A surface plasmon resonance biosensor assay for the simultaneous determination of thiamphenicol, florefenicol, florefenicol amine and chloramphenicol residues in shrimps

A rapid and sensitive screening qualitative method using a surface plasmon resonance (SPR) biosensor was developed which can detect of all fenicol antibiotic residues in shrimps from a single sample extract. This method requires ethyl acetate extraction followed by a single wash with isooctane/chlorofonrm. Each sample extract is injected over the surfaces of two biosensor chip flow cells, one surface having the capability to detect florefenicol amine (FF amine), florefenicol (FF), and thiamphenicol (TAP) and the second surface for chloramphenicol (CAP) detection. The estimated detection capabilities (CC beta) were 0. 1, 0.2, 250, and 0.5 ppb for CAP, FF, FF amine, and TAP, respectively. This quick, simple test allowed the detection of CAP residues in shrimps at the minimum required performance limit (MRPL) of 0.1 mu g kg(-1) for this compound and of FF, FF amine, and TAP below their maximum residue limits (MRLs). (c) 2006 Elsevier B.V. All rights reserved.

Characterisation of antibodies to chloramphenicol, produced in different species by enzyme-linked immunosorbent assay and biosensor technologies

Six polyclonal antisera to chloramphenicol (CAP) were successfully raised in camels, donkeys and goats. As a comparison of sensitivity, IC50 values ranged from 0.3 ng mL(-1) to 5.5 ng mL(-1) by enzyme-linked immunosorbent assay (ELISA) and from 0.7 ng mL(-1) to 1.7 ng mL(-1) by biosensor assay. The introduction of bovine milk extract improved the sensitivity of four of the antisera by ELISA and two by biosensor assay; a reduction in sensitivity of the remaining antisera ranged by a factor of 1.1-2.6. Porcine kidney extract reduced the sensitivity of all the antisera by a factor ranging from 1.1 to 7 by ELISA and a factor of 1.5 to 4 by biosensor. A low cross-reactivity with thiamphenicol (TAP) and florfenicol (FF) was displayed by antiserurn G2 (1.2% and 18%, respectively) when a homologous ELISA assay format was employed. No cross-reactivity was displayed by any of the antisera when a homologous biosensor assay format was employed. Switching to a heterologous ELISA format prompted three of the antisera to display more significant cross-reactivity with TAP and FF (53% and 82%, respectively, using Dl). The heterologous biosensor assay also increased the cross-reactivity of D1 for TAP and FF (56% and 129%, respectively) and of one other antiserum (Gl) to a lesser degree. However, unlike the ELISA, the heterologous biosensor assay produced a substantial reduction in sensitivity (by a factor of 6 for D1). (C) 2007 Elsevier B.V. All rights reserved.

A nonradioactive method for the assay of polyphosphate kinase activity and its application in the study of polyphosphate metabolism in Burkholderia cepacia

Studies of polyphosphate (polyP) metabolism in microorganisms have been hampered by the lack of a convenient method for the assay in cell extracts of the activity of polyphosphate kinase (PPK), the enzyme principally responsible for microbial polyP biosynthesis. We report the development of such an assay, based on the well-established metachromatic reaction, with toluidine blue, of the polyP formed during the PPK-catalyzed reaction. The method was successfully used in the characterization of PPK activity in crude extracts of an environmental Burkholderia cepacia isolate. The development of a protocol for the physical recovery of polyP from solution is also reported. (C) 2002 Elsevier Science (USA). All rights reserved.

Specific and sensitive detection of Nosema bombi (Microsporidia : Nosematidae) in bumble bees (Bombus spp.; Hymenoptera : Apidae) by PCR of partial rRNA gene sequences

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees. Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N. bombi (323 bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 107 down to 10 spores diluted in 100 mu l of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 10(6) spores down to 1 spore per PCR). Though the N. bombi-specific primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi-non-specific primer pair ITS-f2/r2, which amplifies a short fragment of similar to 120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to b more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy. (c) 2005 Elsevier Inc. All rights reserved.

Systematic random sampling of the comet assay

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory ‘tail’ DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the ‘randomness’ of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

Modelling the comet assay

The single-cell gel electrophoresis technique or comet assay is widely regarded as a quick and reliable method of analysing DNA damage in individual cells. It has a proven track record from the fields of biomonitoring to nutritional studies. The assay operates by subjecting cells that are fixed in agarose to high salt and detergent lysis, thus removing all the cellular content except the DNA. By relaxing the DNA in an alkaline buffer, strands containing breaks are released from supercoiling. Upon electrophoresis, these strands are pulled out into the agarose, forming a tail which, when stained with a fluorescent dye, can be analysed by fluorescence microscopy. The intensity of this tail reflects the amount of DNA damage sustained. Despite being such an established and widely used assay, there are still many aspects of the comet assay which are not fully understood. The present review looks at how the comet assay is being used, and highlights some of its limitations. The protocol itself varies among laboratories, so results from similar studies may vary. Given such discrepancies, it would be attractive to break the assay into components to generate a mathematical model to investigate specific parameters.

Inhibition of Advanced Glycation and Absence of Galectin-3 Prevent Blood-Retinal Barrier Dysfunction during Short-Term Diabetes

Breakdown of the inner blood-retinal barrier (iBRB) occurs early in diabetes and is central to the development of sight-threatening diabetic macular edema (DME) as retinopathy progresses. In the current study, we examined how advanced glycation end products (AGEs) forming early in diabetes could modulate vasopermeability factor expression in the diabetic retina and alter inter-endothelial cell tight junction (TJ) integrity leading to iBRB dysfunction. We also investigated the potential for an AGE inhibitor to prevent this acute pathology and examined a role of the AGE-binding protein galectin-3 (Gal-3) in AGE-mediated cell retinal pathophysiology. Diabetes was induced in C57/BL6 wild-type (WT) mice and in Gal-3(-/-) transgenic mice. Blood glucose was monitored and AGE levels were quantified by ELISA and immunohistochemistry. The diabetic groups were subdivided, and one group was treated with the AGE-inhibitor pyridoxamine (PM) while separate groups of WT and Gal-3(-/-) mice were maintained as nondiabetic controls. iBRB integrity was assessed by Evans blue assay alongside visualisation of TJ protein complexes via occludin-1 immunolocalization in retinal flat mounts. Retinal expression levels of the vasopermeability factor VEGF were quantified using real-time RT-PCR and ELISA. WT diabetic mice showed significant AGE -immunoreactivity in the retinal microvasculature and also showed significant iBRB breakdown (P < .005). These diabetics had higher VEGF mRNA and protein expression in comparison to controls (P < .01). PM-treated diabetics had normal iBRB function and significantly reduced diabetes-mediated VEGF expression. Diabetic retinal vessels showed disrupted TJ integrity when compared to controls, while PM-treated diabetics demonstrated near-normal configuration. Gal-3(-/-) mice showed significantly less diabetes-mediated iBRB dysfunction, junctional disruption, and VEGF expression changes than their WT counterparts. The data suggests an AGE-mediated disruption of iBRB via upregulation of VEGF in the diabetic retina, possibly modulating disruption of TJ integrity, even after acute diabetes. Prevention of AGE formation or genetic deletion of Gal-3 can effectively prevent these acute diabetic retinopathy changes.