Background In the context of a potential bioterrorist attack sensitive and
Background In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. sensitive detection was decided after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from organic matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in discovering active ricin in environmental sample materials, as shown for organic fertilizer made up of material. Findings/Significance The cell-electrode Belnacasan impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is usually monitored online, the assay can be standardized more efficiently than previous methods based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices. Introduction Ricin, one of the most poisonous toxins known, is usually a glycoprotein produced from the seeds of the castor herb agglutinin (abbreviated in the text as agglutinin), which is usually highly homologous to ricin, but less harmful . Agglutinin is made up of a dimer of two associated ricin-like molecules, each of which contains A- and B-chains. The homology at the amino acid level between agglutinin and ricin is usually around 93% for the A-chains and around 84% for the B-chains . The mortality in ricin poisoning is usually dependent on the route of administration. In mice, the Belnacasan median lethal doses (LD50) for injection, inhalation or ingestion are reported as 2C10 g ricin/kg body excess weight, 3C5 g/kg or 20 000C30 000 g/kg, respectively. In humans, the oral LD50 of 1 000C20 000 g ricin/kg body excess weight is usually estimated from accidental ingestion of castor beans , . For agglutinin, it is usually approximated from animal studies that the toxicity is usually about two orders of magnitude less than that for ricin , . Comparable to other toxins, ricin functions in the absence of the generating herb and its genetic information. Therefore, it is usually necessary to detect the protein itself, not only the plant’s nucleic acid. Currently, the analysis of ricin is usually mainly based on immunological methods , , mass spectrometry analysis , , or functional and assays (for an overview of the second option observe Table H1). In the case of an intentional release of ricin into the environment, the Rabbit polyclonal to ZFP28 discrimination of functionally active and denatured ricin is usually important, especially with regard to emergency operating activities, forensic analysis and therapy. This information can only be obtained from functional assays, which can be principally differentiated into Belnacasan assays discovering the A-chain activity, the B-chain activity, or both. assays, like adenine-release assays or cell-free translation assays based on rabbit reticulocyte lysate, analyze the enzymatic activity of the A-subunit , , , , , , . Glycan binding of the ricin B-subunit is usually detected by enzyme-linked lectin assays . However, the detection of the activity of the isolated subchains provides no information on the activity of the intact 64 kDa ricin molecule. Therefore, the Belnacasan detection of active ricin requires or assays for both subchains. assays have the advantage that whole-organism responses can be monitored but, on the basis of different species and strain susceptibilities that have been reported for ricin, animal bioassays seem to be hard to standardize and raise ethical issues , , . An alternate are immunocapture assays combined with adenine release measurement , , or cell-based functional assays. Current cell assays use different endpoint read-outs of cell death via biochemical, fluorescent or radioactive detection , , , . The detection limits for ricin analysis in cell-based bioassays have been described as being between 0.01 ng/mL and 0.8 ng/mL from complex matrices. Cytotoxicity is detected at the end of the assay after different cell cultivation times (ranging from 4 to 28 h),.