Development of a rapid low cost fluorescent biosensor for the detection of food contaminants

A prototype fluorescent based biosensor has been developed for the antibody based detection of food related contaminants. Its performance was characterised and showed a typical antibody binding signal of 200-2000 mV, a short term noise of 9.1 mV, and baseline slope of -0.016 mV/s over 4 h. Bulk signal detection repeatability (n=23) and reproducibility (n=3) were less than 2.4%CV. The biosensor detection unit was evaluated using two food related model systems proving its ability to monitor both binding using commercial products and inhibition through the development of an assay. This assay development potential was evaluated by observing the biosensor's performance whilst appraising several labelled antibody and glass slide configurations. The molecular interaction between biotin and an anti-biotin antibody was shown to be inhibited by 41% due to the presence of biotin in a sample. A food toxin (domoic acid) calibration curve was produced, with %CVs ranging from 2.7 to 7.8%, and a midpoint of approximately 17 ng/ml with further optimisation possible. The ultimate aim of this study was to demonstrate the working principles of this innovative biosensor as a potential portable tool with the opportunity of interchangeable assays. The biosensor design is applicable for the requirements of routine food contaminant analysis, with respect to performance, functionality and cost. (C) 2012 Elsevier B.V. All rights reserved.

Ligand arsenic complexation and immunoperoxidase detection of metallothionein in the earthworm Lumbricus rubellus inhabiting arsenic-rich soil

Although earthworms have been found to inhabit arsenic-rich soils in the U.K., the mode of arsenic detoxification is currently unknown. Biochemical analyses and subcellular localization studies have indicated that As3+-thiol complexes may be involved; however, it is not known whether arsenic is capable of inducing the expression of metallothionein (MT) in earthworms. The specific aims of this paper were (a) to detect and gain an atomic characterization of ligand complexing by X-ray absorption spectrometry (XAS), and (b) to employ a polyclonal antibody raised against an earthworm MT isoform (w-MT2) to detect and localize the metalloprotein by immunoperoxidase histochemistry in the tissues of earthworms sampled from arsenic-rich soil. Data suggested that the proportion of arsenate to sulfur-bound species varies within specific earthworm tissues. Although some arsenic appeared to be in the form of arsenobetaine, the arsenic within the chlorogogenous tissue was predominantly coordinated with S in the form of -SH groups. This suggests the presence of an As::MT complex. Indeed, MT was detectable with a distinctly localized tissue and cellular distribution. While MT was not detectable in the surface epithelium or in the body wall musculature, immunoperoxidase histochemistry identified the presence of MT in chloragocytes around blood vessels, within the typhlosolar fold, and in the peri-intestinal region. Focal immunostaining was also detectable in a cohort of cells in the intestinal wall. The results of this study support the hypothesis that arsenic induces MT expression and is sequestered by the metalloprotein in certain target cells and tissues.

Detection of intrastrain antigenic variation of Bacteroides fragilis surface polysaccharides by monoclonal antibody labelling

Bacteroides fragilis is a constituent of the normal resident microbiota of the human intestine and is the gram-negative obligately anaerobic bacterium most frequently isolated from clinical infection. Surface polysaccharides are implicated as potential virulence determinants. We present evidence of within strain immunochemical variation of surface polysaccharides in populations that are noncapsulate by light microscopy as determined by monoclonal antibody labelling. Expression of individual epitopes can be enriched from a population of an individual strain by use of immunomagnetic beads. Also, individual colonies in which either >94% or 94% of the bacteria carry a given epitope, there is no enrichment for other epitopes recognized by different polysaccharide-specific monoclonal antibodies. This intrastrain variation has important implications for the development of potential vaccines or immunodiagnostic tests.

Logic feels so good-I like it! Evidence for intuitive detection of logicality in syllogistic reasoning

When people evaluate syllogisms, their judgments of validity are often biased by the believability of the conclusions of the problems. Thus, it has been suggested that syllogistic reasoning performance is based on an interplay between a conscious and effortful evaluation of logicality and an intuitive appreciation of the believability of the conclusions (e.g., Evans, Newstead, Allen, & Pollard, 1994). However, logic effects in syllogistic reasoning emerge even when participants are unlikely to carry out a full logical analysis of the problems (e.g., Shynkaruk & Thompson, 2006). There is also evidence that people can implicitly detect the conflict between their beliefs and the validity of the problems, even if they are unable to consciously produce a logical response (e.g., De Neys, Moyens, & Vansteenwegen, 2010). In 4 experiments we demonstrate that people intuitively detect the logicality of syllogisms, and this effect emerges independently of participants' conscious mindset and their cognitive capacity. This logic effect is also unrelated to the superficial structure of the problems. Additionally, we provide evidence that the logicality of the syllogisms is detected through slight changes in participants' affective states. In fact, subliminal affective priming had an effect on participants' subjective evaluations of the problems. Finally, when participants misattributed their emotional reactions to background music, this significantly reduced the logic effect.

Crack detection of steel girders using Brillouin optical time domain analysis

This article presents the results from an experimental program designed to evaluate the performance of a system consisting of a readout unit and a ribbon type Fiber Optic Sensor (FOS) based on Brillouin Optical Time Domain Analysis (BOTDA). The system is intended for the detection of cracks as well as the monitoring of long-term performance for steel bridge girders. The program consisted of introducing a crack at the center of a 3-m-long steel beam and monitoring its progression using static loading tests performed at ambient and sub-zero temperatures. For sensor lengths similar to those used in the field, the resonant frequency shifts per unit increase in crack width were found to decrease from 114 MHz/mm at ambient temperature (~25C) to 65 MHz/mm at -10C. Results also revealed nonlinearity and variability, which can be attributed to an incompatibility between the settings of the laser pump in the readout unit and the sensor length. Significant losses were detected along the bonded segments of the sensor and were attributed to the presence of ripples along the sensor. These undulations worsen with a reduction in temperature and are induced by the bonding procedure as well as the slack provided in the plastic sleeves containing the splices.

Improved Detection of Mycobacterium bovis Infection in Bovine Lymph Node Tissue Using Immunomagnetic Separation (IMS)-Based Methods

Immunomagnetic separation (IMS) can selectively isolate and concentrate Mycobacterium bovis cells from lymph node tissue to facilitate subsequent detection by PCR (IMS-PCR) or culture (IMS-MGIT). This study describes application of these novel IMS-based methods to test for M. bovis in a survey of 280 bovine lymph nodes (206 visibly lesioned (VL), 74 non-visibly lesioned (NVL)) collected at slaughter as part of the Northern Ireland bovine TB eradication programme. Their performance was evaluated relative to culture. Overall, 174 (62.1%) lymph node samples tested positive by culture, 162 (57.8%) by IMS-PCR (targeting IS6110), and 196 (70.0%) by IMS-MGIT culture. Twelve (6.9%) of the 174 culture positive lymph node samples were not detected by either of the IMS-based methods. However, an additional 78 M. bovis positive lymph node samples (26 (12.6%) VL and 54 (73.0%) NVL) were detected by the IMS-based methods and not by culture. When low numbers of viable M. bovis are present in lymph nodes (e.g. in NVLs of skin test reactor cattle) decontamination prior to culture may adversely affect viability, leading to false negative culture results. In contrast, IMS specifically captures whole M. bovis cells (live, dead or potentially dormant) which are not subject to any deleterious treatment before detection by PCR or MGIT culture. During this study only 2.7% of NVL lymph nodes tested culture positive, whereas 73% of the same samples tested M. bovis positive by the IMS-based tests. Results clearly demonstrate that not only are the IMS-based methods more rapid but they have greater detection sensitivity than the culture approach currently used for the detection of M. bovis infection in cattle.. Adoption of the IMS-based methods for lymph node testing would have the potential to improve M. bovis detection in clinical samples.

Multiplex Detection of Plant Pathogens Using a Microsphere Immunoassay Technology

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. © 2013 Charlermroj et al.

Effect-based proteomic detection of growth promoter abuse

Unregulated growth promoter use in food-producing animals is an issue of concern both from food safety and animal welfare perspectives. However, the monitoring of such practices is analytically challenging due to the concerted actions of users to evade detection. Techniques based on the monitoring of biological responses to exogenous administrations have been proposed as more sensitive methods to identify treated animals. This study has, for the first time, profiled plasma proteome responses in bovine animals to treatment with nortestosterone decanoate and 17 beta-oestradiol benzoate, followed by dexamethasone administration. Two-dimensional fluorescence differential in-gel electrophoresis analysis revealed a series of hepatic and acute-phase proteins within plasma whose levels were up- or down-regulated within phases of the treatment regime. Surface plasmon resonance (SPR) immuno-assays were developed to quantify responses of identified protein markers during the experimental treatment study with a view to developing methods which can be used as screening tools for growth promoter abuse detection. SPR analysis demonstrated the potential for plasma proteins to be used as indicative measures of growth promoter administrations and concludes that the sensitivity and robustness of any detection approach based on plasma proteome analysis would benefit from examination of a range of proteins representative of diverse biological processes rather being reliant on specific individual markers.

Detection of magnetic dipole lines of Fe XII in the ultraviolet spectrum of the dwarf star ε Eri

We report observations of the dwarf star e Eri (K2V) made with the Space Telescope Imaging Spectrograph (STIS) on the Hubble Space Telescope. The high sensitivity of the STIS instrument has allowed us to detect the magnetic dipole transitions of Fe XII at 1242.00 and 1349 38 Å for the first time in a star other than the Sun. The width of the stronger line at 1242.00 Å has also been measured; such measurements are not possible for the permitted lines of Fe XII in the extreme-ultraviolet. To within the accuracy of the measurements the N v and the Fe XII lines occur at their rest wavelengths. Electron densities and linewidths have been measured from other transition region lines. Together, these can be used to investigate the non-thermal energy flux in the lower and upper transition regions, which is useful in constraining possible heating processes. The Fe XII lines are also present in archival STIS spectra of other G/K-type dwarfs.

Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2 V617F and other relevant mutations

Molecular genetic assays for the detection of the JAK2 V617F (c.1849G>T) and other pathogenetic mutations within JAK2 exon 12 and MPL exon 10 are part of the routine diagnostic workup for patients presenting with erythrocytosis, thrombocytosis or otherwise suspected to have a myeloproliferative neoplasm. A wide choice of techniques are available for the detection of these mutations, leading to potential difficulties for clinical laboratories in deciding upon the most appropriate assay, which can lead to problems with inter-laboratory standardization. Here, we discuss the most important issues for a clinical diagnostic laboratory in choosing a technique, particularly for detection of the JAK2 V617F mutation at diagnosis. The JAK2 V617F detection assay should be both specific and sensitive enough to detect a mutant allele burden as low as 13%. Indeed, the use of sensitive assays increases the detection rate of the JAK2 V617F mutation within myeloproliferative neoplasms. Given their diagnostic relevance, it is also beneficial and relatively straightforward to screen JAK2 V617F negative patients for JAK2 exon 12 mutations (in the case of erythrocytosis) or MPL exon 10 mutations (thrombocytosis or myelofibrosis) using appropriate assays. Molecular results should be considered in the context of clinical findings and other haematological or laboratory results.

Next generation planar waveguide detection of microcystins in freshwater and cyanobacterial extracts, utilising a novel lysis method for portable sample preparation and analysis

The study details the development of a fully validated, rapid and portable sensor based method for the on-site analysis of microcystins in freshwater samples. The process employs a novel lysis method for the mechanical lysis of cyanobacterial cells, with glass beads and a handheld frother in only 10min. The assay utilises an innovative planar waveguide device that, via an evanescent wave excites fluorescent probes, for amplification of signal in a competitive immunoassay, using an anti-microcystin monoclonal with cross-reactivity against the most common, and toxic variants. Validation of the assay showed the limit of detection (LOD) to be 0.78ngmL and the CCß to be 1ngmL. Robustness of the assay was demonstrated by intra- and inter-assay testing. Intra-assay analysis had % C.V.s between 8 and 26% and recoveries between 73 and 101%, with inter-assay analysis demonstrating % C.V.s between 5 and 14% and recoveries between 78 and 91%. Comparison with LC-MS/MS showed a high correlation (R=0.9954) between the calculated concentrations of 5 different Microcystis aeruginosa cultures for total microcystin content. Total microcystin content was ascertained by the individual measurement of free and cell-bound microcystins. Free microcystins can be measured to 1ngmL, and with a 10-fold concentration step in the intracellular microcystin protocol (which brings the sample within the range of the calibration curve), intracellular pools may be determined to 0.1ngmL. This allows the determination of microcystins at and below the World Health Organisation (WHO) guideline value of 1µgL. This sensor represents a major advancement in portable analysis capabilities and has the potential for numerous other applications.

Detection of visual-field deterioration by Glaucoma Progression Analysis and Threshold Noiseless Trend programs

BACKGROUND: To compare the ability of Glaucoma Progression Analysis (GPA) and Threshold Noiseless Trend (TNT) programs to detect visual-field deterioration.

METHODS: Patients with open-angle glaucoma followed for a minimum of 2 years and a minimum of seven reliable visual fields were included. Progression was assessed subjectively by four masked glaucoma experts, and compared with GPA and TNT results. Each case was judged to be stable, deteriorated or suspicious of deterioration

RESULTS: A total of 56 eyes of 42 patients were followed with a mean of 7.8 (SD 1.0) tests over an average of 5.5 (1.04) years. Interobserver agreement to detect progression was good (mean kappa = 0.57). Progression was detected in 10-19 eyes by the experts, in six by GPA and in 24 by TNT. Using the consensus expert opinion as the gold standard (four clinicians detected progression), the GPA sensitivity and specificity were 75% and 83%, respectively, while the TNT sensitivity and specificity was 100% and 77%, respectively.

CONCLUSION: TNT showed greater concordance with the experts than GPA in the detection of visual-field deterioration. GPA showed a high specificity but lower sensitivity, mainly detecting cases of high focality and pronounced mean defect slopes.

Clinical agreement among glaucoma experts in the detection of glaucomatous changes of the optic disk using simultaneous stereoscopic photographs

PURPOSE: To evaluate the clinical agreement in the detection of optic disk changes in patients with glaucoma using simultaneous stereophotographs. DESIGN: Masked-observer variability study. METHODS: Ten glaucoma specialists examined pairs of simultaneous stereophotographs of glaucomatous and control optic disks to determine whether there were changes compatible with progression of glaucomatous damage. RESULTS: Intraobserver agreement had a kappa value ranging from 0.55 to 0.78. Interobserver agreement among the glaucoma specialists had a kappa value ranging from 0.34 to 0.68. CONCLUSION: Clinical examination of stereophotographs to detect optic disk changes in glaucoma patients has limitations associated with suboptimal reproducibility. © 2003 by Elsevier Inc. All rights reserved.

Detection of changes of the optic disc in glaucomatous eyes:Clinical examination and image analysis with the Topcon Imagenet system

Purpose: This study was designed to evaluate the clinical agreement in the detection of optic disc changes and the ability of computerized image analysis to detect glaucomatous deterioration of the optic disc. Methods: Pairs of stereophotographs of 35 glaucomatous optic discs taken 5 years apart and of 5 glaucomatous discs photographed twice on the same day. Two glaucoma specialists examined the pairs of stereophotographs (35 cases and 5 controls) in a masked manner and judged whether the optic disc showed changes in the optic disc compatible with progression of glaucomatous damage. The stereophotographs of the five optic discs photographed twice on the same day (which by definition did not change) and of five cases judged to have deteriorated by both glaucoma specialists were analyzed by computerized image analysis with the Topcon ImageNet system. Intra- and inter-observer agreement in the detection of optic disc changes (evaluated using kappa statistic), and changes in the rim area to disc area ratio (evaluated using descriptive statistics and paired t-test). Results: Intra-observer agreement had a kappa value of 0.75 for observer 1 and 0.60 for the observer 2. Inter-observer agreement between the glaucoma specialists had a kappa value of 0.60. The image analyzer did not discriminate between controls and cases with clinically apparent glaucomatous change of the optic disc. Conclusion: Clinical agreement in detecting changes in the optic disc was moderate to substantial. Computerized image analysis with the Topcon ImageNet system appeared not to be useful in detecting glaucomatous changes of the optic disc.

Development of a novel phage-mediated immunoassay for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis

Aims: The objective of this study was to develop a novel screening method for detection of viable Mycobacterium avium subsp. paratuberculosis (Map) in milk and faeces, as a rapid alternative to Map culture.
Methods and results: The new method couples Map-specific peptide-mediated magnetic separation technique with an optimised phage amplification assay followed by detection of released progeny phage by ELISA in a competition assay format using polyclonal antibody produced against the D29 mycobacteriophage involved in the phage assay. Sample matrices were found not to interfere with the developed method and the dynamic range of the assay was 3 X 102 – 6 X 108 phage ml-1. When low numbers of Map were present (102 CFU ml-1) the burst size of a single host Map cell was maximal (103 phage per cell) resulting in a highly sensitive screening assay.
Conclusion: A rapid, sensitive immuno-based screening method suitable for the detection of viable Map in milk and faeces was developed.
Significance and impact of study: The novel PMS-phage-ELISA permits sensitive, qualitative detection of viable Map in milk or faeces samples within 48 h, representing a substantial decrease in time to detection compared to current culture methods for Map.