No abstract

Summary. The reactivity of four different monoclonal antibodies (MAbs) with populations of Bacteroides fragilis NCTC 9343, enriched by density gradient centrifugation for a large capsule, small capsule and electron-dense layer (EDL) only visible by electronmicroscopy, was examined. The MAbs reacted strongly with polysaccharides present in both the large capsule-and EDL-enriched populations but not in the small capsule-enriched populations. The pattern of labelling was determined by immunoblotting, immunofluorescence and immuno-electronmicroscopy, and flow cytometry. The MAbs labelled cell membraneassociated epitopes in the large capsule-and EDL-enriched populations and cell-free material in the EDL population. By immunoblotting, ladders of repeating polysaccharide subunits were evident in the EDL population but not in the large capsule population. The proportion of cells labelled within each population was determined by flow cytometry. The reactivity of another MAb with the small capsule population was confirmed by flow cytometry. A qualitative indication of epitope expression was obtained by examination of the flow cytometric profiles. Differential expression of the same saccharide epitope was observed both between and within structurally distinct B. fragilis populations. The MAbs were speciesspecific and cross-reacted with several recent clinical isolates. These polysaccharides may be relevant to the virulence of B. fragilis.

The mouse bioassay is the methodology that is most widely used to detect okadaic acid (OA) in shellfish samples. This is one of the best-known toxins, and it belongs to the family of marine biotoxins referred to as the diarrhetic shellfish poisons (DSP). Due to animal welfare concerns, alternative methods of toxin detection are being sought. A rapid and specific biosensor immunoassay method was developed and validated for the detection of OA. An optical sensor instrument based on the surface plasmon resonance (SPR) phenomenon was utilised. A polyclonal antibody to OA was raised against OA-bovine thyroglobulin conjugate and OA-N-hydroxy succinimide ester was immobilised onto an amine sensor chip surface. The assay parameters selected for the analysis of the samples were: antibody dilution, 1/750; ratio of antibody to standard, 1:1; volume of sample injected, 25 microl min(-1); flow rate, 25 microl min(-1). An assay action limit of 126 ng g(-1) was established by analysing of 20 shellfish samples spiked with OA at the critical concentration of 160 ng g(-1), which is the action limit established by the European Union (EU). At this concentration of OA, the assay delivered coefficient of variations (CVs) of <10%. The chip surface developed was shown to be highly stable, allowing more than 50 analyses per channel. When the concentrations of OA determined with the biosensor method were compared with the values obtained by LC-MS in contaminated shellfish samples, the correlation between the two analytical methods was found to be highly satisfactory (r(2) = 0.991).

a b s t r a c tOkadaic acid (OA) and structurally related toxins dinophysistoxin-1 (DTX-1), and DTX-2, are lipophilic marine biotoxins. The current reference method for the analysis of these toxins is the mouse bioassay (MBA). This method is under increasing criticism both from an ethical point of view and because of its limited sensitivity and specificity. Alternative replacement methods must be rapid, robust, cost effective, specific and sensitive. Although published immuno-based detection techniques have good sensitivities, they are restricted in their use because of their inability to: (i) detect all of the OA toxins that contribute to contamination; and (ii) factor in the relative toxicities of each contaminant. Monoclonal antibodies (MAbs) were produced to OA and an automated biosensor screening assay developed and compared with ELISA techniques. The screening assay was designed to increase the probability of identifying a MAb capable of detecting all OA toxins. The result was the generation of a unique MAb which not only cross-reacted with both DTX-1 and DTX-2 but had a cross-reactivity profile in buffer that reflected exactly the intrinsic toxic potency of the OA group of toxins. Preliminary matrix studies reflected these results. This antibody is an excellent candidate for the development of a range of functional immunochemical-based detection assays for this group of toxins.

This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate