Marian Kane

Characterisation of an antibody coated microcantilever as a potential immuno-based biosensor

In this study, we investigated the activity, stability, lifetime and re-usability of monoclonal antibodies to myoglobin covalently immobilised onto microfabricated cantilever surfaces. These sensing surfaces are of interest to us in the development of novel cantilever-based immunosensors. For such sensors the antibody layer represents the sensing element while the microcantilever acts as a mechanical transducer. A procedure for producing re-usable biological coatings has been tested with different independent techniques. An Enzyme Linked Immunosorbent Assay (ELISA) was used to determine the presence of an active antibody coating, and to monitor the lifetime and stability of the immobilised antibody. Through this analysis, the activity of the immobilised antibody layer was found to be more stable with the introduction of sucrose, as a stabilising agent. Sucrose was applied to the immobilised antibody layer after each regeneration step. The immobilised antibody was found to have a stable active lifetime for up to 7 weeks. Fluorescence microscopy was used to give information on the distribution of the coating on the gold and silicon nitride sides of the cantilever. Atomic Force Microscopy was used to determine the presence of the biological coating on the cantilever and to obtain information on the surface morphology of the biological element of the sensor. The combined results provide valuable information on the development of an optimised sensing element and demonstrate a set of methods to use for future sensor-to-sensor characterisation. Preliminary experimental results showing the antibody activity against myoglobin, detected with a microcantilever based sensor prototype confirmed the motivations and potentialities of the proposed immunosensing technique.

Development and validation of a biosensor-based immunoassay for progesterone in bovine milk

We have developed a rapid automated immunoassay, using the BIACORE surface plasmon resonance (SPR) biosensor, to measure progesterone in bovine milk. The assay was designed as an inhibition assay with progesterone covalently immobilised to the carboxymethyl dextran matrix of a CM5 sensor chip. A fixed amount of monoclonal anti-progesterone antibody 39C5H7 was mixed 9:1 with the sample and the amount of free antibody was then determined using biomolecular interaction analysis (BIA) by injection of the mixture over the immobilised progesterone sensor surface. The assay was designed to cover the concentration range 0.5 to 50 ng/ml. The limit of detection (LOD) was 3.56 ng/ml. Reproducibility of the assay was very good with both intra-assay and inter-assay coefficients of variation <5%. As results become available within minutes of injection and the procedure involves fully automated instrumentation, we believe that this BIA assay for progesterone in milk could be used in-line in the milking parlour and, thus, provide an important tool for reproductive management of dairy cattle to detect heat and predict pregnancy.

Enzymatic recycling-based amperometric immunosensor for the ultrasensitive detection of okadaic acid in shellfish.

Electrochemical immunosensors based on a competitive indirect enzyme-linked immunosorbent assay (ciELISA) and an enzymatic recycling system were developed for the detection of okadaic acid (OA). OA-ovalbumin (OA-OVA) conjugate was immobilised on screen-printed electrodes (SPEs) and competition of a newly generated monoclonal antibody (MAb) for free and immobilised OA was subsequently performed. Secondary antibodies labelled with alkaline phosphatase (ALP) or horseradish peroxidase (HRP) were used for signal generation. Experimental parameters were firstly optimised by colorimetric ELISA on microtiter wells and on SPEs. The ELISA system was then tested by amperometry at +300 mV vs. Ag/AgCl (detection of p-aminophenol produced by the reaction of p-aminophenyl phosphate with ALP) or -200 mV vs. Ag/AgCl (detection of 5-methyl-phenazinium methyl sulfate, redox mediator in the HRP bioelectrocatalysis). The limits of detection (LODs) with standard solutions were 1.07 and 1.98 microgL(-1) when using ALP and HRP labels, respectively. An electrochemical signal amplification system based on diaphorase (DI) recycling was integrated into the ALP-based immunosensor, decreasing the LOD to 0.03 microgL(-1) and enlarging the working range by two orders of magnitude. Preliminary results with mussel and oyster extracts were obtained and compared with the colorimetric immunoassay, the colorimetric protein phosphatase inhibition assay (PPIA) and LC-MS/MS.

Cross-feeding by Bifidobacterium breve UCC2003 during co-cultivation with Bifidobacterium bifidum PRL2010 in a mucin-based medium.

BackgroundBifidobacteria constitute a specific group of commensal bacteria that commonly inhabit the mammalian gastrointestinal tract. Bifidobacterium breve UCC2003 was previously shown to utilize a variety of plant/diet/host-derived carbohydrates, including cellodextrin, starch and galactan, as well as the mucin and HMO-derived monosaccharide, sialic acid. In the current study, we investigated the ability of this strain to utilize parts of a host-derived source of carbohydrate, namely the mucin glycoprotein, when grown in co-culture with the mucin-degrading Bifidobacterium bifidum PRL2010.ResultsB. breve UCC2003 was shown to exhibit growth properties in a mucin-based medium, but only when grown in the presence of B. bifidum PRL2010, which is known to metabolize mucin. A combination of HPAEC-PAD and transcriptome analyses identified some of the possible monosaccharides and oligosaccharides which support this enhanced co-cultivation growth/viability phenotype.ConclusionThis study describes the potential existence of a gut commensal relationship between two bifidobacterial species. We demonstrate the in vitro ability of B. breve UCC2003 to cross-feed on sugars released by the mucin-degrading activity of B. bifidum PRL2010, thus advancing our knowledge on the metabolic adaptability which allows the former strain to colonize the (infant) gut by its extensive metabolic abilities to (co-)utilize available carbohydrate sources.

A cytotoxicity assay for the detection and differentiation of two families of shellfish toxins

There is an urgent need for an alternative to the mouse bioassay for the detection of algal toxins in shellfish on both analytical and animal welfare grounds. Several alternative methodologies have been described, but have not gained widespread acceptance to date, because each assay measures only one or a small number of related phycotoxins out of the increasing range that needs to be detected. A simple cytotoxicity assay using either the HepG2 or ECV-304 cell lines is described with two end-point measurements, which can detect and distinguish between two unrelated classes of phycotoxins. Morphological examination following 3h exposure to the sample enables the detection of the diarrhetic shellfish poisons, including okadaic acid and related toxins. Viability testing using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), following 24h exposure of the same cells to the sample, reveals a second class of toxin, which is most probably the newly-described toxin, azaspiracid. This assay should play an important role in shellfish monitoring in the future.

Structural and functional characteristics of bovine milk protein glycosylation

Most secreted and cell membrane proteins in mammals are glycosylated. Many of these glycoproteins are also prevalent in milk and play key roles in the biomodulatory properties of milk and ultimately in determining milks nutritional quality. Although a significant amount of information exists on the types and roles of free oligosaccharides in milk, very little is known about the glycans associated with milk glycoproteins, in particular, the biological properties that are linked to their presence. The main glycoproteins found in bovine milk are lactoferrin, the immunoglobulins, glycomacropeptide, a glycopeptide derived from κ-casein, and the glycoproteins of the milk fat globule membrane. Here, we review the glycoproteins present in bovine milk, the information currently available on their glycosylation and the biological significance of their oligosaccharide chains.

Generation of high-affinity chicken single-chain Fv antibody fragments for measurement of the Pseudonitzschia pungens toxin domoic acid.

ABSTRACT Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single VH and VL genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.

Construction of a Natural Mucin Microarray and Interrogation for Biologically Relevant Glyco-Epitopes

Mucins are the principal components of mucus, and mucin glycosylation has important roles in defense, microbial adhesion, immunomodulation, inflammation, and cancer. Mucin expression and glycosylation are dynamic, responding to changes in local environment and disease. Potentially hundreds of heterogeneous glycans can substitute one mucin molecule, and it is difficult to identify biologically accessible glyco-epitopes. Thirty-seven mucins, from the reproductive and gastrointestinal (GI) tracts of six species (bovine, ovine, equine, porcine, chicken, and deer) and from two human-derived cell lines, were purified. Following optimization of mucin printing and construction of a novel mucin microarray, the glycoprofiles of the whole mucins on the microarray were compared using a panel of lectins and one antibody. Accessible glyco-motifs of GI mucins varied according to species and localization of mucin origin, with terminal fucose, the sialyl T-antigen, and N-linked oligosaccharides identified as potentially important. The occurrence of T- and sialyl T-antigen varied in bovine and ovine reproductive tract mucins, and terminal N-acetylgalactosamine (GalNAc) and sulfated carbohydrates were detected. This study introduces natural mucin microarrays as an effective tool for profiling mucin glyco-epitopes and highlights their potential for discovery of biologically important motifs in bacterial-host interactions and fertility.

Dietary Caffeine, Performance and Mood: Enhancing and Restorative Effects after Controlling for Withdrawal Reversal

This study aimed to determine whether sustained (i.e. dietary) use of caffeine has net effects on performance and mood compared with sustained abstinence, and whether dietary caffeine restores performance and mood adversely affected by sleep restriction. Participants (n = 96) alternated weekly between ingesting placebo and caffeine (1.75 mg/kg) three times daily for 4 consecutive weeks, while either rested or sleep restricted. Performance involved either a single task requiring sustained vigilance or a varied battery of brief psychomotor and cognitive tasks, and mood was assessed using the Profile of Mood States. Caffeine had no significant net enhancing effects for either performance or mood when participants were rested, and produced no net restorative effects when performance and mood were degraded by sleep restriction.

Rapid Polymerase Chain Reaction/DNA Probe Membrane-Based Assay for the Detection of Listeria and Listeria monocytogenes in Food

We describe the development of polymerase chain reaction (PCR)/DNA probe membrane-based colorimetric assays for the detection and identification of Listeria and L. monocytogenes. PCR primers designed from the 16S to 23S rRNA intergenic spacer region amplified products that were reverse hybridized to membrane-bound oligonucleotide probes specific for Listeria and L. monocytogenes with a detection limit of 1 to 10 CFU/25 ml in inoculated raw and pasteurized milk samples. These qualitative assays have the potential to be integrated into testing laboratories for monitoring the microbiological quality of foods.