Abstract

BackgroundIn 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. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits.Methodology/FindingsThis work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index–time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined 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 complex 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 detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material.Conclusions/SignificanceThe cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches 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.

Highlights

  • Ricin, one of the most poisonous toxins known, is a glycoprotein derived from the seeds of the castor plant Ricinus communis

  • We describe an online functional ricin cytotoxicity assay based on a real-time cell electronic sensing (RTCES) system

  • Cell growth was recorded as cell index (CI), which corresponds to the electrical impedance of a well measured by the RT-CES system [34]

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Summary

Introduction

One of the most poisonous toxins known, is a glycoprotein derived from the seeds of the castor plant Ricinus communis. This cytotoxin is highly toxic both to humans and animals [1,2]. Active ricin consists of two ,32-kDa subunits, the A-chain and the B-chain, which are linked by a disulfide bond. Both chains are needed for toxic action in vivo. Castor seeds contain a second lectin, Ricinus communis agglutinin (abbreviated in the text as agglutinin), which is highly homologous to ricin, but less toxic [8]. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits

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