† Electronic supplementary information (ESI) available: comparison of multiplex toxin detection techniques, exemplary immunisation schema for rabbit and mouse, and titer development of rabbit.
AbstractProteotoxins such as ricin, abrin, botulinum neurotoxins type A and B (BoNT/A, BoNT/B) and staphylococcal enterotoxin B (SEB) are regarded as potential biological warfare agents which could be used for bioterrorism attacks on the food chain. In this study we used a novel immunisation strategy to generate high-affinity monoclonal and polyclonal antibodies against native ricin, BoNT/A, and BoNT/B. The antibodies were used along with antibodies against SEB and abrin to establish a highly sensitive magnetic and fluorescent multiplex bead array with excellent sensitivities between 2 ng/L and 546 ng/L from a minimal sample volume of 50 mL. The assay was validated using 20 different related analytes and the assay precision was determined. Advancing the existing bead array technology, the novel magnetic and fluorescent microbeads proved amenable to enrichment procedures, by further increasing sensitivity to 0.3-85 ng/L, starting from a sample volume of 500 mL. Furthermore, the method was successfully applied for the simultaneous identification of the target toxins spiked into complex food matrices like milk, baby food and yoghurt. On the basis of our results, the assay appears to be a good tool for large-scale screening of samples from the food supply chain.
In L2 mice, a high expression level of the transgenic λ2315 L chain results in nearly complete exclusion of endogenous L chains and a predominance of B-1a cells. In this study, we show that splenic and peritoneal B-1a cells differ considerably in their Ab repertoire and gene expression profile. Splenic B-1a cells exhibit a more diversified repertoire under L chain limitation. Despite oligoclonal overlaps between both B-1a compartments, some B cell receptor specificities are clearly restricted to the peritoneum. The capacity of peritoneal B-1a cells to enter the splenic B-1a compartment was found to be very limited. Gene expression profiling revealed genes up-regulated in splenic B-1a cells that are involved in mediating specialized first-line-of-defense effector functions and interaction with T cells. Thus, splenic and peritoneal B-1a cells differ not only in their developmental program but also in functional properties.
B‐1a cells are found mainly in the peritoneal cavity of mice but are also present in the spleen. Gene expression profiling defined many genes differentially expressed in B‐1a cells from these two sites. To see whether this gene expression pattern was imprinted by the particular microenvironment, peritoneal or spleen cells from recombinant L2 mice mainly consisting of B‐1a cells were adoptively transferred into Rag1–/– mice. Re‐isolated peritoneal and splenic B‐1a cells were analyzed for expression of three indicator genes – vcam‐1, adamdec1 and spi‐c. The expression of these genes was up‐regulated in splenic and down‐regulated in peritoneal cells. This particular pattern was observed for peritoneal or splenic donor cells transferred either intraperitoneally or intravenously. Similar results were obtained when levels of surface IgM or frequencies of Mac‐1+ B‐1 cells were compared after transfer. This suggests that the environment induces the particular genetic program of B‐1a cells and argues against an independent ontogeny.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.