A Glyco-chip for the Detection of Ricin by an Automated Chemiluminescence Read-out System

Abstract

The plant toxin ricin is a lectin that binds to D-galactose or lactose moieties by multivalent interactions. In the present work, this avidity was used to develop a novel sandwich glyco-immunoassay using a carbohydrate microarray. For realization, 6-azidohexyl-lactose was immobilized on an alkyne silane surface by Cu(I) catalyzed click chemistry. This procedure is fast, and prevents any nonspecific binding on the microarray surface. Ricin binds via its B-chain to the lactose moieties, and is detected by the biotinylated anti-ricin A-chain. By adding a horseradish peroxidase-labeled streptavidin, a chemiluminescence signal can be generated. This method is described as a sandwich-type glyco-immunoassay. The signal on the glyco-chip can be regenerated for at least 10 measurements. The limit of detection was estimated to be 80 ng mL -1 . The assay was carried out on the automated microarray readout platform MCR 3. In this way, it took 20 min for one measurement, including regeneration of the chip surface.

Ricin detection: Tracking active toxin

“…The use of microarrays, which include panels of antibodies for simultaneous detection of a variety of antigenic targets of interest, allowed multiplexed detection of ricin in parallel with other harmful toxic agents, such as cholera toxin, staphylococcal enterotoxins A and B, Bacillus globigii, botulinum toxin A, Yersinia pestis, and heat labile toxin of Escherichia coli, and provided dramatic improvements in assay utility and flexibility (Delehanty and Ligler, 2002;Garber et al, 2010;Simonova et al, 2012;Wadkins et al, 1998;Weingart et al, 2012). In other works, the toxin capture antibodies used as receptors were substituted by DNA/RNA aptamers (Haes et al, 2006;Kirby et al, 2004;Lamont et al, 2011), single domain antibodies (Anderson et al, 2013;Shia and Bailey, 2013;Stine et al, 2005), and sugar-conjugated materials (Huebner et al, 2013;Liu et al, 2011). By bypassing traditional antibodies, improvements can be made in regard to reagent stability and storage life.…”

“…Notably, by complementing ricin-specific sample enrichment steps with RTA enzyme activity assays, ricin could be selectively identified while confirming A-chain activity in parallel (Becher et al, 2007;Bevilacqua et al, 2010;He et al, 2010;May et al, 1989), allowing distinction from nontoxic monomeric type 1 RIP toxins. In addition to anti-ricin antibodies, ricin-specific RNA aptamers (Haes et al, 2006;Kirby et al, 2004;Lamont et al, 2011) and carbohydrate compounds (Huebner et al, 2013;Liu et al, 2011) have also been evaluated for recovering ricin from suspect contaminated samples (Blome and Schengrund, 2008;Stine et al, 2005;Uzawa et al, 2008). Carbohydrate ligands selectively bind to the ricin B-chain.…”

Glycan Arrays: From Basic Biochemical Research to Bioanalytical and Biomedical Applications

“…Glycan arrays can be used to detect toxic proteins such as ricin, a lectin from the castor oil plant Ricinus communis, for which a rapid detection assay was demonstrated on an automated microarray platform with chemiluminescent detection (77). Although the detection limit of 80 ng/mL was sufficient to identify lethal doses of ricin, other detection platforms are several orders of magnitude more sensitive (78).…”

Biotoxin sensing in food and environment via microchip

“…Beside toxins mentioned above (SEB, CT, BoNT), microchips can be used for sensing other bacterial toxins, such as staphylococcal enterotoxin A antibody , coeliac disease toxic gliadin , E. coli O157:H7 , ricin , bacillus anthracis , clostridium difficile toxin A .…”