Ricin detection

In the absence of an unambiguous history of ricin exposure, the preferred diagnostic method is specific immunoassay of ricin in serum, respiratory secretions, or other clinical samples associated with poisoning. Most of the methods described for ricin detection are experimental or are under development. The CDC and the Federal Laboratory Response Network have the capability to detect ricin in environmental specimens using validated polymerase chain reaction (PCR) tests and time-resolved immunofluorescence assays, with cell-based bioassays to confirm ricin activity. The U.S. Department of Defense has produced experimental field immunoassays, but commercial distribution of field test kits currently is limited. 

The detection of ricin to confirm exposure is difficult, not least because the toxin is metabolized and eliminated rapidly. The most commonly used method is the enzyme-linked immunosorbant assay (ELISA) which can be applied to environmental and biological samples and allows ricin detection for up to 48 h after exposure with a detection limit of approximately 200 pg/ml (Griffiths et al, 1986 Leith et al, 1988). More recently, an ELISA that uses monoclonal antibodies with distinct specificities for ricin A and B chains has been developed (Shyu et al, 2002a) which offers high specificity but is too slow to be useful for rapid diagnosis. The same group has now developed a sensitive and rapid... 

Figure 10. Ricin detection with poly(60% 10/20% POPC/10% 11/10% DPPT) attached liposomes with 0.5% 1 incorporated and anti-ricin antibodies conjugated via BS3 O.lngricinj/) ), Ing ricin (Qj lOngricin (A) lOOngricin (O) lOOng bovine IgG (9). 100 pL/well. Control subtracted.

To demonstrate ricin detection, a series of concentrations of ricin solutions are prepared from a stock solution of 35 pM. Then, 2 pL of each ricin sample is added into 6 pL of the reaction solution in the reaction chamber in Figure lb. The volume of the feeding solution remained at 80 pL. For the positive controls in the same device, 2 pL of water is added. The negative controls contain no luciferase vector, providing with the background signal. 

To detect ricin, we studied its inhibitory effects on luciferase expression by adding a series of concentrations of ricin into the IVT reactions in the array device. To achieve a lower detection limit, 4-hr protein expression was used, though ricin detection can be achieved in as short as 5 minutes. Ricin sample size was 2 pL. As shown in Figure 5a, the expression yield of luciferase, indicated by luminescence, decreases with the concentration of ricin (solid circles). However, the expression yield remained the same when the ricin is heat denatured and its toxicity is deactivated (open circles). The error bar of each data point indicates the standard deviation that is obtained from three repeat experiments. The calibration curve is obtained by plotting the detection... 

Figure 5 (a) The inhibitory effects of ricin on the production yield of luciferase. The expression yield of luciferase is plotted as a function of the concentration of ricin (solid circles) and heat-denatured ricin (open circles), (b) Calibration curve for ricin detection. (Reproducedfrom reference 14. Copyright 2006American Chemical Society)... 

A ricin detection method has been developed that couples LC-MS/MS with an enzyme-linked immunosorbent assay (ELISA).The analyte target for MS detection is adenine, which is released from ricin during the assay. Adenine detection limits were achieved at 0.1 ng/mL or 1.56 pM in a 500-pL sample volume with this method. This LC-MS method may be chosen over other techniques because milk and drinking water are the intended real-world application for this protocol [77]. Another MS/MS technique detects tryptic peptide fragments from ricin produced by an immunoassay technique. This technique utilizes the inherent speed of MS analysis to confirm peptide identity. This technique is applied to the detection of ricin in food and body fluids such as blood serum and saliva [78]. A similar LC-MS/MS method uses an organic solvent-assisted tryptic digest to prepare samples of crude ricin extracts. This sample preparation... 

Due especially to concerns about the use of ricin as a bioweapon, numerous approaches for ricin detection and qnantitation have been developed. These methods fall into fonr basic categories activity/toxicity, chemical instrumentation, immunodetection, and indirect detection. 

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