Detection of Botulinum Neurotoxin Serotype B at Sub Mouse LD50 Levels by a Sandwich Immunoassay and Its Application to Toxin Detection in Milk

Botulinum neurotoxin (BoNT), the causative agent of botulism, a serious neuroparylatic disease, is produced by the anaerobic bacterium Clostridium botulinum and consists of a family of seven serotypes (A-H). We previously reported production of high-affinity monoclonal antibodies to BoNT serotype A.Recombinant peptide fragments of the light chain, the transmembrane and receptor-binding domains of the heavy chain of botulinum neurotoxin type B (BoNT/B) were expressed in Escherichia coli as GST-fusion proteins and purified. These proteins were used to immunize BALB/cJ mice for the generation of monoclonal antibodies (mAbs). Antibody-producing hybridomas were detected using either a direct binding ELISA binding to plate-immobilized BoNT/B, or with a capture-capture ELISA whereby the capacity of the antibody to capture BoNT/B from solution was tested. A total of five mAbs were selected, two of which bound the toxin light chain and three bound the receptor-binding domain of BoNT/B heavy chain. MAb MCS6-27 was identified via capture-capture ELISA and was the only mAb able to bind BoNT/B in solution under physiological conditions. MAbs F24-1, F26-16, F27-33 and F29-40 were identified via direct binding ELISA, and were able to capture BoNT/B in solution only in the presence of 0.5-0.9 mM sodium dodecyl sulphate (SDS). MAb MCS6-27 and an anti-BoNT/B polyclonal antibody were incorporated into a sandwich ELISA that did not require SDS.We report here the generation of monoclonal antibodies to serotype B and the subsequent development of a sensitive sandwich immunoassay. This immunoassay has a detection limit of 100 fg BoNT/B, fifty times more sensitive than the mouse bioassay detection limit of 5 pg BoNT/B. Additionally, this assay detected as little as 39 pg/mL of toxin in skim, 2% and whole milk

Immune-Mobilizing Monoclonal T Cell Receptors Mediate Specific and Rapid Elimination of Hepatitis B-Infected Cells

Background and Aims: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV‐specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity‐enhanced T Cell receptor with an anti‐CD3 T Cell‐activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus‐derived peptides presented by human leukocyte antigen (HLA). Approach and Results: ImmTAV molecules specific for HLA‐A*02:01‐restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV‐Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging‐based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV‐Env can redirect T cells from healthy and HBV‐infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV‐Env redirection of T cells induced cytolysis of antigen‐positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA. Conclusions: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non‐HBV‐specific T cells, bypassing exhausted HBV‐specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials

The peatland map of Europe

Based on the ‘European Mires Book’ of the International Mire Conservation Group (IMCG), this article provides a composite map of national datasets as the first comprehensive peatland map for the whole of Europe. We also present estimates of the extent of peatlands and mires in each European country individually and for the entire continent. A minimum peat thickness criterion has not been strictly applied, to allow for (often historically determined) country-specific definitions. Our ‘peatland’ concept includes all ‘mires’, which are peatlands where peat is being formed. The map was constructed by merging national datasets in GIS while maintaining the mapping scales of the original input data. This ‘bottom-up’ approach indicates that the overall area of peatland in Europe is 593,727 km². Mires were found to cover more than 320,000 km² (around 54 % of the total peatland area). If shallow-peat lands (< 30 cm peat) in European Russia are also taken into account, the total peatland area in Europe is more than 1,000,000 km2, which is almost 10 % of the total surface area. Composite inventories of national peatland information, as presented here for Europe, may serve to identify gaps and priority areas for field survey, and help to cross-check and calibrate remote sensing based mapping approaches

Sustainability Reporting Playbook

Sustainability reporting can allow internal stakeholders to be more effective in their roles. It can increase employee engagement and inform management decision-making, among other benefits. Yet companies often fail to connect sustainability reporting to the core operations of the business, and so fail to realize the potential benefits. This playbook identifies opportunities for enhancing the effectiveness and impact of sustainability reporting