011-02-01

    Full Text Available Disease ecology is a new approach to the understanding of the spread and dynamics of pathogens in natural and man-made environments. Defining and describing the ecological niche of the pathogens is one of the major tasks for ecological theory, as well as for practitioners preoccupied with the control and forecasting of established and emerging diseases. Niche theory has been periodically revised, not including in an explicit way the pathogens. However, many progresses have been achieved in niche modeling of disease spread, but few attempts were made to construct a theoretical frame for the ecological niche of pathogens. The paper is a review of the knowledge accumulated during last decades in the niche theory of pathogens and proposes an ecological approach in research. It quest for new control methods in what concerns forest plant pathogens, with a special emphasis on fungi like organisms of the genus Phytophthora. Species of Phytophthora are the most successful plant pathogens of the moment, affecting forest and agricultural systems worldwide, many of them being invasive alien organisms in many ecosystems. The hyperspace of their ecological niche is defined by hosts, environment and human interference, as main axes. To select most important variables within the hyperspace, is important for the understanding of the complex role of pathogens in the ecosystems as well as for control programs. Biotic relationships within ecosystem of host-pathogen couple are depicted by ecological network and specific metrics attached to this. The star shaped network is characterized by few high degree nodes, by short path lengths and relatively low connectivity, premises for a rapid disturbance spread.

  16. Plant-Derived Natural Products as Sources of Anti-Quorum Sensing Compounds

    OpenAIRE

    Koh, Chong-Lek; Sam, Choon-Kook; Yin, Wai-Fong; Tan, Li Ying; Krishnan, Thiba; Chong, Yee Meng; Chan, Kok-Gan

    2013-01-01

    Quorum sensing is a system of stimuli and responses in relation to bacterial cell population density that regulates gene expression, including virulence determinants. Consequently, quorum sensing has been an attractive target for the development of novel anti-infective measures that do not rely on the use of antibiotics. Anti-quorum sensing has been a promising strategy to combat bacterial infections as it is unlikely to develop multidrug resistant pathogens since it does not impose any selec...

  17. Host pathogen relations: exploring animal models for fungal pathogens.

    Science.gov (United States)

    Harwood, Catherine G; Rao, Reeta P

    2014-06-30

    Pathogenic fungi cause superficial infections but pose a significant public health risk when infections spread to deeper tissues, such as the lung. Within the last three decades, fungi have been identified as the leading cause of nosocomial infections making them the focus of research. This review outlines the model systems such as the mouse, zebrafish larvae, flies, and nematodes, as well as ex vivo and in vitro systems available to study common fungal pathogens.

  18. Host Pathogen Relations: Exploring Animal Models for Fungal Pathogens

    Directory of Open Access Journals (Sweden)

    Catherine G. Harwood

    2014-06-01

    Full Text Available Pathogenic fungi cause superficial infections but pose a significant public health risk when infections spread to deeper tissues, such as the lung. Within the last three decades, fungi have been identified as the leading cause of nosocomial infections making them the focus of research. This review outlines the model systems such as the mouse, zebrafish larvae, flies, and nematodes, as well as ex vivo and in vitro systems available to study common fungal pathogens.

  19. Nanobody-based electrochemical immunoassay for Bacillus thuringiensis Cry1Ab toxin by detecting the enzymatic formation of polyaniline

    International Nuclear Information System (INIS)

    Zhu, Min; Li, Guanghui; Li, Min; Zhou, Zikai; Liu, Hong; Lei, Hongtao; Shen, Yanfei; Wan, Yakun

    2015-01-01

    We describe an electrochemical immunoassay for the Cry1Ab toxin that is produced by Bacillus thuringiensis. It is making use of a nanobody (a heavy-chain only antibody) that was selected from an immune phage displayed library. A biotinylated primary nanobody and a HRP-conjugated secondary nanobody were applied in a sandwich immunoassay where horseradish peroxidase (HRP) is used to produce polyaniline (PANI) from aniline. PANI can be easily detected by differential pulse voltammetry at a working voltage as low as 40 mV (vs. Ag/AgCl) which makes the assay fairly selective. This immunoassay for Cry1Ab has an analytical range from 0.1 to 1000 ng∙mL -1 and a 0.07 ng∙mL -1 lower limit of detection. The average recoveries of the toxin from spiked samples are in the range from 102 to 114 %, with a relative standard deviation of <7.5 %. The results demonstrated that the assay represented an attractive alternative to existing immunoassays in enabling affordable, sensitive, robust and specific determination of this toxin. (author)

  20. Tuberculin purified protein derivative (PPD) immunoassay as an in vitro alternative assay for identity and confirmation of potency.

    Science.gov (United States)

    Ho, Mei M; Kairo, Satnam K; Corbel, Michael J

    2006-01-01

    Tuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.

  1. Detection of hidden hazelnut protein in food by IgY-based indirect competitive enzyme-immunoassay

    NARCIS (Netherlands)

    Baumgartner, S.; Bremer, M.G.E.G.; Kemmers - Voncken, A.E.M.; Smits, N.G.E.; Haasnoot, W.; Banks, J.; Reece, P.; Danks, C.; Tomkies, V.; Immer, U.; Schmitt, K.; Krska, R.

    2004-01-01

    The development of an indirect competitive enzyme-immunoassay for the detection of hidden hazelnut protein in complex food matrices is described. A sensitive and selective polyclonal antibody was raised by immunisation of laying hens with protein extracts from roasted hazelnuts. In contrast to

  2. Plasmon enhanced fluoro-immunoassay using egg yolk antibodies for ultra-sensitive detection of herbicide diuron.

    Science.gov (United States)

    Sharma, Priyanka; Kukkar, Manil; Ganguli, Ashok K; Bhasin, Aman; Suri, C Raman

    2013-08-07

    Plasmon enhanced fluorescence immunoassay (PEFI) format has been reported in developing a sensitive heterogeneous fluoroimmunoassay for monitoring the phenylurea herbicide diuron. Computer-assisted molecular modeling was carried out to study the conformational and electrostatic effects of synthesized hapten for producing highly specific egg yolk antibody against a phenyl urea herbicide diuron. The generated antibodies were labeled with fluorescein isothiocyanate at different molar ratios and used as tracer in the developed fluorescence based immunoassay. The sensitivity of the assay format was enhanced by using silver nanoparticles tagged with bovine serum albumin as a new blocking reagent in the developed PEFI format. Enhancer treatment on the developed immunoassay showed a significant improvement of fluorescence signal intensity with approximately 10 fold increase in assay sensitivity. The immunoassay has a detection limit of 0.01 ng mL(-1) with good signal precision (~2%) in the optimum working concentration range between 1 pg mL(-1) to 10 μg mL(-1) of diuron. These findings facilitate high throughput fluorescence-based processes that could be useful in biology, drug discovery and compound screening applications.

  3. Evaluation of a fluorescence polarographic immunoassay with increased sensitivity for measurement of low concentrations of tobramycin in serum

    NARCIS (Netherlands)

    Touw, D J; de Graaf, A I; de Goede, P

    The limits of quantitation of the assay of tobramycin in serum by the fluorescence polarization immunoassay system marketed by Abbott Laboratories (TDxFLx system) are 0.1 and 10.0 mg/L. For some pharmacokinetic studies, however, a more sensitive analysis is needed. The sensitivity of the TDxFLx

  4. Development of a nucleic acid lateral flow immunoassay for simultaneous detection of Listeria spp. and Listeriamonocytogenes in food

    NARCIS (Netherlands)

    Blazkova, M.; Koets, M.; Rauch, P.; Amerongen, van A.

    2009-01-01

    We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24 h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and

  5. Fabrication and characterization of tosyl-activated magnetic and nonmagnetic monodisperse microspheres for use in microfluic-based ferritin immunoassay

    Czech Academy of Sciences Publication Activity Database

    Reymond, F.; Vollet, Ch.; Plichta, Zdeněk; Horák, Daniel

    2013-01-01

    Roč. 29, č. 2 (2013), s. 532-542 ISSN 8756-7938 R&D Projects: GA MŠk 7E12053; GA ČR GAP503/10/0664 EU Projects: European Commission(XE) 246513 - NADINE Institutional support: RVO:61389013 Keywords : biosensors * electrochemistry * immunoassays Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.883, year: 2013

  6. Strip-based immunoassay for the simultaneous detection of the neonicotinoid insecticides imidacloprid and thiamethoxam in agricultural products

    Science.gov (United States)

    A semiquantitative strip immunoassay was developed for the rapid detection of imidacloprid and thiamethoxam in agricultural products using specific nanocolloidal gold-labeled monoclonal antibodies. The conjugates of imidacloprid-BSA and thiamethoxam-BSA and goat anti-mouse IgG were coated on the ni...

  7. Evaluation of two automated enzyme-immunoassays for detection of thermophilic campylobacters in faecal samples from cattle and swine

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Nielsen, E.M.; Stryhn, H.

    1999-01-01

    We evaluated the performance of two enzyme-immunoassays (EIA) for the detection of naturally occurring, thermophilic Campylobacter spp. found in faecal samples from cattle (n = 21 and n = 26) and swine (n = 43) relative to the standard culture method, and also assuming that none of the tests...

  8. Quantification of patient specific assay interference in different formats of enzyme linked immunoassays for therapeutic monoclonal antibodies

    NARCIS (Netherlands)

    Grebenchtchikov, N.J.; Geurts-Moespot, A.; Heijmen, L.; Laarhoven, H.W.M. van; Herpen, C.M.L. van; Thijs, A.M.J.; Span, P.N.; Sweep, F.C.

    2014-01-01

    BackgroundThe use of therapeutic monoclonal antibodies for clinical purposes has significantly increased in recent years, and so has the need to monitor antibody concentrations. This may be achieved using the well-established enzyme linked immunoassay (ELISA) methods; however, these assays are

  9. Quantification of patient-specific assay interference in different formats of enzyme-linked immunoassays for therapeutic monoclonal antibodies

    NARCIS (Netherlands)

    Grebenchtchikov, Nicolai; Geurts-Moespot, Anneke J.; Heijmen, Linda; van Laarhoven, Hanneke W. M.; van Herpen, Carla M. L.; Thijs, Annemarie M. J.; Span, Paul N.; Sweep, Fred C. G. J.

    2014-01-01

    The use of therapeutic monoclonal antibodies for clinical purposes has significantly increased in recent years, and so has the need to monitor antibody concentrations. This may be achieved using the well-established enzyme linked immunoassay (ELISA) methods; however, these assays are subject to a

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

    Science.gov (United States)

    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

  11. Rapid and Highly Sensitive Non-Competitive Immunoassay for Specific Detection of Nodularin

    Directory of Open Access Journals (Sweden)

    Sultana Akter

    2017-09-01

    Full Text Available Nodularin (NOD is a cyclic penta-peptide hepatotoxin mainly produced by Nodularia spumigena, reported from the brackish water bodies of various parts of the world. It can accumulate in the food chain and, for safety reasons, levels of NOD not only in water bodies but also in food matrices are of interest. Here, we report on a non-competitive immunoassay for the specific detection of NOD. A phage display technique was utilized to interrogate a synthetic antibody phage library for binders recognizing NOD bound to an anti-ADDA (3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E,6(E-dienoic acid monoclonal antibody (Mab. One of the obtained immunocomplex binders, designated SA32C11, showed very high specificity towards nodularin-R (NOD-R over to the tested 10 different microcystins (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF, -LW, -LA, -WR. It was expressed in Escherichia coli as a single chain antibody fragment (scFv fusion protein and used to establish a time-resolved fluorometry-based assay in combination with the anti-ADDA Mab. The detection limit (blank + 3SD of the immunoassay, with a total assay time of 1 h 10 min, is 0.03 µg/L of NOD-R. This represents the most sensitive immunoassay method for the specific detection of NOD reported so far. The assay was tested for its performance to detect NOD using spiked (0.1 to 3 µg/L of NOD-R water samples including brackish sea and coastal water and the recovery ranged from 79 to 127%. Furthermore, a panel of environmental samples, including water from different sources, fish and other marine tissue specimens, were analyzed for NOD using the assay. The assay has potential as a rapid screening tool for the analysis of a large number of water samples for the presence of NOD. It can also find applications in the analysis of the bioaccumulation of NOD in marine organisms and in the food chain.

  12. Sequential injection chemiluminescence immunoassay for nonionic surfactants by using magnetic microbeads

    International Nuclear Information System (INIS)

    Zhang Ruiq; Nakajima, Hizuru; Soh, Nobuaki; Nakano, Koji; Masadome, Takashi; Nagata, Kazumi; Sakamoto, Kazuhira; Imato, Toshihiko

    2007-01-01

    A rapid and sensitive immunoassay based on a sequential injection analysis (SIA) using magnetic microbeads for the determination of alkylphenol polyethoxylates (APnEOs) is described. An SIA system was constructed from a syringe pump, a switching valve, a flow-through type immunoreaction cell equipped with a photon counting unit and a neodymium magnet. Magnetic beads, to which an anti-APnEOs monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in and from the immunoreaction cell were controlled by means of a neodymium magnet and adjusting the flow of a carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-APnEOs monoclonal antibody immobilized on the magnetic beads with a sample APnEOs and a horseradish peroxidase (HRP)-labeled APnEOs in the same sample solution, and was based on the subsequent chemiluminscence reaction of HRP on the magnetic microbeads with a luminol solution containing hydrogen peroxide and p-iodophenol. The anti-APnEOs antibody was immobilized on the magnetic microbeads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of the magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced in the immunoreaction cell and trapped in it by the neodymium magnet, which was equipped beneath the immunoreaction cell. An APnEOs sample solution containing the HRP-labeled APnEOs at a constant concentration, and a luminol solution containing hydrogen peroxide and p-iodophenol were sequentially introduced into the immunoreaction cell, according to an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the immunoreaction cell by collecting the emitted light with a lens. A typical sigmoidal calibration curve was obtained, when the logarithm

  13. Rapid Salmonella detection in experimentally inoculated equine faecal and veterinary hospital environmental samples using commercially available lateral flow immunoassays.

    Science.gov (United States)

    Burgess, B A; Noyes, N R; Bolte, D S; Hyatt, D R; van Metre, D C; Morley, P S

    2015-01-01

    Salmonella enterica is the most commonly reported cause of outbreaks of nosocomial infections in large animal veterinary teaching hospitals and the closure of equine hospitals. Rapid detection may facilitate effective control practices in equine populations. Shipping and laboratory testing typically require ≥48 h to obtain results. Lateral flow immunoassays developed for use in food-safety microbiology provide an alternative that has not been evaluated for use with faeces or environmental samples. We aimed to identify enrichment methods that would allow commercially available rapid Salmonella detection systems (lateral flow immunoassays) to be used in clinical practice with equine faecal and environmental samples, providing test results in 18-24 h. In vitro experiment. Equine faecal and environmental samples were inoculated with known quantities of S. enterica serotype Typhimurium and cultured using 2 different enrichment techniques for faeces and 4 enrichment techniques for environmental samples. Samples were tested blindly using 2 different lateral flow immunoassays and plated on agar media for confirmatory testing. In general, commercial lateral flow immunoassays resulted in fewer false-negative test results with enrichment of 1 g faecal samples in tetrathionate for 18 h, while all environmental sample enrichment techniques resulted in similar detection rates. The limit of detection from spiked samples, ∼4 colony-forming units/g, was similar for all methods evaluated. The lateral flow immunoassays evaluated could reliably detect S. enterica within 18 h, indicating that they may be useful for rapid point-of-care testing in equine practice applications. Additional evaluation is needed using samples from naturally infected cases and the environment to gain an accurate estimate of test sensitivity and specificity and to substantiate further the true value of these tests in clinical practice. © 2014 EVJ Ltd.

  14. Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

    Science.gov (United States)

    Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

    2016-01-01

    A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

  15. Comparison of infrared-excited up-converting phosphors and europium nanoparticles as labels in a two-site immunoassay

    International Nuclear Information System (INIS)

    Ukonaho, Telle; Rantanen, Terhi; Jaemsen, Laura; Kuningas, Katri; Paekkilae, Henna; Loevgren, Timo; Soukka, Tero

    2007-01-01

    Research in the field of immunoassays and labels used in the detection has been recently focused on particulate reporters, which possess very high specific activity that excludes the label as a sensitivity limiting factor. However, the large size and shape of the particulate labels may produce additional problems to immunoassay performance. The aim of this work was to study with two identical non-competitive two-site immunoassays whether up-converting phosphor (UCP) particles are comparable in performance with europium(III) chelate-dyed nanoparticles as particulate labels. In addition we strived to verify the common assumption of the photostability of up-converting phosphor particles supporting their potential applicability in imaging. Detection limits in two-site immunoassay for free prostate-specific antigen (free-PSA) were 0.53 ng L -1 and 1.3 ng L -1 using two different up-converting phosphors and 0.16 ng L -1 using europium(III) nanoparticle. Large size distribution and non-specific binding of up-converting phosphor particles caused assay variation in low analyte concentrations and limited the analytical detection limit. The non-specific binding was the major factor limiting the analytical sensitivity of the immunoassay. The results suggests the need for nanoscaled and uniformely sized UCP-particles to increace the sensitivity and applicability of up-converting phosphor particles. Anti-Stokes photoluminescence of up-converting phosphor particles did not photobleach when measured repeatedly, on the contrary, the time-resolved fluorescence of europium nanoparticles photobleached relatively rapidly

  16. Tropism and pathogenicity of rickettsiae

    Directory of Open Access Journals (Sweden)

    Tsuneo eUchiyama

    2012-06-01

    Full Text Available Rickettsiae are obligate intracellular parasitic bacteria that cause febrile exanthematous illnesses such as Rocky Mountain spotted fever, Mediterranean spotted fever, epidemic and murine typhus, etc. Although the vector ranges of each Rickettsia species are rather restricted; i.e., ticks belonging to Arachnida and lice and fleas belonging to Insecta usually act as vectors for spotted fever group and typhus group rickettsiae, respectively, it would be interesting to elucidate the mechanisms controlling the vector tropism of rickettsiae. This review discusses the factors determining the vector tropism of rickettsiae. In brief, the vector tropism of rickettsiae species is basically consistent with their tropism towards cultured tick and insect cells. The mechanisms responsible for rickettsiae pathogenicity are also described. Recently, genomic analyses of rickettsiae have revealed that they possess several genes that are homologous to those affecting the pathogenicity of other bacteria. Analyses comparing the genomes of pathogenic and nonpathogenic strains of rickettsiae have detected many factors that are related to rickettsial pathogenicity. It is also known that a reduction in the rickettsial genome has occurred during the course of its evolution. Interestingly, Rickettsia species with small genomes, such as Rickettsia prowazekii, are more pathogenic to humans than those with larger genomes. This review also examines the growth kinetics of pathogenic and nonpathogenic species of spotted fever group rickettsiae in mammalian cells. The growth of nonpathogenic species is restricted in these cells, which is mediated, at least in part, by autophagy. The superinfection of nonpathogenic rickettsiae-infected cells with pathogenic rickettsiae results in an elevated yield of the nonpathogenic rickettsiae and the growth of the pathogenic rickettsiae. Autophagy is restricted in these cells. These results are discussed in this review.

  17. Pathogen avoidance by insect predators

    OpenAIRE

    Meyling, Nicolai V.; Ormond, Emma; Roy, Helen E.; Pell, Judith K.

    2008-01-01

    Insects can detect cues related to the risk of attack by their natural enemies. Pathogens are among the natural enemies of insects and entomopathogenic fungi attack a wide array of host species. Evidence documents that social insects in particular have adapted behavioural mechanisms to avoid infection by fungal pathogens. These mechanisms are referred to as 'behavioural resistance'. However, there is little evidence for similar adaptations in non-social insects. We have conducted experime...

  18. Antibiotic Resistance in Foodborne Pathogens

    OpenAIRE

    Walsh, Ciara; Duffy, Geraldine

    2013-01-01

    Wide-spread antibiotic resistance among bacterial pathogens is now a serious public health issue and multi-antibiotic resistance has been reported in many foodborne pathogens including Salmonella and E. coli. A study to determine antibiotic resistance profiles of a range of Salmonella and Verocytotoxigenic E.coli (VTEC) isolated from Irish foods revealed significant levels of antibiotic resistance in the strains. S. typhimurium DT104 were multiantibiotic resistant with 97% resistant to 7 anti...

  19. Molecular detection of human bacterial pathogens

    National Research Council Canada - National Science Library

    Liu, Dongyou

    2011-01-01

    .... Molecular Detection of Human Bacterial Pathogens addresses this issue, with international scientists in respective bacterial pathogen research and diagnosis providing expert summaries on current...

  20. Development of Fully Automated Low-Cost Immunoassay System for Research Applications.

    Science.gov (United States)

    Wang, Guochun; Das, Champak; Ledden, Bradley; Sun, Qian; Nguyen, Chien

    2017-10-01

    Enzyme-linked immunosorbent assay (ELISA) automation for routine operation in a small research environment would be very attractive. A portable fully automated low-cost immunoassay system was designed, developed, and evaluated with several protein analytes. It features disposable capillary columns as the reaction sites and uses real-time calibration for improved accuracy. It reduces the overall assay time to less than 75 min with the ability of easy adaptation of new testing targets. The running cost is extremely low due to the nature of automation, as well as reduced material requirements. Details about system configuration, components selection, disposable fabrication, system assembly, and operation are reported. The performance of the system was initially established with a rabbit immunoglobulin G (IgG) assay, and an example of assay adaptation with an interleukin 6 (IL6) assay is shown. This system is ideal for research use, but could work for broader testing applications with further optimization.

  1. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability

    DEFF Research Database (Denmark)

    Assarsson, Erika; Lundberg, Martin; Holmquist, Göran

    2014-01-01

    reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput...... detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes......, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research....

  2. Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens.

    Science.gov (United States)

    Wang, Zhanhui; Zhang, Huiyan; Ni, Hengjia; Zhang, Suxia; Shen, Jianzhong

    2014-04-11

    In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC50 of the ELISA for enrofloxacin from 1.3 μg L(-1) to as low as 0.07 μg L(-1). The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Optimization of condition for conjugation of enrofloxacin to enzymes in chemiluminescence enzyme immunoassay

    Science.gov (United States)

    Yu, Songcheng; Yu, Fei; Zhang, Hongquan; Qu, Lingbo; Wu, Yongjun

    2014-06-01

    In this study, in order to find out a proper method for conjugation of enrofloxacin to label enzymes, two methods were compared and carbodiimide condensation was proved to be better. The results showed that the binding ratio of enrofloxacin and alkaline phosphatase (ALP) was 8:1 and that of enrofloxacin and horseradish peroxidase (HRP) was 5:1. This indicated that conjugate synthesized by carbodiimide condensation was fit for chemiluminescence enzyme immunoassay (CLEIA). Furthermore, data revealed that dialysis time was an important parameter for conjugation and 6 days was best. Buffer to dilute conjugate had little effect on CLEIA. The storage condition for conjugates was also studied and it was shown that the conjugate was stable at 4 °C with no additive up to 30 days. These data were valuable for establishing CLEIA to quantify enrofloxacin.

  4. Detection of Pesticides and Pesticide Metabolites Using the Cross Reactivity of Enzyme Immunoassays

    Science.gov (United States)

    Thurman, E.M.; Aga, D.S.

    2001-01-01

    Enzyme immunoassay is an important environmental analysis method that may be used to identify many pesticide analytes in water samples. Because of similarities in chemical structure between various members of a pesticide class, there often may be an unwanted response that is characterized by a percentage of cross reactivity. Also, there may be cross reactivity caused by degradation products of the target analyte that may be present in the sample. In this paper, the concept of cross reactivity caused by degradation products or by nontarget analytes is explored as a tool for identification of metabolites or structurally similar compounds not previously known to be present in water samples. Two examples are examined in this paper from various water quality studies. They are alachlor and its metabolite, alachlor ethane sulfonic acid, and atrazine and its class members, prometryn and propazine. A method for using cross reactivity for the detection of these compounds is explained in this paper.

  5. Vertical microreactor stack consist of poly-(tetrafluoroethylene) microfluidics for immunoassay

    International Nuclear Information System (INIS)

    Ukita, Yoshiaki; Kondo, Saki; Utsumi, Yuichi; Takeo, Masahiro; Negoro, Seiji; Kataoka, Chiwa

    2010-01-01

    This paper reports the first application of high-aspect ratio PTFE microstruscute, which fabricated by synchrotron radiation induced photo-evaporation process, to enzyme-linked immunosorvent assay. The advantages of PTFE microstructure for the development of lab-on-a-chip due to the extremely high-aspect ratio microstructure and chemical stability of PTFE is discussed. The results of immunoassay shows the successful detection of analyte (mouse IgG) with detection range with 0-100ng/ml. This result suggests the successful immobilization of antibody (anti-mouse IgG goat antibody) onto the x-ray exposed surface of PTFE microstructure and successful demonstration of antigen-antibody reaction in the PTFE high-aspect ratio microstructure. We also demonstrated the detection of polychlorinated biphenyl (PCB). As the result of demonstration, we successfully detected PCB with ranging analyte concentration of 0.1-10 ng/ml. (author)

  6. Rapid bead-based immunoassay for measurement of mannose-binding lectin

    DEFF Research Database (Denmark)

    Bay, J T; Garred, P

    2009-01-01

    have been developed more automated platforms for MBL analysis is urgently needed. To pursue this, we set out to develop a flexible bead-based MBL immunoassay. Serum was obtained from 98 healthy individuals and 50 patients investigated for possible immunodeficiencies. We used the Luminex xMAP bead array...... coefficient were found be 7.88% and 5.70%, respectively. A close correlation between the new assay and a reference MBL measurement ELISA was found (rho 0.9381, P bead-based assay was less sensitive to interfering anti-murine antibodies in the blood samples than when the antibodies employed were...... used in the reference polystyrene-based ELISA. The new assay could be performed in 3 h with less than 25 microl serum required of each sample. These results show that MBL can be measured readily using a bead-based platform, which may form an efficient basis for a multiplex approach to measure different...

  7. Mapping of Epitopes Occurring in Bovine α(s1)-Casein Variants by Peptide Microarray Immunoassay.

    Science.gov (United States)

    Lisson, Maria; Erhardt, Georg

    2016-01-01

    Immunoglobulin E epitope mapping of milk proteins reveals important information about their immunologic properties. Genetic variants of αS1-casein, one of the major allergens in bovine milk, are until now not considered when discussing the allergenic potential. Here we describe the complete procedure to assess the allergenicity of αS1-casein variants B and C, which are frequent in most breeds, starting from milk with identification and purification of casein variants by isoelectric focusing (IEF) and anion-exchange chromatography, followed by in vitro gastrointestinal digestion of the casein variants, identification of the resulting peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), in silico analysis of the variant-specific peptides as allergenic epitopes, and determination of their IgE-binding properties by microarray immunoassay with cow's milk allergic human sera.

  8. Time-Resolved Fluorescence Immunoassay for C-Reactive Protein Using Colloidal Semiconducting Nanoparticles

    Directory of Open Access Journals (Sweden)

    Pekka Hänninen

    2011-11-01

    Full Text Available Besides the typical short-lived fluorescence with decay times in the nanosecond range, colloidal II/VI semiconductor nanoparticles dispersed in buffer also possess a long-lived fluorescence component with decay times in the microsecond range. Here, the signal intensity of the long-lived luminescence at microsecond range is shown to increase 1,000-fold for CdTe nanoparticles in PBS buffer. This long-lived fluorescence can be conveniently employed for time-gated fluorescence detection, which allows for improved signal-to-noise ratio and thus the use of low concentrations of nanoparticles. The detection principle is demonstrated with a time-resolved fluorescence immunoassay for the detection of C-reactive protein (CRP using CdSe-ZnS nanoparticles and green light excitation.

  9. Synthesis-based approach toward direct sandwich immunoassay for ciguatoxin CTX3C.

    Science.gov (United States)

    Oguri, Hiroki; Hirama, Masahiro; Tsumuraya, Takeshi; Fujii, Ikuo; Maruyama, Megumi; Uehara, Hisatoshi; Nagumo, Yoko

    2003-06-25

    Ciguatoxins are the major causative toxins of ciguatera seafood poisoning. Limited availability of ciguatoxins has hampered the development of a reliable and specific immunoassay for detecting these toxins in contaminated fish. Monoclonal antibodies (mAbs) specific against both ends of ciguatoxin CTX3C were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens, 3 and 4, in place of the natural toxin. Haptenic groups that possess a surface area larger than 400 A(2) were required to produce mAbs that can bind strongly to CTX3C itself. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was established to detect CTX3C at the ppb level with no cross-reactivity against other related marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin.

  10. Developments of sensitive immunoassays for detection of antibodies against hepatitis B surface antigen

    Energy Technology Data Exchange (ETDEWEB)

    Ionescu-Matiu, I; Sanchez, Y; Dreesman, G R [Baylor Univ., Houston, TX (USA). Coll. of Medicine; Fields, H A [Centers for Disease Control, Public Health Service, Department of Health and Human Services, Phoenix, AZ (USA)

    1983-01-01

    Three micro solid phase immunoassays (a micro-SPRIA and two ELISA techniques) were developed and tested for the detection of anti-HBs antibodies. Two different crosslinkers (glutaraldehyde and N-succinimidyl 3-(2-pyridyldithio) propionate) were used to couple a goat anti-mouse IgG reagent to alkaline phosphatase for use as enzyme-labeled probes in the two ELISA tests. With the latter cross-linker, a defined conjugate with a 1 : 1 antibody-enzyme molar ratio was obtained. The sensitivities of micro-SPRIA and the two types of ELISA were compared to that of the commercial solid phase radioimmunoassay AUSAB test. All three microtests were significantly more sensitive than the AUSAB test. The ELISA using the glutaraldehyde cross-linked conjugate was 3-5 times less sensitive than micro-SPRIA, while the ELISA using the disulfide-linked conjugate was 2.6-4.0 times more sensitive than micro-SPRIA.

  11. Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Brock, I; Weldingh, K; Leyten, EM

    2004-01-01

    Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection.Brock I, Weldingh K, Leyten EM, Arend SM, Ravn P, Andersen P. Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. The currently used...... method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four...... recently identified antigens (Rv2653, Rv2654, Rv3873, and Rv3878) from genomic regions that are lacking from the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine strains as well as from the most common nontuberculous mycobacteria. The fine specificity of potential epitopes in these molecules...

  12. Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Lyndrup, J

    1995-01-01

    We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA......, and cytosolic progesterone receptors (PR) measured by LBA were also studied. While ERc concentrations determined by LBA and EIA were highly correlated (r: 0.94), ERc values detected by LBA were approximately twice those found by EIA (median values of ERc: 155 vs. 64 fmol/mg cytosol protein, DCC vs. EIA......). The percentages of ERc positive tumors were 89% by LBA and 77% by EIA. The median fraction of total ER present as ERn was 63%. PR levels correlated positively with ERn concentrations (r: 0.73). We explore possible reasons why greater concentrations of ERc are determined by estradiol binding than by the ER-EIA kit...

  13. Concordance Between Different Amyloid Immunoassays and Visual Amyloid Positron Emission Tomographic Assessment.

    Science.gov (United States)

    Janelidze, Shorena; Pannee, Josef; Mikulskis, Alvydas; Chiao, Ping; Zetterberg, Henrik; Blennow, Kaj; Hansson, Oskar

    2017-12-01

    Visual assessment of amyloid positron emission tomographic (PET) images has been approved by regulatory authorities for clinical use. Several immunoassays have been developed to measure β-amyloid (Aβ) 42 in cerebrospinal fluid (CSF). The agreement between CSF Aβ42 measures from different immunoassays and visual PET readings may influence the use of CSF biomarkers and/or amyloid PET assessment in clinical practice and trials. To determine the concordance between CSF Aβ42 levels measured using 5 different immunoassays and visual amyloid PET analysis. The study included 262 patients with mild cognitive impairment or subjective cognitive decline from the Swedish BioFINDER (Biomarkers for Identifying Neurodegenerative Disorders Early and Reliably) cohort (recruited from September 1, 2010, through December 31, 2014) who had undergone flutemetamol F 18 ([18F]flutemetamol)-labeled PET. Levels of CSF Aβ42 were analyzed using the classic INNOTEST and the newer modified INNOTEST, fully automated Lumipulse (FL), EUROIMMUN (EI), and Meso Scale Discovery (MSD) assays. Concentrations of CSF Aβ were assessed using an antibody-independent mass spectrometry-based reference measurement procedure. The concordance of CSF Aβ42 levels and Aβ42:Aβ40 and Aβ42:tau ratios with visual [18F]flutemetamol PET status. Of 262 participants (mean [SD] age, 70.9 [5.5] years), 108 were women (41.2%) and 154 were men (58.8%). The mass spectrometry-derived Aβ42 values showed higher correlations with the modified Aβ42-INNOTEST (r = 0.97), Aβ42-FL (r = 0.93), Aβ42-EI (r = 0.93), and Aβ42-MSD (r = 0.95) assays compared with the classic Aβ42-INNOTEST assay (r = 0.88; P ≤ .01). The signal in the classic Aβ42-INNOTEST assay was partly quenched by recombinant Aβ1-40 peptide. However, the classic Aβ42-INNOTEST assay showed better concordance with visual [18F]flutemetamol PET status (area under the receiver operating characteristic curve [AUC], 0.92) compared with the

  14. Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Brock, I; Weldingh, K; Leyten, EM

    2004-01-01

    Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection.Brock I, Weldingh K, Leyten EM, Arend SM, Ravn P, Andersen P. Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. The currently used...... method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four...... selected and combined the specific peptide stretches from the four proteins not recognized by M. bovis BCG-vaccinated individuals. These peptide stretches were tested with peripheral blood mononuclear cells obtained from patients with microscopy- or culture-confirmed tuberculosis and from healthy M. bovis...

  15. A Homogeneous Time-Resolved Fluorescence Immunoassay Method for the Measurement of Compound W.

    Science.gov (United States)

    Huang, Biao; Yu, Huixin; Bao, Jiandong; Zhang, Manda; Green, William L; Wu, Sing-Yung

    2018-01-01

    Using compound W (a 3,3'-diiodothyronine sulfate [T 2 S] immuno-crossreactive material)-specific polyclonal antibodies and homogeneous time-resolved fluorescence immunoassay assay techniques (AlphaLISA) to establish an indirect competitive compound W (ICW) quantitative detection method. Photosensitive particles (donor beads) coated with compound W or T 2 S and rabbit anti-W antibody were incubated with biotinylated goat anti-rabbit antibody. This constitutes a detection system with streptavidin-coated acceptor particle. We have optimized the test conditions and evaluated the detection performance. The sensitivity of the method was 5 pg/mL, and the detection range was 5 to 10 000 pg/mL. The intra-assay coefficient of variation averages W levels in extracts of maternal serum samples. This may have clinical application to screen congenital hypothyroidism in utero.

  16. A reactor/separator device for use in automated solid phase immunoassay

    International Nuclear Information System (INIS)

    Farina, P.R.; Ordonez, K.P.; Siewers, I.J.

    1979-01-01

    A reactor/separator device is described for use in automated solid phase immunoassay, including radioimmunoassays. The device is a column fitted at the bottom portion with a water impermeable disc which can hold, for example, immunoabsorbents, immobilized antisera or ion exchange resins. When the contents of the column supported by the disc are brought into contact with an aqueous phase containing reagents or reactants, a chemical reaction is initiated. After the reaction, centrifugally applied pressure forces the aqueous phase through the filter disc making it water permeable and separating a desired component for subsequent analysis. The reactor/separator device of the present invention permits kinetic solid phase assays (non-equilibrium conditions) to be carried out which would be difficult to perform by other conventional methods. (author)

  17. Optical remote sensing

    CERN Document Server

    Prasad, Saurabh; Chanussot, Jocelyn

    2011-01-01

    Optical remote sensing relies on exploiting multispectral and hyper spectral imagery possessing high spatial and spectral resolutions respectively. These modalities, although useful for most remote sensing tasks, often present challenges that must be addressed for their effective exploitation. This book presents current state-of-the-art algorithms that address the following key challenges encountered in representation and analysis of such optical remotely sensed data: challenges in pre-processing images, storing and representing high dimensional data, fusing different sensor modalities, patter

  18. REMOTE SENSING IN OCEANOGRAPHY.

    Science.gov (United States)

    remote sensing from satellites. Sensing of oceanographic variables from aircraft began with the photographing of waves and ice. Since then remote measurement of sea surface temperatures and wave heights have become routine. Sensors tested for oceanographic applications include multi-band color cameras, radar scatterometers, infrared spectrometers and scanners, passive microwave radiometers, and radar imagers. Remote sensing has found its greatest application in providing rapid coverage of large oceanographic areas for synoptic and analysis and

  19. Analytical validation of Gentian NGAL particle-enhanced enhanced turbidimetric immunoassay (PETIA

    Directory of Open Access Journals (Sweden)

    Gian Luca Salvagno

    2017-08-01

    Full Text Available Objectives: This study was designed to validate the analytical performance of the new Gentian particle-enhanced enhanced turbidimetric immunoassay (PETIA for measuring neutrophil gelatinase-associated lipocalin (NGAL in serum samples. Design and methods: Analytical validation of the Gentian NGAL assay was carried out on a Roche Cobas c501 and was based on assessment of limit of blank (LOB, limit of detection (LOD, functional sensitivity, imprecision, linearity and concordance with the BioPorto NGAL test. Results: The LOB and LOD of Gentian NGAL were found to be 3.8 ng/mL and 6.3 ng/mL, respectively. An analytical coefficient of variation (CV of 20% corresponded to a NGAL value of 10 ng/mL. The intra-assay and inter-assay imprecision (CV was between 0.4 and 5.2% and 0.6 and 7.1% and the total imprecision (CV was 3.7%. The linearity was optimal at NGAL concentrations between 37 and 1420 ng/mL (r=1.00; p<0.001. An excellent correlation was observed between values measured with Gentian NGAL and BioPorto NGAL in 74 routine serum samples (r=0.993. The mean percentage bias of the Gentian assay versus the Bioporto assay was +3.1% (95% CI, +1.6% to +4.5%. Conclusions: These results show that Gentian NGAL may be a viable option to other commercial immunoassays for both routine and urgent assessment of serum NGAL. Keywords: Neutrophil gelatinase-associated lipocalin, NGAL, Analytical validation, Acute kidney injury

  20. Development and statistical assessment of a paper-based immunoassay for detection of tumor markers

    Energy Technology Data Exchange (ETDEWEB)

    Mazzu-Nascimento, Thiago [Instituto de Química de São Carlos, Universidade de São Paulo, 13566-590, São Carlos, SP (Brazil); Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Campinas, SP (Brazil); Morbioli, Giorgio Gianini [Instituto de Química de São Carlos, Universidade de São Paulo, 13566-590, São Carlos, SP (Brazil); Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Campinas, SP (Brazil); School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332 (United States); Milan, Luis Aparecido [Departamento de Estatística, Universidade Federal de São Carlos, São Carlos, SP (Brazil); Donofrio, Fabiana Cristina [Instituto de Ciências da Saúde, Universidade Federal de Mato Grosso, 78557-267, Sinop, MT (Brazil); Mestriner, Carlos Alberto [Wama Produtos para Laboratório Ltda, 13560-971, São Carlos, SP (Brazil); Carrilho, Emanuel, E-mail: emanuel@iqsc.usp.br [Instituto de Química de São Carlos, Universidade de São Paulo, 13566-590, São Carlos, SP (Brazil); Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Campinas, SP (Brazil)

    2017-01-15

    Paper-based assays are an attractive low-cost option for clinical chemistry testing, due to characteristics such as short time of analysis, low consumption of samples and reagents, and high portability of assays. However, little attention has been given to the evaluation of the performance of these simple tests, which should include the use of a statistical approach to define the choice of best cut-off value for the test. The choice of the cut-off value impacts on the sensitivity and specificity of the bioassay. Here, we developed a paper-based immunoassay for the detection of the carcinoembryonic antigen (CEA) and performed a statistical assessment to establish the assay's cut-off value using the Youden's J index (68.28 A.U.), what allowed for a gain in sensibility (0.86) and specificity (1.0). We also discuss about the importance of defining a gray zone as a safety margin for test (±12% over the cut-off value), eliminating all false positives and false negatives outcomes and avoiding misleading results. The test accuracy was calculated as the area under the curve (AUC) of the receiver operating characteristic (ROC) curve, presenting a value of 0.97, what classifies this test as highly accurate. We propose here a low-cost method capable of detecting carcinoembryonic antigen (CEA) in human serum samples, highlighting the importance of statistical tools to evaluate a new low-cost diagnostic method. - Highlights: • A paper-based sandwich immunoassay protocol for detection of tumor markers. • A statistical approach to define cut-off values and measuring test's sensitivity, specificity and accuracy. • A simple way to create a gray zone, avoiding false positive and false negative outcomes.

  1. System-on-fluidics immunoassay device integrating wireless radio-frequency-identification sensor chips.

    Science.gov (United States)

    Yazawa, Yoshiaki; Oonishi, Tadashi; Watanabe, Kazuki; Shiratori, Akiko; Funaoka, Sohei; Fukushima, Masao

    2014-09-01

    A simple and sensitive point-of-care-test (POCT) device for chemiluminescence (CL) immunoassay was devised and tested. The device consists of a plastic flow-channel reactor and two wireless-communication sensor chips, namely, a photo-sensor chip and a temperature-sensor chip. In the flow-channel reactor, a target antigen is captured by an antibody immobilized on the inner wall of the flow-channel and detected with enzyme labeled antibody by using CL substrate. The CL signal corresponding to the amount of antigen is measured by a newly developed radio-frequency-identification (RFID) sensor, which enables batteryless operation and wireless data communication with an external reader. As for the POCT device, its usage environment, especially temperature, varies for each measurement. Hence, temperature compensation is a key issue in regard to eliminating dark-signal fluctuation, which is a major factor in deterioration of the precision of the POCT device. A two-stage temperature-compensation scheme was adopted. As for the first stage, the signals of two photodiodes, one with an open window and one with a sealed window, integrated on the photo-sensor chip are differentiated to delete the dark signal. As for the second stage, the differentiated signal fluctuation caused by a temperature variation is compensated by using the other sensor chip (equipped with a temperature sensor). The dark-level fluctuation caused by temperature was reduced from 0.24 to 0.02 pA/°C. The POCT device was evaluated as a CL immunoassay of thyroid-stimulating hormone (TSH). The flow rate of the CL reagent in the flow channel was optimized. As a result, the detection limit of the POCT device was 0.08 ng/ml (i.e., 0.4 μIU/ml). Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. A Nanoparticle-Lectin Immunoassay Improves Discrimination of Serum CA125 from Malignant and Benign Sources.

    Science.gov (United States)

    Gidwani, Kamlesh; Huhtinen, Kaisa; Kekki, Henna; van Vliet, Sandra; Hynninen, Johanna; Koivuviita, Niina; Perheentupa, Antti; Poutanen, Matti; Auranen, Annika; Grenman, Seija; Lamminmäki, Urpo; Carpen, Olli; van Kooyk, Yvette; Pettersson, Kim

    2016-10-01

    Measurement of serum cancer antigen 125 (CA125) is the standard approach for epithelial ovarian cancer (EOC) diagnostics and follow-up. However, the clinical specificity is not optimal because increased values are also detected in healthy controls and in benign diseases. CA125 is known to be differentially glycosylated in EOC, potentially offering a way to construct CA125 assays with improved cancer specificity. Our goal was to identify carbohydrate-reactive lectins for discriminating between CA125 originating from EOC and noncancerous sources. CA125 from the OVCAR-3 cancer cell line, placental homogenate, and ascites fluid from patients with cirrhosis were captured on anti-CA125 antibody immobilized on microtitration wells. A panel of lectins, each coated onto fluorescent europium-chelate-doped 97-nm nanoparticles (Eu(+3)-NPs), was tested for detection of the immobilized CA125. Serum samples from high-grade serous EOC or patients with endometriosis and healthy controls were analyzed. By using macrophage galactose-type lectin (MGL)-coated Eu(+3)-NPs, an analytically sensitive CA125 assay (CA125(MGL)) was achieved that specifically recognized the CA125 isoform produced by EOC, whereas the recognition of CA125 from nonmalignant conditions was reduced. Serum CA125(MGL) measurement better discriminated patients with EOC from endometriosis compared to conventional immunoassay. The discrimination was particularly improved for marginally increased CA125 values and for earlier detection of EOC progression. The new CA125(MGL) assay concept could help reduce the false-positive rates of conventional CA125 immunoassays. The improved analytical specificity of this test approach is dependent on a discriminating lectin immobilized in large numbers on Eu(+3)-NPs, providing both an avidity effect and signal amplification. © 2016 American Association for Clinical Chemistry.

  3. Mobile Phone Ratiometric Imaging Enables Highly Sensitive Fluorescence Lateral Flow Immunoassays without External Optical Filters.

    Science.gov (United States)

    Shah, Kamal G; Singh, Vidhi; Kauffman, Peter C; Abe, Koji; Yager, Paul

    2018-05-14

    Paper-based diagnostic tests based on the lateral flow immunoassay concept promise low-cost, point-of-care detection of infectious diseases, but such assays suffer from poor limits of detection. One factor that contributes to poor analytical performance is a reliance on low-contrast chromophoric optical labels such as gold nanoparticles. Previous attempts to improve the sensitivity of paper-based diagnostics include replacing chromophoric labels with enzymes, fluorophores, or phosphors at the expense of increased fluidic complexity or the need for device readers with costly optoelectronics. Several groups, including our own, have proposed mobile phones as suitable point-of-care readers due to their low cost, ease of use, and ubiquity. However, extant mobile phone fluorescence readers require costly optical filters and were typically validated with only one camera sensor module, which is inappropriate for potential point-of-care use. In response, we propose to couple low-cost ultraviolet light-emitting diodes with long Stokes-shift quantum dots to enable ratiometric mobile phone fluorescence measurements without optical filters. Ratiometric imaging with unmodified smartphone cameras improves the contrast and attenuates the impact of excitation intensity variability by 15×. Practical application was shown with a lateral flow immunoassay for influenza A with nucleoproteins spiked into simulated nasal matrix. Limits of detection of 1.5 and 2.6 fmol were attained on two mobile phones, which are comparable to a gel imager (1.9 fmol), 10× better than imaging gold nanoparticles on a scanner (18 fmol), and >2 orders of magnitude better than gold nanoparticle-labeled assays imaged with mobile phones. Use of the proposed filter-free mobile phone imaging scheme is a first step toward enabling a new generation of highly sensitive, point-of-care fluorescence assays.

  4. Development of monoclonal antibody-based immunoassays for the analysis of bisphenol A in canned vegetables.

    Science.gov (United States)

    Moreno, María J; D'Arienzo, Pasquale; Manclús, Juan J; Montoya, Angel

    2011-01-01

    The aim of this work was the development of monoclonal antibodies (MAbs) and highly sensitive immunoassays (ELISAs) to bisphenol A (BPA), a well-known endocrine disruptor able to migrate from the internal coating of cans to food contained inside, particularly vegetables. To produce MAbs to BPA, four synthetic compounds were conjugated to proteins and used as immunizing haptens in mice. By applying hybridoma technology, several MAbs were produced and selected. These antibodies were characterized in the conjugate-coated and in the antibody-coated formats, using both homologous and heterologous conjugates. Three indirect ELISA based on the MAbs showing the highest affinity to BPA were selected. The limit of detection of the most sensitive ELISA was 0.22 nM (0.05 ng/mL), with an I₅₀ value of around 1 nM (0.23 ng/mL). An homologous ELISA based on the MAb BPAB-11 was applied to the simple, direct determination of BPA in the liquid portion of canned artichoke, peas, and sweet corn. Only sample dilution in an appropriate saline buffer was required to minimize matrix effects and to enter the ELISA working range. Recovery and precision of the method were evaluated by spiking the liquid portion of these cans with BPA at 20, 50, and 100 ng/mL. Coefficients of variation were below 20% in most cases. With regard to recovery, the analytical data obtained were also acceptable. This immunoassay has therefore proved its potential as a new tool for the rapid, sensitive and accurate determination of BPA in canned food.

  5. A Portable Immunoassay Platform for Multiplexed Detection of Biotoxins in Clinical and Environmental Samples

    Energy Technology Data Exchange (ETDEWEB)

    Koh, Chung-Yan [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Piccini, Matthew Ernest [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Cepheid, Sunnyvale, CA (United States); Schaff, Ulrich Y. [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Sandstone Diagnostics, Livermore, CA (United States); Stanker, Larry H. [US Dept. of Agriculture, Albany, CA (United States). Western Regional Research Center, Foodborne Contaminants Research Unit; Cheng, Luisa W. [US Dept. of Agriculture, Albany, CA (United States). Western Regional Research Center, Foodborne Contaminants Research Unit; Ravichandran, Easwaran [Univ. of Massachusetts, Dartmouth, MA (United States); Singh, Bal-Ram [Univ. of Massachusetts, Dartmouth, MA (United States); Sommer, Greg J. [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Sandstone Diagnostics, Livermore, CA (United States); Singh, Anup K. [Sandia National Lab. (SNL-CA), Livermore, CA (United States)

    2015-01-01

    Multiple cases of attempted bioterrorism events using biotoxins have highlighted the urgent need for tools capable of rapid screening of suspect samples in the field (e.g., mailroom and public events). We present a portable microfluidic device capable of analyzing environmental (e.g., white powder), food (e.g., milk) and clinical (e.g., blood) samples for multiplexed detection of biotoxins. The device is rapid (<15-30 min sample-to-answer), sensitive (< 0.08 pg/mL detection limit for botulinum toxin), multiplexed (up to 64 parallel assays) and capable of analyzing small volume samples (< 20 μL total sample input). The immunoassay approach (SpinDx) is based on binding of toxins in a sample to antibody-laden capture particles followed by sedimentation of particles through a density-media in a microfluidic disk and quantification using a laser-induced fluorescence detector. A direct, blinded comparison with a gold standard ELISA revealed a 5-fold more sensitive detection limit for botulinum toxin while requiring 250-fold less sample volume and a 30 minute assay time with a near unity correlation. A key advantage of the technique is its compatibility with a variety of sample matrices with no additional sample preparation required. Ultrasensitive quantification has been demonstrated from direct analysis of multiple clinical, environmental and food samples, including white powder, whole blood, saliva, salad dressing, whole milk, peanut butter, half and half, honey, and canned meat. We believe that this device can met an urgent need in screening both potentially exposed people as well as suspicious samples in mail-rooms, airports, public sporting venues and emergency rooms. The general-purpose immunodiagnostics device can also find applications in screening of infectious and systemic diseases or serve as a lab device for conducting rapid immunoassays.

  6. Hyperspectral sensing of forests

    Science.gov (United States)

    Goodenough, David G.; Dyk, Andrew; Chen, Hao; Hobart, Geordie; Niemann, K. Olaf; Richardson, Ash

    2007-11-01

    Canada contains 10% of the world's forests covering an area of 418 million hectares. The sustainable management of these forest resources has become increasingly complex. Hyperspectral remote sensing can provide a wealth of new and improved information products to resource managers to make more informed decisions. Research in this area has demonstrated that hyperspectral remote sensing can be used to create more accurate products for forest inventory, forest health, foliar biochemistry, biomass, and aboveground carbon than are currently available. This paper surveys recent methods and results in hyperspectral sensing of forests and describes space initiatives for hyperspectral sensing.

  7. LIGO sensing system performance

    CERN Document Server

    Landry, M

    2002-01-01

    The optical sensing subsystem of a LIGO interferometer is described. The system includes two complex interferometric sensing schemes to control test masses in length and alignment. The length sensing system is currently employed on all LIGO interferometers to lock coupled cavities on resonance. Auto-alignment is to be accomplished by a wavefront-sensing scheme which automatically corrects for angular fluctuations of the test masses. Improvements in lock stability and duration are noted when the wavefront auto-alignment system is employed. Preliminary results from the commissioning of the 2 km detector in Washington are shown.

  8. Intelligent environmental sensing

    CERN Document Server

    Mukhopadhyay, Subhas

    2015-01-01

    Developing environmental sensing and monitoring technologies become essential especially for industries that may cause severe contamination. Intelligent environmental sensing uses novel sensor techniques, intelligent signal and data processing algorithms, and wireless sensor networks to enhance environmental sensing and monitoring. It finds applications in many environmental problems such as oil and gas, water quality, and agriculture. This book addresses issues related to three main approaches to intelligent environmental sensing and discusses their latest technological developments. Key contents of the book include:   Agricultural monitoring Classification, detection, and estimation Data fusion Geological monitoring Motor monitoring Multi-sensor systems Oil reservoirs monitoring Sensor motes Water quality monitoring Wireless sensor network protocol  

  9. Advanced Remote Sensing Research

    Science.gov (United States)

    Slonecker, Terrence; Jones, John W.; Price, Susan D.; Hogan, Dianna

    2008-01-01

    'Remote sensing' is a generic term for monitoring techniques that collect information without being in physical contact with the object of study. Overhead imagery from aircraft and satellite sensors provides the most common form of remotely sensed data and records the interaction of electromagnetic energy (usually visible light) with matter, such as the Earth's surface. Remotely sensed data are fundamental to geographic science. The Eastern Geographic Science Center (EGSC) of the U.S. Geological Survey (USGS) is currently conducting and promoting the research and development of three different aspects of remote sensing science: spectral analysis, automated orthorectification of historical imagery, and long wave infrared (LWIR) polarimetric imagery (PI).

  10. Optical Remote Sensing Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — The Optical Remote Sensing Laboratory deploys rugged, cutting-edge electro-optical instrumentation for the collection of various event signatures, with expertise in...

  11. Targeting quorum sensing in Pseudomonas aeruginosa biofilms

    DEFF Research Database (Denmark)

    Jakobsen, Tim Holm; Bjarnsholt, Thomas; Jensen, Peter Østrup

    2013-01-01

    Bacterial resistance to conventional antibiotics combined with an increasing acknowledgement of the role of biofilms in chronic infections has led to a growing interest in new antimicrobial strategies that target the biofilm mode of growth. In the aggregated biofilm mode, cell-to-cell communication...... alternative antibacterial strategies. Here, we review state of the art research of quorum sensing inhibitors against the opportunistic human pathogen Pseudomonas aeruginosa, which is found in a number of biofilm-associated infections and identified as the predominant organism infecting the lungs of cystic...

  12. Calcium sensing in exocytosis

    DEFF Research Database (Denmark)

    Gustavsson, Natalia; Wu, Bingbing; Han, Weiping

    2012-01-01

    an increase in intracellular calcium levels. Besides the triggering role, calcium signaling modulates the precise amount and kinetics of vesicle release. Thus, it is a central question to understand the molecular machineries responsible for calcium sensing in exocytosis. Here we provide an overview of our...... current understanding of calcium sensing in neurotransmitter release and hormone secretion....

  13. Sense and Sensibility

    NARCIS (Netherlands)

    Austen, Jane

    2005-01-01

    Two sisters of opposing temperament but who share the pangs of tragic love provide the subjects for Sense and Sensibility. Elinor, practical and conventional, the epitome of sense, desires a man who is promised to another woman. Marianne, emotional and sentimental, the epitome of sensibility, loses

  14. Mobile teleoperator remote sensing

    International Nuclear Information System (INIS)

    Hall, E.L.

    1986-01-01

    Sensing systems are an important element of mobile teleoperators and robots. This paper discusses certain problems and limitations of vision and other sensing systems with respect to operations in a radiological accident environment. Methods which appear promising for near-term improvements to sensor technology are described. 3 refs

  15. Deterministic Compressed Sensing

    Science.gov (United States)

    2011-11-01

    39 4.3 Digital Communications . . . . . . . . . . . . . . . . . . . . . . . . . 40 4.4 Group Testing ...deterministic de - sign matrices. All bounds ignore the O() constants. . . . . . . . . . . 131 xvi List of Algorithms 1 Iterative Hard Thresholding Algorithm...sensing is information theoretically possible using any (2k, )-RIP sensing matrix . The following celebrated results of Candès, Romberg and Tao [54

  16. Hyperspectral remote sensing

    CERN Document Server

    Eismann, Michael

    2012-01-01

    Hyperspectral remote sensing is an emerging, multidisciplinary field with diverse applications that builds on the principles of material spectroscopy, radiative transfer, imaging spectrometry, and hyperspectral data processing. This book provides a holistic treatment that captures its multidisciplinary nature, emphasizing the physical principles of hyperspectral remote sensing.

  17. Mapping sense(s) of place

    DEFF Research Database (Denmark)

    Skovse, Astrid Ravn; Hovy, Dirk; Johannsen, Anders Trærup

    2016-01-01

    , the question of how to tap into this constitutes a methodological challenge to researchers (Latham 2003, Hall 2009). This paper presents an experimental method aimed at eliciting data on sense of place and everyday mobility in a feasible and low-tech manner through the use of mental maps and mobility maps...... for answering questions about the relationship between places, speakers and linguistic practice....

  18. Multiplex immunoassay of lower genital tract mucosal fluid from women attending an urban STD clinic shows broadly increased IL1ß and lactoferrin.

    Directory of Open Access Journals (Sweden)

    Gregory T Spear

    Full Text Available BACKGROUND: More than one million new cases of sexually transmitted diseases (STDs occur each day. The immune responses and inflammation induced by STDs and other frequent non-STD microbial colonizations (i.e. Candida and bacterial vaginosis can have serious pathologic consequences in women including adverse pregnancy outcomes, infertility and increased susceptibility to infection by other pathogens. Understanding the types of immune mediators that are elicited in the lower genital tract by these infections/colonizations can give important insights into the innate and adaptive immune pathways that are activated and lead to strategies for preventing pathologic effects. METHODOLOGY/PRINCIPAL FINDINGS: 32 immune mediators were measured by multiplexed immunoassays to assess the immune environment of the lower genital tract mucosa in 84 women attending an urban STD clinic. IL-3, IL-1ß, VEGF, angiogenin, IL-8, ß2Defensin and ß3Defensin were detected in all subjects, Interferon-α was detected in none, while the remaining mediators were detected in 40% to 93% of subjects. Angiogenin, VEGF, FGF, IL-9, IL-7, lymphotoxin-α and IL-3 had not been previously reported in genital mucosal fluid from women. Strong correlations were observed between levels of TNF-α, IL-1ß and IL-6, between chemokines IP-10 and MIG and between myeloperoxidase, IL-8 and G-CSF. Samples from women with any STD/colonization had significantly higher levels of IL-8, IL-3, IL-7, IL-1ß, lactoferrin and myeloperoxidase. IL-1ß and lactoferrin were significantly increased in gonorrhea, Chlamydia, cervicitis, bacterial vaginosis and trichomoniasis. CONCLUSIONS/SIGNIFICANCE: These studies show that mucosal fluid in general appears to be an environment that is rich in immune mediators. Importantly, IL-1ß and lactoferrin are biomarkers for STDs/colonizations providing insights into immune responses and pathogenesis at this mucosal site.

  19. Comparison of clinical performance of antigen basedenzyme immunoassay (EIA and major outer membrane protein (MOMP-PCR for detection of genital Chlamydia trachomatis infection

    Directory of Open Access Journals (Sweden)

    Mahmoud Nateghi Rostami

    2016-06-01

    Full Text Available Background: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Objective: Performances of enzyme immunoassay (EIA and major outer membrane protein (MOMP-polymerase chain reaction (PCR for diagnosis of genital C.trachomatis infection in women were compared. Materials and Methods: In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV and negative predictive values (NPV were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. Results: In total, 37 (7.14% cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMPPCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48. Conclusion: C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.

  20. Comparison of clinical performance of antigen based-enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection.

    Science.gov (United States)

    Nateghi Rostami, Mahmoud; Hossein Rashidi, Batool; Aghsaghloo, Fatemeh; Nazari, Razieh

    2016-06-01

    Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.

  1. Hydroball string sensing system

    International Nuclear Information System (INIS)

    Hurwitz, M.J.; Ekeroth, D.E.; Squarer, D.

    1991-01-01

    This patent describes a hydroball string sensing system for a nuclear reactor having a core containing a fluid at a fluid pressure. It comprises a tube connectable to the nuclear reactor so that the fluid can flow within the tube at a fluid pressure that is substantially the same as the fluid pressure of the nuclear reactor core; a hydroball string including - a string member having objects positioned therealong with a specified spacing, the object including a plurality of hydroballs, and bullet members positioned at opposing ends of the string member; first sensor means, positioned outside a first segment of the tube, for sensing one of the objects being positioned within the first segment, and for providing a sensing signal responsive to the sensing of the first sensing means

  2. Adaptation to the Host Environment by Plant-Pathogenic Fungi.

    Science.gov (United States)

    van der Does, H Charlotte; Rep, Martijn

    2017-08-04

    Many fungi can live both saprophytically and as endophyte or pathogen inside a living plant. In both environments, complex organic polymers are used as sources of nutrients. Propagation inside a living host also requires the ability to respond to immune responses of the host. We review current knowledge of how plant-pathogenic fungi do this. First, we look at how fungi change their global gene expression upon recognition of the host environment, leading to secretion of effectors, enzymes, and secondary metabolites; changes in metabolism; and defense against toxic compounds. Second, we look at what is known about the various cues that enable fungi to sense the presence of living plant cells. Finally, we review literature on transcription factors that participate in gene expression in planta or are suspected to be involved in that process because they are required for the ability to cause disease.

  3. Chemical signaling between plants and plant-pathogenic bacteria.

    Science.gov (United States)

    Venturi, Vittorio; Fuqua, Clay

    2013-01-01

    Studies of chemical signaling between plants and bacteria in the past have been largely confined to two models: the rhizobial-legume symbiotic association and pathogenesis between agrobacteria and their host plants. Recent studies are beginning to provide evidence that many plant-associated bacteria undergo chemical signaling with the plant host via low-molecular-weight compounds. Plant-produced compounds interact with bacterial regulatory proteins that then affect gene expression. Similarly, bacterial quorum-sensing signals result in a range of functional responses in plants. This review attempts to highlight current knowledge in chemical signaling that takes place between pathogenic bacteria and plants. This chemical communication between plant and bacteria, also referred to as interkingdom signaling, will likely become a major research field in the future, as it allows the design of specific strategies to create plants that are resistant to plant pathogens.

  4. Respiratory Pathogens Adopt a Chronic Lifestyle in Response to Bile

    Science.gov (United States)

    Reen, F. Jerry; Woods, David F.; Mooij, Marlies J.; Adams, Claire; O'Gara, Fergal

    2012-01-01

    Chronic respiratory infections are a major cause of morbidity and mortality, most particularly in Cystic Fibrosis (CF) patients. The recent finding that gastro-esophageal reflux (GER) frequently occurs in CF patients led us to investigate the impact of bile on the behaviour of Pseudomonas aeruginosa and other CF-associated respiratory pathogens. Bile increased biofilm formation, Type Six Secretion, and quorum sensing in P. aeruginosa, all of which are associated with the switch from acute to persistent infection. Furthermore, bile negatively influenced Type Three Secretion and swarming motility in P. aeruginosa, phenotypes associated with acute infection. Bile also modulated biofilm formation in a range of other CF-associated respiratory pathogens, including Burkholderia cepacia and Staphylococcus aureus. Therefore, our results suggest that GER-derived bile may be a host determinant contributing to chronic respiratory infection. PMID:23049911

  5. Understanding the Pathogenicity of Burkholderia contaminans, an Emerging Pathogen in Cystic Fibrosis.

    Science.gov (United States)

    Nunvar, Jaroslav; Kalferstova, Lucie; Bloodworth, Ruhi A M; Kolar, Michal; Degrossi, Jose; Lubovich, Silvina; Cardona, Silvia T; Drevinek, Pavel

    2016-01-01

    Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence

  6. Microfluidic devices for sample preparation and rapid detection of foodborne pathogens

    DEFF Research Database (Denmark)

    Kant, Krishna; Shahbazi, Mohammad-Ali; Dave, Vivek Priy

    2018-01-01

    and improve the limit of detections. Integration of pathogen capturing bio-receptors on microfluidic devices is a crucial step, which can facilitate recognition abilities in harsh chemical and physical conditions, offering a great commercial benefit to the food-manufacturing sector. This article reviews...... diagnosis competences. This has prompted researchers to call the current status of detection approaches into question and leverage new technologies for superior pathogen sensing outcomes. Novel strategies mainly rely on incorporating all the steps from sample preparation to detection in miniaturized devices...... recent advances in current state-of-the-art of sample preparation and concentration from food matrices with focus on bacterial capturing methods and sensing technologies, along with their advantages and limitations when integrated into microfluidic devices for online rapid detection of pathogens in foods...

  7. Microbiological food safety issues in Brazil: bacterial pathogens.

    Science.gov (United States)

    Gomes, Bruna Carrer; Franco, Bernadette Dora Gombossy de Melo; De Martinis, Elaine Cristina Pereira

    2013-03-01

    The globalization of food supply impacts patterns of foodborne disease outbreaks worldwide, and consumers are having increased concern about microbiological food safety. In this sense, the assessment of epidemiological data of foodborne diseases in different countries has not only local impact, but it can also be of general interest, especially in the case of major global producers and exporters of several agricultural food products, such as Brazil. In this review, the most common agents of foodborne illnesses registered in Brazil will be presented, compiled mainly from official databases made available to the public. In addition, some representative examples of studies on foodborne bacterial pathogens commonly found in Brazilian foods are provided.

  8. Dynamics of serum testosterone during the menstrual cycle evaluated by daily measurements with an ID-LC-MS/MS method and a 2nd generation automated immunoassay

    NARCIS (Netherlands)

    Bui, Hong N.; Sluss, Patrick M.; Blincko, Stuart; Knol, Dirk L.; Blankenstein, Marinus A.; Heijboer, Annemieke C.

    2013-01-01

    Testosterone concentrations in normally cycling women are assumed to be elevated around the time of ovulation. The clinical relevance of changing testosterone concentrations during the menstrual cycle, however, is unclear. Poor performance of current direct immunoassays for testosterone at low

  9. Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons

    DEFF Research Database (Denmark)

    Georgiadis, Panagiotis; Kovács, Katalin; Kaila, Stella

    2012-01-01

    We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated...

  10. The Multitrophic Plant-Herbivore-Parasitoid-Pathogen System

    DEFF Research Database (Denmark)

    Bruni, Luis Emilio

    2011-01-01

    . The models for multitrophic interactions and above and below ground interactions furnish a picture based on material exchanges, i.e., trophic webs, between the participating taxa. On the other hand, in most disciplines of biology there is an incipient trend that considers biology as a science of “sensing......”, that is, biologists in different sub-disciplines are assigning increasing importance to the informational processes in living systems and are paying more attention to the “context” (e.g., from quorum sensing to info-chemicals to signal transduction in general). There is a new and exciting epistemological...... in many different empirical studies of what we could call the “multitrophic plant–herbivore–parasitoid–pathogen system”....

  11. Pathogenicity of highly pathogenic avian influenza virus in mammals

    NARCIS (Netherlands)

    de Wit, Emmie; Kawaoka, Yoshihiro; de Jong, Menno D.; Fouchier, Ron A. M.

    2008-01-01

    In recent years, there has been an increase in outbreaks of highly pathogenic avian influenza (HPAI) in poultry. Occasionally, these outbreaks have resulted in transmission of influenza viruses to humans and other mammals, with symptoms ranging from conjunctivitis to pneumonia and death. Here, the

  12. Applied Genomics of Foodborne Pathogens

    DEFF Research Database (Denmark)

    and customized source of information designed for and accessible to microbiologists interested in applying cutting-edge genomics in food safety and public health research. This book fills this void with a well-selected collection of topics, case studies, and bioinformatics tools contributed by experts......This book provides a timely and thorough snapshot into the emerging and fast evolving area of applied genomics of foodborne pathogens. Driven by the drastic advance of whole genome shot gun sequencing (WGS) technologies, genomics applications are becoming increasingly valuable and even essential...... at the forefront of foodborne pathogen genomics research....

  13. Transcriptome analysis of acyl-homoserine lactone-based quorum sensing regulation in Yersinia pestis [corrected].

    Directory of Open Access Journals (Sweden)

    Christopher N LaRock

    Full Text Available The etiologic agent of bubonic plague, Yersinia pestis, senses self-produced, secreted chemical signals in a process named quorum sensing. Though the closely related enteric pathogen Y. pseudotuberculosis uses quorum sensing system to regulate motility, the role of quorum sensing in Y. pestis has been unclear. In this study we performed transcriptional profiling experiments to identify Y. pestis quorum sensing regulated functions. Our analysis revealed that acyl-homoserine lactone-based quorum sensing controls the expression of several metabolic functions. Maltose fermentation and the glyoxylate bypass are induced by acyl-homoserine lactone signaling. This effect was observed at 30°C, indicating a potential role for quorum sensing regulation of metabolism at temperatures below the normal mammalian temperature. It is proposed that utilization of alternative carbon sources may enhance growth and/or survival during prolonged periods in natural habitats with limited nutrient sources, contributing to maintenance of plague in nature.

  14. The application of bioflocs technology to protect brine shrimp (Artemia franciscana) from pathogenic Vibrio harveyi.

    Science.gov (United States)

    Crab, R; Lambert, A; Defoirdt, T; Bossier, P; Verstraete, W

    2010-11-01

    To study the potential biocontrol activity of bioflocs technology. Glycerol-grown bioflocs were investigated for their antimicrobial and antipathogenic properties against the opportunistic pathogen Vibrio harveyi. The bioflocs did not produce growth-inhibitory substances. However, bioflocs and biofloc supernatants decreased quorum sensing-regulated bioluminescence of V. harveyi. This suggested that the bioflocs had biocontrol activity against this pathogen because quorum sensing regulates virulence of vibrios towards different hosts. Interestingly, the addition of live bioflocs significantly increased the survival of gnotobiotic brine shrimp (Artemia franciscana) larvae challenged to V. harveyi. Bioflocs grown on glycerol as carbon source inhibit quorum sensing-regulated bioluminescence in V. harveyi and protect brine shrimp larvae from vibriosis. The results presented in this study indicate that in addition to water quality control and in situ feed production, bioflocs technology could help in controlling bacterial infections within the aquaculture pond. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.

  15. Performance of hepatitis B assays on the Bayer ADVIA Centaur Immunoassay System.

    Science.gov (United States)

    van Helden, Josef; Denoyel, Gérard; Karwowska, Sylwia; Reamer, Randy; Schmalz, John; Wright, Ted; Preisel-Simmons, Barbara

    2004-01-01

    Bayer HealthCare LLC, Diagnostics Division, has developed several new assays on the ADVIA Centaur immunoassay system for the detection of markers of hepatitis B virus infection in human serum and plasma. This panel includes assays for: hepatitis B surface antigen (HBsAg), a confirmatory test method for HBsAg, antibodies to hepatitis B surface antigen (anti-HBs), IgM and IgG antibodies to hepatitis B core antigen (anti-HBc Total) and IgM antibodies to hepatitis B core antigen (anti-HBc IgM). These assays employ magnetic particle separation technology with direct chemiluminescence for optimal assay performance. All of the assays are fully automated, require sample volumes ranging from 15 microl to 100 microl (with the exception of the ADVIA Centaur HBsAg Confirmatory Assay, which requires 2 x 100 microl), and have throughputs of up to 240 tests per hour. The five ADVIA Centaur HBV assays were tested in extensive performance evaluations conducted at two sites in Europe. The performance evaluations, which included samples from HBV-infected individuals, blood donors, hospitalized/clinical patients, and HBV vaccinees (for Anti-HBs evaluation), generated performance data in support of obtaining the Communautés Européennes (CE) mark for European market distribution. The HBV performance evaluations resulted in an overall diagnostic specificity > 99%, i.e. 99.94% for the ADVIA Centaur HBsAg Assay, 100% for the ADVIA Centaur Anti-HBs Assay, 100% for the ADVIA Centaur HBc IgM Assay and 99.94% for the ADVIA Centaur HBc Total Assay. All of the ADVIA Centaur assays showed a very good diagnostic sensitivity on these populations with 100% for the ADVIA Centaur HBsAg Assay, 99.0% for the ADVIA Centaur Anti-HBs Assay, 98.53% for the ADVIA Centaur HBc IgM Assay and 100% for the ADVIA Centaur HBc Total Assay. The ADVIA Centaur HBsAg Confirmatory Test confirmed 100% of the positive HBsAg samples. Testing of interfering substances and potential cross-reacting samples for all ADVIA

  16. Compressed sensing & sparse filtering

    CERN Document Server

    Carmi, Avishy Y; Godsill, Simon J

    2013-01-01

    This book is aimed at presenting concepts, methods and algorithms ableto cope with undersampled and limited data. One such trend that recently gained popularity and to some extent revolutionised signal processing is compressed sensing. Compressed sensing builds upon the observation that many signals in nature are nearly sparse (or compressible, as they are normally referred to) in some domain, and consequently they can be reconstructed to within high accuracy from far fewer observations than traditionally held to be necessary. Apart from compressed sensing this book contains other related app

  17. 340nm UV LED excitation in time-resolved fluorescence system for europium-based immunoassays detection

    DEFF Research Database (Denmark)

    Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter

    2017-01-01

    In immunoassay analyzers for in-vitro diagnostics, Xenon flash lamps have been widely used as excitation light sources. Recent advancements in UV LED technology and its advantages over the flash lamps such as smaller footprint, better wall-plug efficiency, narrow emission spectrum......, and no significant afterglow, have made them attractive light sources for gated detection systems. In this paper, we report on the implementation of a 340 nm UV LED based time-resolved fluorescence system based on europium chelate as a fluorescent marker. The system performance was tested with the immunoassay based...... on the cardiac marker, TnI. The same signal-to-noise ratio as for the flash lamp based system was obtained, operating the LED below specified maximum current. The background counts of the system and its main contributors were measured and analyzed. The background of the system of the LED based unit was improved...

  18. Giant unilamellar vesicles containing Rhodamine 6G as a marker for immunoassay of bovine serum albumin and lipocalin-2.

    Science.gov (United States)

    Sakamoto, Misato; Shoji, Atsushi; Sugawara, Masao

    2016-07-15

    Functionalized giant unilamellar vesicles (GUVs) containing a fluorescence dye Rhodamine 6G is proposed as a marker in sandwich-type immunoassay for bovine serum albumin (BSA) and lipocalin-2 (LCN2). The GUVs were prepared by the electroformation method and functionalized with anti-BSA antibody and anti-LCN2 antibody, respectively. The purification of antibody-modified GUVs was achieved by conventional centrifugation and a washing step in a flow system. To antigen on an antibody slip, antibody-modified GUVs were added as a marker and incubated. After wash-out of excess reagents and lysis of the bound GUVs with Triton X-100, the fluorescence image was captured. The fluorometric immunoassays for BSA and LCN2 exhibited lower detection limits of 4 and 80 fg ml(-)(1), respectively. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Increasing of sensitivity of fluorescent immunoassay analysis of alpha-fetoprotein by means of plasmonical silver nanoparticles

    International Nuclear Information System (INIS)

    Vashchenko, S.V.; Min'ko, A.A.; Romanenko, A.A.; Gaponenko, S.V.; Kulakovich, O.S.

    2014-01-01

    A test system is proposed based on metal enhanced fluorescence to analyze low concentrations of alpha-fetoprotein (AFP), a tumor marker. Antigen-antibody reaction was performed on polystyrene plates coated with silver nanoparticles to increase sensitivity of fluorescent immunoassay and signal-to-noise ratio as compared to silver-free system. As compared to widely used ELISA technique and other immunoassay techniques the proposed approach is characterized by smaller probe volume, fast analysis and simplicity. The proposed test system uses layer-by-layer assembly approach, LED excitation and nanowatt photodetection set-up. The proposed test system offers AFP detection at concentrations used in clinical practice. Fluorescence enhancement for labeled AFP antibodies on a silver substrate was found to depend on antibodies concentration and was up to 6 times. (authors)

  20. The fabrication of magnetic particle-based chemiluminescence immunoassay for human epididymis protein-4 detection in ovarian cancer.

    Science.gov (United States)

    Fu, Xiaoling; Liu, Yangyang; Qiu, Ruiyun; Foda, Mohamed F; Zhang, Yong; Wang, Tao; Li, Jinshan

    2018-03-01

    The magnetic particles have a significant influence on the immunoassay detection and cancer therapy. Herein, the chemiluminescence immunoassay combined with the magnetic particles (MPCLIA) was presented for the clinical determination and analysis of human epididymis protein 4 (HE4) in the human serum. Under the optimized experiment conditions, the secure MPCLIA method can detect HE4 in the broader range of 0-1000 pmol/L, with a lower detection limit of 1.35 pmol/L. The satisfactory recovery rate of the method in the serum ranged from 83.62% to 105.10%, which was well within the requirement of clinical analysis. Moreover, the results showed the good correlation with enzyme-linked immunosorbent assay (ELISA), with the correlation coefficient of 0.9589. This proposed method has been successfully applied to the clinical determination of HE4 in the human serum.

  1. Performance characteristics of bioassay, radioenzymatic assay, homogeneous enzyme immunoassay, and high-performance liquid chromatographic determination of serum gentamicin

    International Nuclear Information System (INIS)

    Delaney, C.J.; Opheim, K.E.; Smith, A.L.; Plorde, J.J.

    1982-01-01

    We compared the accuracy, precision, and between-method error of the microbiological assay, the radioenzymatic assay, the homogeneous enzyme immunoassay, and the high-performance liquid chromatographic assay for the quantitation of gentamicin in serum. Precision and accuracy were evaluated by reference samples prepared to contain 0.0 to 32.7 micrograms of gentamicin per ml. Correlations between the methods utilized patient sera with gentamicin concentrations ranging from 0.6 to 13.3 micrograms/ml. All methods were reliable within acceptable limits for routine clinical use; intermethod correlation coefficients exceeded 0.96. Relative to the microbiological assay, the alternative methods offer the advantage of rapid analysis. The elapsed times for acquiring data on a set of 10 specimens under routine operating conditions were 0.5 h by the enzyme immunoassay, 4 h by the radioenzymatic assay, 5 h by the high-performance liquid chromatographic assay, and 10 h by the microbiological assay

  2. Color encoded microbeads-based flow cytometric immunoassay for polycyclic aromatic hydrocarbons in food

    International Nuclear Information System (INIS)

    Meimaridou, Anastasia; Haasnoot, Willem; Noteboom, Linda; Mintzas, Dimitrios; Pulkrabova, Jana; Hajslova, Jana; Nielen, Michel W.F.

    2010-01-01

    Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC 50 of 0.3 μg L -1 using a monoclonal antibody (Mab22F12) against BaP, similar to the IC 50 of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography-mass spectrometry (GC-MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction procedure which are

  3. Optimization and validation of CEDIA drugs of abuse immunoassay tests in serum on Hitachi 912.

    Science.gov (United States)

    Kirschbaum, Katrin M; Musshoff, Frank; Schmithausen, Ricarda; Stockhausen, Sarah; Madea, Burkhard

    2011-10-10

    Due to sensitive limits of detection of chromatographic methods and low limit values regarding the screening of drugs under the terms of impairment in safe driving (§ 24a StVG, Street Traffic Law in Germany), preliminary immunoassay (IA) tests should be able to detect also low concentrations of legal and illegal drugs in serum in forensic cases. False-negatives should be avoided, the rate of false-positive samples should be low due to cost and time. An optimization of IA cutoff values and a validation of the assay is required for each laboratory. In a retrospective study results for serum samples containing amphetamine, methylenedioxy derivatives, cannabinoids, benzodiazepines, cocaine (metabolites), methadone and opiates obtained with CEDIA drugs of abuse reagents on a Hitachi 912 autoanalyzer were compared with quantitative results of chromatographic methods (gas or liquid chromatography coupled with mass spectrometry (GC/MS or LC/MS)). Firstly sensitivity, specificity, positive and negative predictive values and overall misclassification rates were evaluated by contingency tables and compared to ROC-analyses and Youden-Indices. Secondly ideal cutoffs were statistically calculated on the basis of sensitivity and specificity as decisive statistical criteria with focus on a high sensitivity (low rates of false-negatives), i.e. using the Youden-Index. Immunoassay (IA) and confirmatory results were available for 3014 blood samples. Sensitivity was 90% or more for nearly all analytes: amphetamines (IA cutoff 9.5 ng/ml), methylenedioxy derivatives (IA cutoff 5.5 ng/ml), cannabinoids (IA cutoff 14.5 ng/ml), benzodiazepines (IA cutoff >0 ng/ml). Test of opiates showed a sensitivity of 86% for a IA cutoff value of >0 ng/ml. Values for specificity ranged between 33% (methadone, IA cutoff 10 ng/ml) and 90% (cocaine, IA cutoff 20 ng/ml). Lower cutoff values as recommended by ROC analyses were chosen for most tests to decrease the rate of false-negatives. Analyses enabled

  4. Photonic ring resonance is a versatile platform for performing multiplex immunoassays in real time.

    Science.gov (United States)

    Mudumba, Sasi; de Alba, Sophia; Romero, Randy; Cherwien, Carli; Wu, Alice; Wang, Jue; Gleeson, Martin A; Iqbal, Muzammil; Burlingame, Rufus W

    2017-09-01

    Photonic ring resonance is a property of light where in certain circumstances specific wavelengths are trapped in a ring resonator. Sensors based on silicon photonic ring resonators function by detecting the interaction between light circulating inside the sensor and matter deposited on the sensor surface. Binding of biological material results in a localized change in refractive index on the sensor surface, which affects the circulating optical field extending beyond the sensor boundary. That is, the resonant wavelength will change when the refractive index of the medium around the ring resonator changes. Ring resonators can be fabricated onto small silicon chips, allowing development of a miniature multiplex array of ring based biosensors. This paper describes the properties of such a system when responding to the refractive index changed in a simple and precise way by changing the ionic strength of the surrounding media, and in a more useful way by the binding of macromolecules to the surface above the resonators. Specifically, a capture immunoassay is described that measures the change of resonant wavelength as a patient serum sample with anti-SS-A autoantibodies is flowed over a chip spotted with SS-A antigen and amplified with anti-IgG. The technology has been miniaturized and etched into a 4×6mm silicon chip that can measure 32 different reactions in quadruplicate simultaneously. The variability between 128 rings on a chip as measured by 2M salt assays averaged 0.6% CV. The output of the assays is the average shift per cluster of 4 rings, and the assays averaged 0.5% CV between clusters. The variability between chips averaged 1.8%. Running the same array on multiple instruments showed that after some improvements to the wavelength referencing system, the upper boundary of variation was 3% between 13 different instruments. The immunoassay displayed about 2% higher variability than the salt assays. There are several outstanding features of this system. The

  5. Erythema migrans and serodiagnosis by enzyme immunoassay and immunoblot with three borrelia species.

    Science.gov (United States)

    Stanek, G; Breier, F; Menzinger, G; Schaar, B; Hafner, M; Partsch, H

    1999-12-10

    There is wide divergence of opinion between physicians regarding the use of serological measures for the diagnosis and treatment of erythema migrans, the hallmark of Lyme borreliosis. We studied the outcome of an enzyme immunoassay and immunoblot (Western blot) used on the sera of patients who had suffered tick bite and erythema migrans, and had been subsequently treated with various antibiotics. Ninety-nine consecutive patients presenting with erythema migrans after tick bite were prospectively recruited at the outpatient department of two Vienna City hospitals and at the consultation office for Lyme borreliosis of the Institute of Hygiene. University Vienna. Blood samples were taken before antibiotic treatment and 3 and 6 months thereafter. Blood samples from 100 blood donors served as controls. Antibodies against Borrelia burgdorferi sensu lato were determined by enzyme immunoassay (IgG and IgM EIA) and by IgG immunoblot. The latter was performed with isolates of B. alzelii (H2) B. burgdorferi sensu stricto (Le) and B. garinii (W) from Austrian patients. The 4 interpretation criteria for immunoblot results were: A (3 bands out of 8), B (2 bands out of 9), C and D (1 band out of 6). In all patients, the erythema resolved within the treatment period. No complications secondary to the borrelia infection were registered. After treatment there was no significant change in titre, nor was there a difference in the immunoblot pattern between the first, second and third serum samples. Serum antibodies to B. burgdorferi were positive by EIA in 22.9% (IgG) and 2.5% (IgM). Immunoblot results offered by borrelia species and by the interpretation criteria, ranging between 8.3% (criterion A, strain Le) and 44.2% (criterion D, strain H2). By EIA, control samples were IgG and IgM positive in 5% and 1%, respectively. Positive immunoblot results with strain H2 were found in 9%, 13%, 18%, and 20% by the criteria A through D respectively. After antibiotic treatment of erythema

  6. 340nm UV LED excitation in time-resolved fluorescence system for europium-based immunoassays detection

    OpenAIRE

    Rodenko, Olga; Fodgaard, Henrik; Tidemand-Lichtenberg, Peter; Pedersen, Christian

    2017-01-01

    In immunoassay analyzers for in-vitro diagnostics, Xenon flash lamps have been widely used as excitation light sources. Recent advancements in UV LED technology and its advantages over the flash lamps such as smaller footprint, better wall-plug efficiency, narrow emission spectrum, and no significant afterglow, have made them attractive light sources for gated detection systems. In this paper, we report on the implementation of a 340 nm UV LED based time-resolved fluorescence system based on ...

  7. NiCoBP-doped carbon nanotube hybrid: A novel oxidase mimetic system for highly efficient electrochemical immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bing; He, Yu; Liu, Bingqian; Tang, Dianping, E-mail: dianping.tang@fzu.edu.cn

    2014-12-03

    Highlights: • We report a new oxidase mimetic system for highly efficient electrochemical immunoassay. • NiCoBP-doped carbon nanotube hybrids were used as the nanocatalysts. • NiCoBP-doped carbon nanotube hybrids were used as the mimic oxidase. - Abstract: NiCoBP-doped multi-walled carbon nanotube (NiCoBP–MWCNT) was first synthesized by using induced electroless-plating method and functionalized with the biomolecules for highly efficient electrochemical immunoassay of prostate-specific antigen (PSA, used as a model analyte). We discovered that the as-synthesized NiCoBP–MWCNT had the ability to catalyze the glucose oxidization with a stable and well-defined redox peak. The catalytic current increased with the increment of the immobilized NiCoBP–MWCNT on the electrode. Transmission electron microscope (TEM) and energy dispersive X-ray spectrometry (EDX) were employed to characterize the as-prepared NiCoBP–MWCNT. Using the NiCoBP–MWCNT-conjugated anti-PSA antibody as the signal-transduction tag, a new enzyme-free electrochemical immunoassay protocol could be designed for the detection of target PSA on the capture antibody-functionalized immunosensing interface. Experimental results revealed that the designed immunoassay system could exhibit good electrochemical responses toward target PSA, and allowed the detection of PSA at a concentration as low as 0.035 ng mL{sup −1}. More importantly, the NiCoBP-MWCNT-based oxidase mimetic system could be further extended for the monitoring of other low-abundance proteins or disease-related biomarkers by tuning the target antibody.

  8. Positive predictive value of the immunoassay for Clostridium difficile toxin A and B detection at a private hospital.

    Science.gov (United States)

    Pérez-Topete, S E; Miranda-Aquino, T; Hernández-Portales, J A

    Clostridium difficile (C. difficile) is a Gram-positive bacillus that is a common cause of diarrhea in the hospital environment, with a documented incidence of up to 10%. There are different methods to detect it, but a widely used test in our environment is the immunoassay for toxins A and B. The aim of our study was to 1) estimate the positive predictive value of the immunoassay for the detection of the C. difficile toxins A and B, 2) to establish the incidence of C. difficile-associated diarrhea in the hospital, and 3) to know the most common associated factors. A diagnostic test accuracy study was conducted within the time frame of January 2010 to August 2013 at the Hospital Christus Muguerza® Alta Especialidad on patients with symptoms suggestive of C. difficile-associated diarrhea that had a positive immunoassay test and confirmation of C. difficile through colon biopsy and stool culture. The immunoassay for toxins A and B was performed in 360 patients. Fifty-five of the cases had positive results, 35 of which showed the presence of C. difficile. Incidence was 10.2% and the positive predictive value of the test for C. difficile toxins A and B was 0.64 (95% CI, 0.51-0.76). Previous antibiotic therapy (n=29) and proton pump inhibitor use (n=19) were the most common associated factors. C. difficile incidence in our environment is similar to that found in the literature reviewed, but the positive predictive value of the test for toxin A and B detection was low. Copyright © 2016 Asociación Mexicana de Gastroenterología. Publicado por Masson Doyma México S.A. All rights reserved.

  9. High-sensitivity detection of cardiac troponin I with UV LED excitation for use in point-of-care immunoassay

    OpenAIRE

    Rodenko, Olga; Eriksson, Susann; Tidemand-Lichtenberg, Peter; Troldborg, Carl Peder; Fodgaard, Henrik; van Os, Sylvana; Pedersen, Christian

    2017-01-01

    High-sensitivity cardiac troponin assay development enables determination of biological variation in healthy populations, more accurate interpretation of clinical results and points towards earlier diagnosis and rule-out of acute myocardial infarction. In this paper, we report on preliminary tests of an immunoassay analyzer employing an optimized LED excitation to measure on a standard troponin I and a novel research high-sensitivity troponin I assay. The limit of detection is improved by fac...

  10. Comparison between Amnisure Placental Alpha Microglobulin-1 Rapid Immunoassay and Standard Diagnostic Methods for Detection of Rupture of Membranes

    Directory of Open Access Journals (Sweden)

    Beng Kwang Ng

    2013-01-01

    Full Text Available Objective. To determine the diagnostic accuracy of placental alpha microglobulin-1 assay and standard diagnostic methods for detecting rupture of membrane. Study Design. Prospective diagnostic study, between June 2011 to November 2011 at a tertiary centre. Initial evaluation included both the standard diagnostic methods for rupture of membranes and placental alpha microglobulin-1 immunoassay. The actual rupture of membranes was diagnosed on review of the medical records after delivery (absence of membrane or a positive pad chart. Main Outcome Measures. Placental alpha microglobulin-1 immunoassay and standard diagnostic methods for diagnosis of rupture of membrane. Results. A total of 211 patients were recruited. At initial presentation, 187 patients (88.6% had ruptured membranes, while 24 patients (11.4% had intact membranes. Placental alpha microglobulin-1 immunoassay confirmed rupture of membranes at initial presentation with a sensitivity of 95.7% (179 of 187, specificity of 100% (24 of 24, positive predictive value of 100% (179 of 179, and negative predictive value of 75.0% (24 of 32. By comparison, the conventional standard diagnostic methods had a sensitivity of 78.1% (146 of 187, specificity of 100% (24 of 24, positive predictive value of 100% (146 of 146, and negative predictive value of 36.9% (24 of 65 in diagnosing rupture of membrane. Conclusion. Placental alpha-microglobulin-1 immunoassay is a rapid and accurate method for confirming the diagnosis of rupture of membrane. It was superior to conventional standard diagnostic methods (pooling, nitrazine, and ferning, the nitrazine test alone or fern test alone.

  11. Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples.

    Science.gov (United States)

    Li, Y S; Meng, X Y; Zhou, Y; Zhang, Y Y; Meng, X M; Yang, L; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Wang, X R

    2015-04-15

    In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, magnetic beads (MBs), employed as supports for the immobilization of anti-β-casein polyclonal antibody (PAb), were used as the capture probe. Colloidal gold nanoparticles (AuNPs), employed as a bridge for loading anti-β-casein monoclonal antibody (McAb) and horseradish peroxidase (HRP), were used as the amplification probe. The presence of β-casein causes the sandwich structures of MBs-PAb-β-casein-McAb-AuNPs through the interaction between β-casein and the anti-β-casein antibodies. The HRP, used as an enzymatic-amplified tracer, can catalytically oxidize the substrate 3,3',5,5'-tetramethylbenzidine (TMB), generating optical signals that are proportional to the quantity of β-casein. The linear range of the immunoassay was from 6.5 to 1520ngmL(-1). The limit of detection (LOD) was 4.8ngmL(-1) which was 700 times lower than that of MBs-antibody-HRP based immunoassay and 6-7 times lower than that from the microplate-antibody-HRP based assay. The recoveries of β-casein from bovine milk samples were from 95.0% to 104.3% that had a good correlation coefficient (R(2)=0.9956) with those obtained by an official standard Kjeldahl method. For higher sensitivity, simple sample pretreatment and shorter time requirement of the antigen-antibody reaction, the developed immunoassay demonstrated the viability for detection of β-casein in bovine milk samples. Copyright © 2014. Published by Elsevier B.V.

  12. Detection of soluble antigens of Toxoplasma gondii by a four-layer modification of an enzyme immunoassay.

    OpenAIRE

    Turunen, H J

    1983-01-01

    A sensitive four-layer modification of an enzyme immunoassay for the detection of soluble antigens of Toxoplasma gondii is described. Microtiter plates were sensitized with rabbit anti-toxoplasma immunoglobulins (6 micrograms/ml) used as the primary antibodies; guinea pig anti-toxoplasma immunoglobulins (6 micrograms/ml) were used as the secondary trapping antibodies. Horseradish peroxidase-conjugated anti-guinea pig immunoglobulins were used as the indicator antibodies. The specificity of th...

  13. Abbott’s Fluorescence Polarization Immunoassay for Cyclosporine and Metabolites Compared with the Sandoz “Sandimmune” RIA

    OpenAIRE

    Sanghvi, Ajit; Diven, Warren; Seltman, Howard; Starzl, Thomas

    1988-01-01

    A new procedure for measuring cyclosporine in plasma has been introduced by Abbott Laboratories, involving their TDx instrumentation and fluorescence polarization immunoassay. Radioimmunoassay (RIA) and high-performance liquid chromatography are currently the conventional methods for measuring cyclosporine in plasma and whole blood. In an effort to find a method that will decrease the radioactive hazard, the reagent and supply cost, and the labor requirements associated with RIA procedures, w...

  14. Multicentric Evaluation of New Commercial Enzyme Immunoassays for the Detection of Immunoglobulin M and Total Antibodies against Hepatitis A Virus▿

    Science.gov (United States)

    Arcangeletti, M. C.; Dussaix, E.; Ferraglia, F.; Roque-Afonso, A. M.; Graube, A.; Chezzi, C.

    2011-01-01

    A multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively). PMID:21653739

  15. Pathogen disgust and interpersonal personality

    NARCIS (Netherlands)

    Kupfer, Tom R.; Tybur, Joshua M.

    2017-01-01

    The behavioral immune system includes motivational systems for avoiding contact with pathogens, including those transmitted by other people. Motivations to avoid others may depend not only on the perceived risk of infection but also on perceived benefits of social interaction. Based on this idea, we

  16. Lectins in human pathogenic fungi.

    Science.gov (United States)

    Gallegos, Belém; Martínez, Ruth; Pérez, Laura; Del Socorro Pina, María; Perez, Eduardo; Hernández, Pedro

    2014-01-01

    Lectins are carbohydrate-binding proteins widely distributed in nature. They constitute a highly diverse group of proteins consisting of many different protein families that are, in general, structurally unrelated. In the last few years, mushroom and other fungal lectins have attracted wide attention due to their antitumour, antiproliferative and immunomodulatory activities. The present mini-review provides concise information about recent developments in understanding lectins from human pathogenic fungi. A bibliographic search was performed in the Science Direct and PubMed databases, using the following keywords "lectin", "fungi", "human" and "pathogenic". Lectins present in fungi have been classified; however, the role played by lectins derived from human pathogenic fungi in infectious processes remains uncertain; thus, this is a scientific field requiring more research. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  17. Pathogenicity of Shigella in chickens.

    Science.gov (United States)

    Shi, Run; Yang, Xia; Chen, Lu; Chang, Hong-tao; Liu, Hong-ying; Zhao, Jun; Wang, Xin-wei; Wang, Chuan-qing

    2014-01-01

    Shigellosis in chickens was first reported in 2004. This study aimed to determine the pathogenicity of Shigella in chickens and the possibility of cross-infection between humans and chickens. The pathogenicity of Shigella in chickens was examined via infection of three-day-old SPF chickens with Shigella strain ZD02 isolated from a human patient. The virulence and invasiveness were examined by infection of the chicken intestines and primary chicken intestinal epithelial cells. The results showed Shigella can cause death via intraperitoneal injection in SPF chickens, but only induce depression via crop injection. Immunohistochemistry and transmission electron microscopy revealed the Shigella can invade the intestinal epithelia. Immunohistochemistry of the primary chicken intestinal epithelial cells infected with Shigella showed the bacteria were internalized into the epithelial cells. Electron microscopy also confirmed that Shigella invaded primary chicken intestinal epithelia and was encapsulated by phagosome-like membranes. Our data demonstrate that Shigella can invade primary chicken intestinal epithelial cells in vitro and chicken intestinal mucosa in vivo, resulting in pathogenicity and even death. The findings suggest Shigella isolated from human or chicken share similar pathogenicity as well as the possibility of human-poultry cross-infection, which is of public health significance.

  18. Silver nanoparticles deposited on graphene oxide for ultrasensitive surface-enhanced Raman scattering immunoassay of cancer biomarker.

    Science.gov (United States)

    Yang, Lin; Zhen, Shu Jun; Li, Yuan Fang; Huang, Cheng Zhi

    2018-06-14

    Graphene oxide (GO) exhibits distinctive Raman scattering features for its high frequency D (disordered) and tangential modes (G-band), which are characteristically sharp at 1580 cm-1 and 1350 cm-1, respectively, but are too weak for sensitive quantitation purposes. By depositing silver nanoparticles on the surface of GO in this contribution, both D and G bands of GO become enhanced. The enzyme label of this method controls the dissolution of silver nanoparticles on the surface of GO through hydrogen peroxide which is produced by the oxidation of the enzyme substrate. With the dissolution of the silver nanoparticles a greatly decreased SERS signal of GO was obtained. This strategy involves dual signal amplification of the enzyme and nanocomposites to improve the detection sensitivity. As a proof of concept, prostate specific antigen (PSA), a biomarker for prostate cancer, is successfully detected as a target by forming a sandwich structure in immunoassay. The SERS immunoassay possesses excellent analytical performance in the range 0.5 pg mL-1 to 500 pg mL-1 with a limit of detection of 0.23 pg mL-1, making the detection of PSA serum samples from prostate cancer patients satisfactory, demonstrating that the sensitive enzyme-assisted dissolved AgNPs SERS immunoassay of PSA has potential applications in clinical diagnosis.

  19. A sulfhydryl-reactive ruthenium (II complex and its conjugation to protein G as a universal reagent for fluorescent immunoassays.

    Directory of Open Access Journals (Sweden)

    Jing-Tang Lin

    Full Text Available To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2'-dipyridine Ruthenium bis (hexafluorophosphate. The synthesized Ru(II complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. The emission peak wavelength of the Ru(II-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II complex, indicating that Ru(II-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG binding assay was conducted. The result showed that Ru(II-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays.

  20. Accurate Point-of-Care Detection of Ruptured Fetal Membranes: Improved Diagnostic Performance Characteristics with a Monoclonal/Polyclonal Immunoassay

    Directory of Open Access Journals (Sweden)

    Linda C. Rogers

    2016-01-01

    Full Text Available Objective Accurate and timely diagnosis of rupture of membranes (ROM is imperative to allow for gestational age-specific interventions. This study compared the diagnostic performance characteristics between two methods used for the detection of ROM as measured in the same patient. Methods Vaginal secretions were evaluated using the conventional fern test as well as a point-of-care monoclonal/polyclonal immunoassay test (ROM Plus® in 75 pregnant patients who presented to labor and delivery with complaints of leaking amniotic fluid. Both tests were compared to analytical confirmation of ROM using three external laboratory tests. Diagnostic performance characteristics were calculated including sensitivity, specificity, positive predictive value (PPV, negative predictive value (NPV, and accuracy. Results Diagnostic performance characteristics uniformly favored ROM detection using the immunoassay test compared to the fern test: sensitivity (100% vs. 77.8%, specificity (94.8% vs. 79.3%, PPV (75% vs. 36.8%, NPV (100% vs. 95.8%, and accuracy (95.5% vs. 79.1%. Conclusions The point-of-care immunoassay test provides improved diagnostic accuracy for the detection of ROM compared to fern testing. It has the potential of improving patient management decisions, thereby minimizing serious complications and perinatal morbidity.

  1. Homogeneous immunoassay for human IgG using oriented hen egg IgY immobilized on gold sol nanoparticles

    International Nuclear Information System (INIS)

    Yeritsyan, H.E.; Gasparyan, V.K.

    2012-01-01

    Homogeneous immunoassays using (red) gold nanoparticles represent an attractive detection scheme because of the option of photometric readout. We have applied oriented immobilization of hen egg immunoglobulin Y (IgY) on gold nanoparticles when developing a homogeneous immunoassay for human IgG. In oriented immobilization, as opposed to random immobilization, the antigen binding capabilities of the antibodies are retained. It is shown that such immunoassay has significantly better sensitivity in comparison with methods based on conventional immobilization of affinity-purified antibodies. It is also shown that hen egg IgY is better suited than rabbit antibodies, because much more antibody can be immobilized on gold nanoparticles without any destabilization, probably because of the more acidic nature of these antibodies. In addition, hen egg IgY can be supplied in higher quantity and can be prepared more easily than IgG from rabbits. Bleeding and slaughtering of animals is not needed. The assay presented here has a wide detection range (30-500 ng. mL -1 ) and a limit of detection as low as 30 ng. mL -1 of human IgG. (author)

  2. Oriented antibody immobilization to polystyrene macrocarriers for immunoassay modified with hydrazide derivatives of poly(methacrylic acid

    Directory of Open Access Journals (Sweden)

    Vinokurova Ludmila G

    2001-08-01

    Full Text Available Abstract Background Hydrophobic polystyrene is the most common material for solid phase immunoassay. Proteins are immobilized on polystyrene by passive adsorption, which often causes considerable denaturation. Biological macromolecules were found to better retain their functional activity when immobilized on hydrophilic materials. Polyacrylamide is a common material for solid-phase carriers of biological macromolecules, including immunoreagents used in affinity chromatography. New macroformats for immunoassay modified with activated polyacrylamide derivatives seem to be promising. Results New polymeric matrices for immunoassay in the form of 0.63-cm balls which contain hydrazide functional groups on hydrophilic polymer spacer arms at their surface shell are synthesized by modification of aldehyde-containing polystyrene balls with hydrazide derivatives of poly(methacrylic acid. The beads contain up to 0.31 μmol/cm2 active hydrazide groups accessible for covalent reaction with periodate-oxidized antibodies. The matrices obtained allow carrying out the oriented antibody immobilization, which increases the functional activity of immunosorbents. Conclusions An efficient site-directed antibody immobilization on a macrosupport is realized. The polymer hydrophilic spacer arms are the most convenient and effective tools for oriented antibody coupling with molded materials. The suggested scheme can be used for the modification of any other solid supports containing electrophilic groups reacting with hydrazides.

  3. Inkjet-printed point-of-care immunoassay on a nanoscale polymer brush enables subpicomolar detection of analytes in blood

    Science.gov (United States)

    Joh, Daniel Y.; Hucknall, Angus M.; Wei, Qingshan; Mason, Kelly A.; Lund, Margaret L.; Fontes, Cassio M.; Hill, Ryan T.; Blair, Rebecca; Zimmers, Zackary; Achar, Rohan K.; Tseng, Derek; Gordan, Raluca; Freemark, Michael; Ozcan, Aydogan; Chilkoti, Ashutosh

    2017-08-01

    The ELISA is the mainstay for sensitive and quantitative detection of protein analytes. Despite its utility, ELISA is time-consuming, resource-intensive, and infrastructure-dependent, limiting its availability in resource-limited regions. Here, we describe a self-contained immunoassay platform (the “D4 assay”) that converts the sandwich immunoassay into a point-of-care test (POCT). The D4 assay is fabricated by inkjet printing assay reagents as microarrays on nanoscale polymer brushes on glass chips, so that all reagents are “on-chip,” and these chips show durable storage stability without cold storage. The D4 assay can interrogate multiple analytes from a drop of blood, is compatible with a smartphone detector, and displays analytical figures of merit that are comparable to standard laboratory-based ELISA in whole blood. These attributes of the D4 POCT have the potential to democratize access to high-performance immunoassays in resource-limited settings without sacrificing their performance.

  4. Remote Sensing Information Gateway

    Science.gov (United States)

    Remote Sensing Information Gateway, a tool that allows scientists, researchers and decision makers to access a variety of multi-terabyte, environmental datasets and to subset the data and obtain only needed variables, greatly improving the download time.

  5. Hyperspectral remote sensing

    National Research Council Canada - National Science Library

    Eismann, Michael Theodore

    2012-01-01

    ..., and hyperspectral data processing. While there are many resources that suitably cover these areas individually and focus on specific aspects of the hyperspectral remote sensing field, this book provides a holistic treatment...

  6. Evaluation of enzyme immunoassay techniques for diagnosis of the most common intestinal protozoa in fecal samples.

    Science.gov (United States)

    Gaafar, Maha R

    2011-08-01

    This study was designed to evaluate the antigen capture enzyme immunoassays (EIAs) Triage parasite panel and TechLab Entamoeba histolytica II in detecting Giardia intestinalis, Cryptosporidium sp, and Entamoeba histolytica in fecal samples in comparison to microscopy, and in differentiating Entamoeba histolytica from Entamoeba dispar. The Triage EIA was evaluated using 100 stool specimens that were tested by standard ova and parasite examination, including staining with both trichrome and modified acid-fast stains. Differentiation between E. histolytica and E. dispar was performed using TechLab. Microscopic examination revealed that 19% of the samples were positive for Giardia, 4% for Cryptosporidium, and 1% for E. histolytica/E. dispar, and other parasites were found in 5%. By Triage, 23% of the samples were infected with Giardia, 5% with Cryptosporidium, and 2% with E. histolytica/E. dispar. Triage showed a sensitivity of 100% and specificity of 91.5%. The TechLab assay was negative for both samples diagnosed as E. histolytica/E. dispar by Triage, which suggested that they were E. dispar. Both tests showed no cross-reactivity with other intestinal protozoa. These results indicate that antigen detection by EIA has the potential to become a valuable tool, capable of making stool diagnostics more effective. Copyright © 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  7. Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference.

    Science.gov (United States)

    Liu, Xing; Tang, Zongwen; Duan, Zhenhua; He, Zhenyun; Shu, Mei; Wang, Xianxian; Gee, Shirley J; Hammock, Bruce D; Xu, Yang

    2017-03-01

    A sensitive indirect competitive nanobody-based enzyme linked immunosorbent assay (Nb-ELISA) for ochratoxin A (OTA) with high resistance to cereal matrix interference was developed. Nanobodies against OTA (Nb15, Nb28, Nb32, Nb36) were expressed in E. coli cells and their thermal stabilities were compared with that of an OTA-specific monoclonal antibody 6H8. All nanobodies could still retain their antigen-binding activity after exposure to temperature 95°C for 5min or to 90°C for 75min. Nb28 that exhibited the highest sensitivity in ELISA was selected for further research. An indirect competitive ELISA based on Nb28 was developed for OTA, with an IC 50 of 0.64ng/mL and a linear range (IC 20 -IC 80 ) of 0.27-1.47ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSIA. The recovery of spiked cereal samples (rice, oats, barley) ranged from 80% to 105% and the Nb-ELISA results of OTA content in naturally contamined samples were in good agreement with those determined by a commercial ELISA kit. The results indicated the reliablity of nanobody as a promising immunoassay reagent for detection of mycotoxins in food matrix and its potential in biosensor development. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. An efficient enzyme immunoassay for glutamate using glutaraldehyde coupling of the hapten to microtiter plates.

    Science.gov (United States)

    Ordronneau, P; Abdullah, L H; Petrusz, P

    1991-09-13

    In order to coat microtiter plates for enzyme immunoassays (EIAs), amino acids and other haptens are usually coupled to larger protein molecules. The formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, unfavorable orientation of the hapten on the protein and/or well-to-well variation in the concentration of the available hapten. In the assay described here the excitatory amino acid (EAA) Glu is coupled directly to polystyrene microtiter wells with GA. Each step of the assay was tested for maximum efficiency. The resulting EIA with Glu as a competitor gave excellent reproducibility (coefficient of variation = 5.87%), an EC50 of 2.02 X 10(-5) M and a detection limit of 1.26 X 10(-6) M. This EIA method is generally useful for a variety of antisera to amino acids and small peptides and a wide range of competing substances. It can be used to characterize the conformational requirements for antigen binding, to assay for glutamate or to identify compounds with glutamate-like structure in unknown solutions.

  9. Biconically Tapered Fiber Optic Probes for Rapid Label-Free Immunoassays

    Directory of Open Access Journals (Sweden)

    John Miller

    2015-04-01

    Full Text Available We report use of U-shaped biconically tapered optical fibers (BTOF as probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G and tested for detection of anti-IgG at concentrations of 50 ng/mL to 50 µg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G-antigen (rabbit anti-mouse IgG reactions was studied. Hydrofluoric acid treatment makes the sensitive region thinner to enhance sensitivity, which we confirmed by experiments and simulations. The limit of detection for the sensor was estimated to be less than 50 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of the antibody-antigen reaction is proposed as a rapid protein detection method.

  10. A sensitive chemiluminescent immunoassay to detect Chromotrope FB (Chr FB) in foods.

    Science.gov (United States)

    Xu, Kun; Long, Hao; Xing, Rongge; Yin, Yongmei; Eremin, Sergei A; Meng, Meng; Xi, Rimo

    2017-03-01

    Chromotrope FB (Chr FB) is a synthetic azo dye permitted for use in foods and medicines. An acceptable daily intake (ADI) of Chr FB was 0-0.5mg/kg in China. In this study, we synthesized a Chr FB hapten with an amino group to prepare its artificial immunogen. Polyclonal antibodies obtained from New Zealand rabbits were applied to develop an indirect competitive chemiluminescent immunoassay (icCLIA) to detect Chr FB in foods. A horseradish peroxidase (HRP)-luminol-H 2 O 2 system was used to yield CL signal with p-iodophenol as an enhancement reagent. The method showed good specificity towards Chr FB and could detect as low as 0.02ngmL -1 Chr FB in buffer, 0.07ngg -1 in yoghurt candy, 0.07ngg -1 in vitamin drink and 0.13ngg -1 in bread. Compared with HPLC method, the proposed method is more sensitive by two orders of magnitude. The accuracy and precision of this method are acceptable and comparable with HPLC method. Therefore, the proposed method could be used for rapid screening of Chr FB in the mentioned foodstuffs. Copyright © 2016. Published by Elsevier B.V.

  11. Capillary electrochromatography immunoassay for alpha-fetoprotein based on poly(guanidinium ionic liquid) monolithic material.

    Science.gov (United States)

    Liu, Cuicui; Deng, Qiliang; Fang, Guozhen; Dang, Meng; Wang, Shuo

    2017-08-01

    Alpha-fetoprotein (AFP) is widely used as a tumor marker for the serum diagnosis of primary hepatoma. Sensitive detection of AFP level plays an important role in the early diagnosis of disease and highly reliable prediction. In this study, a novel non-competitive immunoassay (IA) based on poly(guanidinium ionic liquid) monolithic material was developed for detecting ultra trace levels of AFP in capillary electrochromatography (CEC) mode. The AFP was mixed with an excess amount of fluorescently labeled antibody. After incubation, the immunocomplex was separated from the free labeled antibody and detected by CEC coupled with laser-induced fluorescence detector. Under the optimized conditions, the developed CEC-IA performed a low detection limit of 0.05 μg L -1  (S/N = 3) and a wide linearity ranging from 0.1 to 1000 μg L -1 for AFP, which can be largely attributed to the high separation and enrichment efficiency of poly(guanidinium ionic liquid) monolithic material for the targets. The application of this method was demonstrated by determining AFP in human serum. Copyright © 2017. Published by Elsevier Inc.

  12. Development of a Smartphone-based reading system for lateral flow immunoassay.

    Science.gov (United States)

    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.

  13. [Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and anatoxins].

    Science.gov (United States)

    Burkin, M A; Gal'vidis, I A; Iakovleva, I V; Sviridov, V V

    2007-01-01

    Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG1 monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse anatoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and anatoxins. The detection sensitivity of diphtheria toxin/anatoxin equaled 0.0005 Lf/ml; tetanus toxin and anatoxin were detected with sensitivities of 20 LD50/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum anatoxins (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E anatoxin (0.001 UI/ml).

  14. Fieldable, real-time enzyme immunoassay kits for drugs on surfaces

    Science.gov (United States)

    Chiappini, Michele W.; Wendel, Gregory J.; Duquette, Peter H.; Hamilton, Martha J.; Chudzik, Stephen J.; Chappa, Ralph A.

    1994-03-01

    Immunoassays (e.g., RIA, EIA) have been demonstrated to be useful for rapid, convenient detection and semiquantitative analysis of drugs. Thermedics Detection, Inc. manufactures a rapid, sensitive, self-contained, disposable, EIA device, developed by Bio-Metric Systems, Inc., designed to allow untrained personnel to perform in field situations. This format has been developed for drugs in biological fluids and on surfaces. The analyte in the test sample competes with an enzyme-analyte conjugate for a limited number of immobilized antibody sites. The AccuPRESS Test format can detect analytes at 10 ppb in biological fluids, water, and soil, and on surfaces, such as suitcases, vehicles, tables and hands, with positive results indicated by clearly visible color development within 5 minutes. This format is designed to have all dry components and to have an ambient shelf life of greater than one year. The format is available for cocaine and opiate derivatives, including heroin, and is readily adaptable for use with numerous other drugs, explosives, and environmental pollutants.

  15. Microsphere-based immunoassay integrated with a microfluidic network to perform logic operations

    International Nuclear Information System (INIS)

    Sabhachandani, Pooja; Cohen, Noa; Sarkar, Saheli; Konry, Tania

    2015-01-01

    Lab on a chip (LOC) intelligent diagnostics can be described by molecular logic-based circuits. We report on the development of an LOC approach with logic capability for screening combinations of antigen and antibody in the same sample. A microsphere-based immunoassay was integrated with a microfluidic network device to perform the logic operations AND and INHIBIT. Using the clinically relevant biomarkers TNF-α cytokine and anti-TNF-α antibody, we obtained a fluorescent output in the presence of both inputs. This results in an AND operation, while the presence of only one specific input results in a different fluorescent signal, thereby indicating the INHIBIT operation. This approach demonstrates the effective use of molecular logic computation for developing portable, point-of-care technologies for diagnostic purposes due to fast detection times, minimal reagent consumption and low costs. This model system may be further expanded to screening of multiple disease markers, combinatorial logic applications, and developing “smart” sensors and therapeutic technologies. (author)

  16. Electrochemical Immunoassay Using Open Circuit Potential Detection Labeled by Platinum Nanoparticles

    Directory of Open Access Journals (Sweden)

    Kanokwan Charoenkitamorn

    2018-02-01

    Full Text Available In this work, a simple electrochemical immunoassay based on platinum nanoparticles (PtNPs using open circuit potential (OCP detection was developed. The detection of human chorionic gonadotropin hormone (hCG as a model analyte, was demonstrated by direct electrical detection of PtNPs in hydrazine solution using OCP measurement without any application of either potential or current to the system. Disposable screen-printed carbon electrodes (SPCEs were utilized for the development of our immunosensor, which required a sample volume as small as 2 μL. After preparation of a sandwich-type immunosystem, hydrazine solution was dropped on the electrode’s surface, which was followed immediately by electrical detection using OCP. The change of the OCP signal originated from electrocatalytic oxidation of the hydrazine on PtNPs. Under the optimal conditions of a pH of 6.0 and a hydrazine concentration of 1 mM, a detection limit of 0.28 ng mL−1 and a linearity of 0–10 ng mL−1 were obtained. The PtNP-based OCP method is a simpler electrochemical detection procedure than those obtained from other electrochemical methods and has an acceptable sensitivity and reproducibility. The simplicity of the detection procedure and the cost-effectiveness of the disposable SPCE illustrate the attractive benefits of this sensor. Moreover, it could be applied to a simplified and miniaturized diagnostic system with minimal user manipulation.

  17. Pharmacokinetic analysis of topical tobramycin in equine tears by automated immunoassay.

    Science.gov (United States)

    Czerwinski, Sarah L; Lyon, Andrew W; Skorobohach, Brian; Léguillette, Renaud

    2012-08-21

    Ophthalmic antibiotic therapy in large animals is often used empirically because of the lack of pharmacokinetics studies. The purpose of the study was to determine the pharmacokinetics of topical tobramycin 0.3% ophthalmic solution in the tears of normal horses using an automated immunoassay analysis. The mean tobramycin concentrations in the tears at 5, 10, 15, 30 minutes and 1, 2, 4, 6 hours after administration were 759 (±414), 489 (±237), 346 (±227), 147 (±264), 27.6 (±28.4), 14.8 (±66.6), 6.7 (±18.6), and 23.4 (±73.4) mg/L. Mean tobramycin concentration was maintained above the MIC90 for commonly isolated bacteria for 68.5 min. A single dose of topical tobramycin resulted in therapeutic concentrations of tobramycin in the tears for 1 h after administration. Therapeutic levels of tobramycin remained in equine tears 6 times longer than was reported in rabbit tears.

  18. The Role of Nanoparticle Design in Determining Analytical Performance of Lateral Flow Immunoassays.

    Science.gov (United States)

    Zhan, Li; Guo, Shuang-Zhuang; Song, Fayi; Gong, Yan; Xu, Feng; Boulware, David R; McAlpine, Michael C; Chan, Warren C W; Bischof, John C

    2017-12-13

    Rapid, simple, and cost-effective diagnostics are needed to improve healthcare at the point of care (POC). However, the most widely used POC diagnostic, the lateral flow immunoassay (LFA), is ∼1000-times less sensitive and has a smaller analytical range than laboratory tests, requiring a confirmatory test to establish truly negative results. Here, a rational and systematic strategy is used to design the LFA contrast label (i.e., gold nanoparticles) to improve the analytical sensitivity, analytical detection range, and antigen quantification of LFAs. Specifically, we discovered that the size (30, 60, or 100 nm) of the gold nanoparticles is a main contributor to the LFA analytical performance through both the degree of receptor interaction and the ultimate visual or thermal contrast signals. Using the optimal LFA design, we demonstrated the ability to improve the analytical sensitivity by 256-fold and expand the analytical detection range from 3 log 10 to 6 log 10 for diagnosing patients with inflammatory conditions by measuring C-reactive protein. This work demonstrates that, with appropriate design of the contrast label, a simple and commonly used diagnostic technology can compete with more expensive state-of-the-art laboratory tests.

  19. Specific detection of Vibrio parahaemolyticus by fluorescence quenching immunoassay based on quantum dots.

    Science.gov (United States)

    Wang, Ling; Zhang, Junxian; Bai, Haili; Li, Xuan; Lv, Pintian; Guo, Ailing

    2014-07-01

    In this study, anti-Vibrio parahaemolyticus polyclonal and monoclonal antibodies were prepared through intradermal injection immune and lymphocyte hybridoma technique respectively. CdTe quantum dots (QDs) were synthesized at pH 9.3, 98 °C for 1 h with stabilizer of 2.7:1. The fluorescence intensity was 586.499, and the yield was 62.43%. QD probes were successfully prepared under the optimized conditions of pH 7.4, 37 °C for 1 h, 250 μL of 50 mg/mL EDC · HCl, 150 μL of 4 mg/mL NHS, buffer system of Na2HPO4-citric acid, and 8 μL of 2.48 mg/mL polyclonal antibodies. As gold nanoparticles could quench fluorescence of quantum dots, the concentration of V. parahaemolyticus could be detected through measuring the reduction of fluorescence intensity in immune sandwich reaction composed of quantum dot probe, gold-labeled antibody, and the sample. For pure culture, fluorescence intensity of the system was proportional with logarithm concentration of antigen, and the correlation coefficient was 99.764%. The fluorescence quenching immunoassay based on quantum dots is established for the first time to detect Vibrio parahaemolyticus. This method may be used as rapid testing procedure due to its high simplicity and sensitivity.

  20. Rapid detection of fumonisin B1 using a colloidal gold immunoassay strip test in corn samples.

    Science.gov (United States)

    Ling, Sumei; Wang, Rongzhi; Gu, Xiaosong; Wen, Can; Chen, Lingling; Chen, Zhibin; Chen, Qing-Ai; Xiao, Shiwei; Yang, Yanling; Zhuang, Zhenhong; Wang, Shihua

    2015-12-15

    Fumonisin B1 (FB1) is the most common and highest toxic of fumonisins species, exists frequently in corn and corn-based foods, leading to several animal and human diseases. Furthermore, FB1 was reported that it was associated with the human esophageal cancer. In view of the harmful of FB1, it is urgent to develop a feasible and accuracy method for rapid detection of FB1. In this study, a competitive immunoassay for FB1 detection was developed based on colloidal gold-antibody conjugate. The FB1-keyhole limpet hemoeyanin (FB1-KLH) conjugate was embedded in the test line, and goat anti-mouse IgG antibody embedded in the control line. The color density of the test line correlated with the concentration of FB1 in the range from 2.5 to 10 ng/mL, and the visual limit detection of test for FB1 was 2.5 ng/mL. The results indicated that the test strip is specific for FB1, and no cross-reactivity to other toxins. The quantitative detection for FB1 was simple, only needing one step without complicated assay performance and expensive equipment, and the total time of visual evaluation was less than 5 min. Hence, the developed colloidal gold-antibody assay can be used as a feasible method for FB1 rapid and quantitative detection in corn samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Multi-sample immunoassay inside optical fiber capillary enabled by evanescent wave detection

    Directory of Open Access Journals (Sweden)

    Chun-Wei Wang

    2016-03-01

    Full Text Available A novel evanescent wave-based (EW microfluidic capillary fiber-optic biosensor (MCFOB has been developed using capillaries as a transducer embedded in a multichannel device to enhance the collection efficiency of the fluorescence signal. The capillary serves dual roles as a waveguide and a container, enabling more straightforward, consistent, and compact biosensor packaging compared to conventional optical fiber biosensors and microfluidic systems. In order to detect multiple samples in one device, the biosensor incorporates a polydimethysiloxane (PDMS multi-channel device, which also serves as cladding for the biosensor. In addition, this biosensor only consumes 10 μl of a sample and does not require hydrofluoric acid etching in the fabrication process. The orientation for signal collection is optimized by comparing the lateral and normal signal directions for detected glyceraldehyde 3-phosphate dehydrogenase (GAPDH. C-reactive protein (CRP is used to validate the MCFOB, and the limit of detection (LOD for CRP in the MCFOB is 1.94 ng/ml (74 pM. Moreover, the real-time measurement is demonstrated to verify that the evanescent wave is the only exciting light source in the MCFOB, which gives the potential for real-time measurement applications. Keywords: C-reactive protein, Capillary, Fiber-optic, Microfluidic, Evanescent wave, Immunoassay

  2. Effects of saliva collection using cotton swab on cortisol enzyme immunoassay.

    Science.gov (United States)

    Kozaki, Tomoaki; Hashiguchi, Nobuko; Kaji, Yumi; Yasukouchi, Akira; Tochihara, Yutaka

    2009-12-01

    Cotton swabs are among the most commonly used devices for collecting saliva, but various studies have reported that their use impacts the results of salivary cortisol assays. These studies, however, estimated this impact by comparing the average of the concentration and/or scatter plots. In the present study, we estimated the impact of cotton swabs on the results of salivary cortisol enzyme immunoassay (EIA) by Bland-Altman plot. Eight healthy males (aged 20-23 years) provided four saliva samples on different days to yield a total of 32 samples. Saliva samples were collected directly in plastic tubes using plastic straws and then pipetted onto cotton swabs (cotton saliva collection) and into clear sterile tubes (passive saliva collection). There was a lower correlation between cotton and passive saliva collection. Individually, four subjects showed a negative correlation between passive and cotton saliva collection. A Bland-Altman plot indicated that cotton swabs causes a proportional bias on the EIA assay result. Our findings indicate a considerable effect of using cotton swabs for saliva collection, and subject-specific variability in the impact. A Bland-Altman plot further suggests possible reasons for this effect.

  3. Toward development of a surface-enhanced Raman scattering (SERS)-based cancer diagnostic immunoassay panel.

    Science.gov (United States)

    Granger, Jennifer H; Granger, Michael C; Firpo, Matthew A; Mulvihill, Sean J; Porter, Marc D

    2013-01-21

    Proteomic analyses of readily obtained human fluids (e.g., serum, urine, and saliva) indicate that the diagnosis of complex diseases will be enhanced by the simultaneous measurement of multiple biomarkers from such samples. This paper describes the development of a nanoparticle-based multiplexed platform that has the potential for simultaneous read-out of large numbers of biomolecules. For this purpose, we have chosen pancreatic adenocarcinoma (PA) as a test bed for diagnosis and prognosis. PA is a devastating form of cancer in which an estimated 86% of diagnoses resulted in death in the United States in 2010. The high mortality rate is due, in part, to the asymptomatic development of the disease and the dearth of sensitive diagnostics available for early detection. One promising route lies in the development of a serum biomarker panel that can generate a signature unique to early stage PA. We describe the design and development of a proof-of-concept PA biomarker immunoassay array coupled with surface-enhanced Raman scattering (SERS) as a sensitive readout method.

  4. Fluorescently labelled multiplex lateral flow immunoassay based on cadmium-free quantum dots.

    Science.gov (United States)

    Beloglazova, Natalia V; Sobolev, Aleksander M; Tessier, Mickael D; Hens, Zeger; Goryacheva, Irina Yu; De Saeger, Sarah

    2017-03-01

    A sensitive tool for simultaneous qualitative detection of two mycotoxins based on use of non-cadmium quantum dots (QDs) is presented for the first time. QDs have proven themselves as promising fluorescent labels for biolabeling and chemical analysis. With an increasing global tendency to regulate and limit the use of hazardous elements, indium phosphide (InP) QDs are highlighted as environmentally-friendly alternatives to the highly efficient and well-studied, but potentially toxic Cd- and Pb-based QDs. Here, we developed water-soluble InP QDs-based fluorescent nanostructures. They consisted of core/shell InP/ZnS QDs enrobed in a silica shell that allowed the water solubility (QD@SiO 2 ). Then we applied the QD@SiO 2 as novel, silica shell-encapsulated fluorescent labels in immunoassays for rapid multiplexed screening. Two mycotoxins, zearalenone and deoxynivalenol, were simultaneously detected in maize and wheat, since the two QD@SiO 2 labelled conjugates emit at two different, individually detectable wavelengths. The cutoff values for the simultaneous determination were 50 and 500μgkg -1 for zearalenone and deoxynivalenol, respectively, in both maize and wheat. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to confirm the result. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Production of monoclonal antibodies for sandwich immunoassay detection of Pacific ciguatoxins.

    Science.gov (United States)

    Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

    2010-10-01

    Ciguatoxins are the major causative toxins of ciguatera seafood poisoning. Limited availability of ciguatoxins has hampered the development of a reliable and specific immunoassay for detecting these toxins in contaminated fish. Monoclonal antibodies (mAbs) specific against both ends of Pacific ciguatoxins CTX3C and 51-hydroxyCTX3C were prepared by immunization of mice with the protein conjugates of rationally designed synthetic haptens in place of the natural toxin. Haptenic groups that possess a surface area larger than 400 A(2) were required to produce mAbs that can bind strongly to CTX3C or 51-hydroxyCTX3C. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was established to detect CTX3C and 51-hydroxyCTX3C at the ppb level with no cross-reactivity against the other marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin. Copyright 2009 Elsevier Ltd. All rights reserved.

  6. Comparative digoxin determination in serum by means of a radioimmuno- and an enzyme immunoassay

    International Nuclear Information System (INIS)

    Reinhard, M.

    1981-01-01

    Two immunologic measuring methods for the quantitative digoxin determination in serum were compared. One method bases on the principle of radioisotope dilution, the second one on the principle of enzyme inhibition. The radioimmunoassay served as reference method. The limit of detection for RIA is 0.23 ng/ml, for EIA 0.40 ng/ml. For both methods the measuring range extends up to approx. 5.5 ng/ml. The degree of precision in series is 8.2% for RIA, 10.8% for EIA. Day-to-day precision is 4.4% for RIA, 15.2% for EIA. On comparison, the 59 serum samples of patients who received digoxin did not show any systematic difference. The results obtained can be transformed by means of the equations Csub(EIA) = 0.041 ng/ml + 0.936 Csub(RIA). In pathologic sera, however, there are significant differences disfavoring EIA, because due to high color concentrations or turbidities these sera do not permit any or any exact extinction measurements. The enzyme immunoassay should not be used with such sera. With regard to practicability the EIA corresponds more or less to RIA. The EIA can essentially be economized by using semi-microcuvettes and applying only the half of the recommended enzyme and antibody volume. (orig.) [de

  7. A sensitive chemiluminescence enzyme immunoassay based on molecularly imprinted polymers solid-phase extraction of parathion.

    Science.gov (United States)

    Chen, Ge; Jin, Maojun; Du, Pengfei; Zhang, Chan; Cui, Xueyan; Zhang, Yudan; She, Yongxin; Shao, Hua; Jin, Fen; Wang, Shanshan; Zheng, Lufei; Wang, Jing

    2017-08-01

    The chemiluminescence enzyme immunoassay (CLEIA) method responds differently to various sample matrices because of the matrix effect. In this work, the CLEIA method was coupled with molecularly imprinted polymers (MIPs) synthesized by precipitation polymerization to study the matrix effect. The sample recoveries ranged from 72.62% to 121.89%, with a relative standard deviation (RSD) of 3.74-18.14%.The ratio of the sample matrix-matched standard curve slope rate to the solvent standard curve slope was 1.21, 1.12, 1.17, and 0.85 for apple, rice, orange and cabbage in samples pretreated with the mixture of PSA and C 18 . However, the ratio of sample (apple, rice, orange, and cabbage) matrix-matched standard-MIPs curve slope rate to the solvent standard curve was 1.05, 0.92, 1.09, and 1.05 in samples pretreated with MIPs, respectively. The results demonstrated that the matrices of the samples greatly interfered with the detection of parathion residues by CLEIA. The MIPs bound specifically to the parathion in the samples and eliminated the matrix interference effect. Therefore, the CLEIA method have successfully applied MIPs in sample pretreatment to eliminate matrix interference effects and provided a new sensitive assay for agro-products. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Comparison of Salivary and Serum Enzyme Immunoassays for the Diagnosis of Helicobacter pylori Infection

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    John M Embil

    1998-01-01

    Full Text Available Infection with Helicobacter pylori has been established as an important risk factor for the development of peptic ulcer disease, gastritis and gastric cancer. The diagnosis of H pylori infection can be established by invasive or noninvasive techniques. Two noninvasive enzyme immunoassays (EIAs for antibody detection – HeliSal and Pylori Stat – were compared with histology. Both assays detect immunoglobulin (Ig G directed against purified H pylori antigen. The test populations consisted of 104 consecutive patients scheduled for upper gastrointestinal endoscopy. Of these patients, 97 (93% had symptoms compatible with peptic ulcer disease. Saliva and serum were collected simultaneously at the time of endoscopy. Salivary EIA had a sensitivity of 66%, specificity of 67%, positive predictive value of 67% and negative predictive value of 66% compared with the serum EIA, where the results were 98%, 48%, 64% and 96%, respectively. Although the salivary EIA is an appealing noninvasive test, it was not a sensitive and specific assay. The serum EIA also lacked specificity, but was highly sensitive with a good negative predictive value. Although a negative serum EIA rules out H pylori infection, a positive result must be interpreted in the clinical context and confirmed with a more specific measure.

  9. Data mining strategies to improve multiplex microbead immunoassay tolerance in a mouse model of infectious diseases.

    Directory of Open Access Journals (Sweden)

    Akshay Mani

    Full Text Available Multiplex methodologies, especially those with high-throughput capabilities generate large volumes of data. Accumulation of such data (e.g., genomics, proteomics, metabolomics etc. is fast becoming more common and thus requires the development and implementation of effective data mining strategies designed for biological and clinical applications. Multiplex microbead immunoassay (MMIA, on xMAP or MagPix platform (Luminex, which is amenable to automation, offers a major advantage over conventional methods such as Western blot or ELISA, for increasing the efficiencies in serodiagnosis of infectious diseases. MMIA allows detection of antibodies and/or antigens efficiently for a wide range of infectious agents simultaneously in host blood samples, in one reaction vessel. In the process, MMIA generates large volumes of data. In this report we demonstrate the application of data mining tools on how the inherent large volume data can improve the assay tolerance (measured in terms of sensitivity and specificity by analysis of experimental data accumulated over a span of two years. The combination of prior knowledge with machine learning tools provides an efficient approach to improve the diagnostic power of the assay in a continuous basis. Furthermore, this study provides an in-depth knowledge base to study pathological trends of infectious agents in mouse colonies on a multivariate scale. Data mining techniques using serodetection of infections in mice, developed in this study, can be used as a general model for more complex applications in epidemiology and clinical translational research.

  10. The clinical application of fluorescent-enzyme immunoassay to detect human thyroid peroxidase autoantibody quantitatively

    International Nuclear Information System (INIS)

    Chai Jinyan; Fang Peihua; Li Ning; Zhang Yanli

    2010-01-01

    Objective: To establish a fluorescent-enzyme immunoassay (FEIA) with recombinant human thyroid peroxidase (hTPO) as the antigen. Methods: Sera of 326 healthy people, 119 cases of Hashimoto's thyroiditis (HT), 116 cases of Graves disease (untreated), 28 cases of nodular goiter, 10 ca-ses of subacute thyroiditis and 6 cases of simple goiter were measured by the FEIA with recombinant hTPO as the antigen. Rrank sum test and χ 2 -test were used in inter-groups. Results: (1) Concentration above 4000 U/L was considered to be positive. (2) The intra-assay coefficient of variation (CV) and the inter-as-say CV were 4.59% ∼ 6.52% and 17.37% ∼ 17.45%. (3) The values measured by the FEIA were positively correlated with the values measured by hTPO antibody (hTPOAb) commercial kit (r=0.80, P 2 = 53.45, 39.30, 15.41 and 21.74, all P < 0.01). Conclusions: The method can be applied in the serum measurement of thyroid disease. HT presented the highest positive rate, therefore, the FEIA was an effective method in diagnosing HT. (authors)

  11. Evaluation of electrochemiluminescene immunoassay and enzyme-linked immunosorbent assay for serum HBsAb detection

    International Nuclear Information System (INIS)

    Ma Caiyun; Jiang Li; Ge Gaoxia; Zhang Xiaojie

    2005-01-01

    Electrochemiluminescene immunoassay (ECLIA) and enzyme-linked immunosorbent (ELISA) were used to detect the different concentrations of serum HBsAb, in order to compare the results of ECLIA and ELISA. The result showed that intra-assay coefficient of variation of ECLIA was about 0.95% for high value, 1.13% for mean values and 2.63% for low value, while that of ELISA was about 5.76%, 12.8% and 15.9%, respectively. The interassay coefficient of ECLIA was about 4.03% for high values, 4.93% for mean values and 7.34% for low values, while that of ELISA was about 10.1%, 19.6% and 25.2%, respectively. The analytical sensitivity of ECLIA was about 4.0IU/L, while that of ELISA is about 19.0IU/L. Only in 3 samples, the results measured by ECLIA and ELISA were different (ECLIA: positive; ELISA: negative) among 165 samples. It is concluded that the met hod of ECLIA is more efficient than ELISA for detection of HBsAb in serum. (authors)

  12. Hierarchical Nanogold Labels to Improve the Sensitivity of Lateral Flow Immunoassay

    Science.gov (United States)

    Serebrennikova, Kseniya; Samsonova, Jeanne; Osipov, Alexander

    2018-06-01

    Lateral flow immunoassay (LFIA) is a widely used express method and offers advantages such as a short analysis time, simplicity of testing and result evaluation. However, an LFIA based on gold nanospheres lacks the desired sensitivity, thereby limiting its wide applications. In this study, spherical nanogold labels along with new types of nanogold labels such as gold nanopopcorns and nanostars were prepared, characterized, and applied for LFIA of model protein antigen procalcitonin. It was found that the label with a structure close to spherical provided more uniform distribution of specific antibodies on its surface, indicative of its suitability for this type of analysis. LFIA using gold nanopopcorns as a label allowed procalcitonin detection over a linear range of 0.5-10 ng mL-1 with the limit of detection of 0.1 ng mL-1, which was fivefold higher than the sensitivity of the assay with gold nanospheres. Another approach to improve the sensitivity of the assay included the silver enhancement method, which was used to compare the amplification of LFIA for procalcitonin detection. The sensitivity of procalcitonin determination by this method was 10 times better the sensitivity of the conventional LFIA with gold nanosphere as a label. The proposed approach of LFIA based on gold nanopopcorns improved the detection sensitivity without additional steps and prevented the increased consumption of specific reagents (antibodies).

  13. Development of a Highly Specific Fluorescence Immunoassay for Detection of Diisobutyl Phthalate in Edible Oil Samples.

    Science.gov (United States)

    Cui, Xiping; Wu, Panpan; Lai, Dan; Zheng, Shengwu; Chen, Yingshan; Eremin, Sergei A; Peng, Wei; Zhao, Suqing

    2015-10-28

    The diisobutyl phthalate (DiBP) hapten containing an amino group was synthesized successfully, and the polyclonal antibody against 4-amino phthalate-bovine serum albumin (BSA) was developed. On the basis of the polyclonal antibody, a rapid and sensitive indirect competitive fluorescence immunoassay (icFIA) has been established to detect DiBP in edible oil samples for the first time. Under the optimized conditions, the quantitative working range of the icFIA was from 10.47 to 357.06 ng/mL (R(2) = 0.991), exhibiting a detection limit of 5.82 ng/mL. In this assay, the specific results showed that other similar phthalates did not significantly interfere with the analysis, with the cross-reactivity less than 1.5%, except for that of DiBAP. Thereafter, DiBP contamination in edible oil samples was detected by icFIA, with the recovery being from 79 to 103%. Furthermore, the reliability of icFIA was validated by gas chromatography-mass spectrometry (GC-MS). Therefore, the developed icFIA is suitable for monitoring DiBP in some edible oil samples.

  14. Preparation and Characteristic of Dextran-BSA Antibody and Establishment of its ELISA Immunoassay.

    Science.gov (United States)

    Xie, Zhen-ming; Yu, Lin; Fang, Li-sha

    2015-01-01

    The enzyme-linked immunosorbent assay (ELISA) is a potential tool for the determination of dextran. In this study, dextran neoglycoprotein antigens were prepared by Reductive Amination method, and were confirmed by SDS-PAGE and free amino detection. The impact factors such as different oxidation degree of dextran, the conjugate reaction time to BSA were investigated. The best preparation conditions were obtained (n(dextran)/n(oxidant) of NaIO4 = 1/120, the reaction time of 24 h), and the antigen with best combination with standard was obtained. The antigens interacted with standard antibody and were evaluated through ELISA. The immunogen was immunized with white rabbits to obtained antibody, respectively. A general and broad class-specific ELISA immunoassay was developed for dextran detection according to ELISA theory. The optimized conditions of assay used coating antigen at 10 μg/mL, reaction time of antibody and rabbit-anti-bovine IgG in 45 min, blocking reagents with 5% calf serum. The developed ELISA detection method with good linear and accuracy was put to use for quantitative analysis of dextran T40 in commercial sugarpractical for detection of dextran.

  15. A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin.

    Science.gov (United States)

    Feng, Xiao-Li; Ren, Hong-Lin; Li, Yan-Song; Hu, Pan; Zhou, Yu; Liu, Zeng-Shan; Yan, Dong-Ming; Hui, Qi; Liu, Dong; Lin, Chao; Liu, Nan-Nan; Liu, Yan-Yan; Lu, Shi-Ying

    2014-08-15

    Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles-chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol-H2O2 chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs-CLEIA method had a linear range from 0.31 to 100ng/ml with a detection limit of 0.24ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Sensitive and rapid immunoassay for parathyroid hormone using magnetic particle labels and magnetic actuation.

    Science.gov (United States)

    Dittmer, W U; de Kievit, P; Prins, M W J; Vissers, J L M; Mersch, M E C; Martens, M F W C

    2008-09-30

    A rapid method for the sensitive detection of proteins using actuated magnetic particle labels, which are measured with a giant magneto-resistive (GMR) biosensor, is described. The technique involves a 1-step sandwich immunoassay with no fluid replacement steps. The various assay binding reactions as well as the bound/free separation are entirely controlled by magnetic forces induced by electromagnets above and below the sensor chip. During the assay, particles conjugated with tracer antibodies are actuated through the sample for target capture, and rapidly brought to the sensor surface where they bind to immobilized capture antibodies. Weakly or unbound labels are removed with a magnetic force oriented away from the GMR sensor surface. For the measurement of parathyroid hormone (PTH), a detection limit in the 10 pM range is obtained with a total assay time of 15 min when 300 nm particles are used. The same sensitivity can be achieved in 5 min when 500 nm particles are used. If 500 nm particles are employed in a 15-minute assay, then 0.8 pM of PTH is detectable. The low sample volume, high analytical performance and high speed of the test coupled with the compact GMR biosensor make the system especially suitable for sensitive testing outside of laboratory environments.

  17. Surface plasmon resonance immunoassay analysis of pituitary hormones in urine and serum samples.

    Science.gov (United States)

    Treviño, Juan; Calle, Ana; Rodríguez-Frade, José Miguel; Mellado, Mario; Lechuga, Laura M

    2009-05-01

    Direct determination of four pituitary peptide hormones: human thyroid stimulating hormone (hTSH), growth hormone (hGH), follicle stimulating hormone (hFSH), and luteinizing hormone (hLH) has been carried out using a portable surface plasmon resonance (SPR) immunosensor. A commercial SPR biosensor was employed. The immobilization of the hormones was optimized and monoclonal antibodies were selected in order to obtain the best sensor performance. Assay parameters as running buffer and regeneration solution composition or antibody concentration were adjusted to achieve a sensitive analyte detection. The performance of the assays was assessed in buffer solution, serum and urine, showing sensitivity in the range from 1 to 6 ng/mL. The covalent attachment of the hormones ensured the stability of the SPR signal through repeated use in up to 100 consecutive assay cycles. Mean intra- and inter-day coefficients of variation were all <7%, while batch-assay variability using different sensor surfaces was <5%. Taking account both the excellent reutilization performance and the outstanding reproducibility, this SPR immunoassay method turns on a highly reliable tool for endocrine monitoring in laboratory and point-of-care (POC) settings.

  18. Electrochemical Sandwich Immunoassay for the Ultrasensitive Detection of Human MUC1 Cancer Biomarker

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    Zahra Taleat

    2013-01-01

    Full Text Available A new electrochemical sandwich immunoassay for the ultrasensitive detection of human MUC1 cancer biomarker using protein G-functionalized magnetic beads (MBs and graphite-based screen-printed electrodes (SPEs was developed. Magnetic beads were employed as the platforms for the immobilization and immunoreaction process. A pair of primary and secondary antibodies was used to capture the MUC1 protein. After labeling with a third antibody conjugated with horseradish peroxidase (HRP, the resulting conjugate was trapped at the surface of the graphite-based SPEs and MUC1 determination was carried out by differential pulse voltammetry (DPV at 0.4 V upon H2O2 addition using acetaminophen (APAP as the redox mediator. A linear relationship was obtained for the detection of human MUC1 over a range of 0–25 ppb with the lowest detection limit of 1.34 ppb when HRP was applied as a label. Preliminary experiments were performed using disposable electrochemical sensors in order to optimize some parameters (i.e., incubation times, concentrations, and blocking agent.

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

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    Xiang-Yang Shao

    2017-02-01

    Full Text Available 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.

  20. Multiplexed capillary microfluidic immunoassay with smartphone data acquisition for parallel mycotoxin detection.

    Science.gov (United States)

    Machado, Jessica M D; Soares, Ruben R G; Chu, Virginia; Conde, João P

    2018-01-15

    The field of microfluidics holds great promise for the development of simple and portable lab-on-a-chip systems. The use of capillarity as a means of fluidic manipulation in lab-on-a-chip systems can potentially reduce the complexity of the instrumentation and allow the development of user-friendly devices for point-of-need analyses. In this work, a PDMS microchannel-based, colorimetric, autonomous capillary chip provides a multiplexed and semi-quantitative immunodetection assay. Results are acquired using a standard smartphone camera and analyzed with a simple gray scale quantification procedure. The performance of this device was tested for the simultaneous detection of the mycotoxins ochratoxin A (OTA), aflatoxin B1 (AFB1) and deoxynivalenol (DON) which are strictly regulated food contaminants with severe detrimental effects on human and animal health. The multiplexed assay was performed approximately within 10min and the achieved sensitivities of<40, 0.1-0.2 and<10ng/mL for OTA, AFB1 and DON, respectively, fall within the majority of currently enforced regulatory and/or recommended limits. Furthermore, to assess the potential of the device to analyze real samples, the immunoassay was successfully validated for these 3 mycotoxins in a corn-based feed sample after a simple sample preparation procedure. Copyright © 2017 Elsevier B.V. All rights reserved.