Ricin detection with

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.

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... 

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... 

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... 

CDC Case Definition A clinically compatible case with (1) detection of urinary ricinine, an alkaloid in the castor bean plant or (2) detection of ricin in environmental samples. The case can be confirmed if laboratory testing is not performed because either a predominant amount of clinical and nonspecific laboratory evidence is present or an absolute certainty of the etiology of the agent is known. 

An evanescent wave fiber optic immunosensor has been used for the detection of ricin in river water.(131) A tapered fiber optic waveguide with covalently bound anti-ricin IgG is used in a sandwich format, with tetramethylrhodamine-labeled antibody as tracer. In a two-step format, ricin in the sample is bound to the fiber first, and then the fiber is exposed to the tracer antibody. Sensitivity is 1 ng/ml for the two-step assay. The one-step assay, in which the fiber optic probe contacts the sample and labeled antibody simultaneously is less sensitive, but more convenient. 

Antibody-toxin conjugates made with ricin A chain, abrin A chain, gelomn, and momordin can be stored for at least 4 yr at -70°C without detectable loss of activity. The bond between the antibody and the RIP breaks down very slowly at 4°C in PBSE but, provided that care is taken to ensure the sterility of the solution, conjugates can be stored under these conditions for up to one year with little deterioration in quality. 

Detection of staphylococcal enterotoxin B (SEB), a causative agent of food poisoning, was achieved by QDs conjugated with polyclonal sheep anti-SEB antibody.57 Moreover, this approach also harbors the possibility of a multiplexed immunoassay (see Fig. 12.3), which was first reported by Goldman et al.58 in 2004 four toxins of interest in food- or water-borne illnesses (cholera toxin, ricin,... 

Ricin as a potential biological threat agent has received much popular press. In 2003, suspects were arrested in London for making ricin from castor seed in their apartment (Risen and van Natta 2003). The popular press has speculated that if ricin were made, it could be used to contaminate food in military mess halls. All of these instances indicate that biological and chemical materials may be a potential terrorist weapon to compromise the safety of the military and civilian food supply or other vulnerable areas. Given the extreme toxicity of ricin, the relative ease with which it can be obtained, and the fact that references to its use have been discovered in terrorist haunts, the ability to accurately and precisely detect ricin is a critical need. 

Injection (i.v.) of laboratory rats with I-ricin resulted in an accumulation of about 70% of the toxin in the spleen, liver, and muscle at 30 m, with detectable levels decreasing to about 11 % of injected toxin by 24 h after exposure (Ramsden et al., 1989). Injection (i.m.) of rats with ricin showed concentration of the toxin at the injection site and the ipsilateral para-aortic lymph nodes beginning as early as 4—8 h after exposure (Griffiths et al., 1986). 

Radioimmunoassays can detect ricin in blood at concentrations as low as 50-100 pg/mL (Godal et al., 1981). Colorimetric ELISA methods can detect ricin spiked into ex vivo human serum or urine samples accurately at levels as low as 1 ng/mL (100 pg/well) with acceptable inter- and intraassay variation (Poli et al., 1994). Chemical luminescence-based ELISA technology improves the detection limit to 0.1-1 ng/mL ricin, but the coefficients of variation may be as high as 50% (Poli et al., 1994). Combination of a sensitive immunological assay with PCR amplification may achieve a limit of detection for ricin at levels as low as 10 fg/mL (Lubelli et al., 2006). 

The use of antibody- or aptamer-based approaches to protect against ricin holds potential as a possible pretreatment for armed forces, or as an adjunct to PPE for civilian first-responders and other special populations required to enter contaminated areas. However, extracellular antitoxin would probably be of limited use in a bioterrorist or civilian mass casualty scenario, because the therapeutic window for administration is likely to be short (a few hours), the delay to onset of symptoms is relatively long, and there presently is no method of immediately detecting ricin exposure. It also remains to be determined whether specific combinations of MAbs are required for optimal in vivo protection against the toxin. Moreover, in some cases, MAbs that bind toxin with high avidity and block enzymatic activity of RTA in vitro actually enhance the toxicity of ricin in vivo (Maddaloni et al., 2004). 

The CDC identified Key Eocus Areas, which go from Preparedness and Prevention to Response and Communication to counteract possible attacks. Recently electronic chips with hve nerve cells have been identified as capable to detect many bacterial toxins. Eiber-optic tubes can detect specific pathogens such as anthrax, botulinum and ricin. 

As for ricin, abrin detection is possible by radioimmunoasay with little cross-reactivity between the two toxins (Godal et al, 1981). 

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