Detection Of Ricin Research Articles

Application of Microwave Irradiation and Heat to Improve Gliadin Detection and Ricin ELISA Throughput with Food Samples.

The utility of microwave irradiation to accelerate the onset of equilibrium and improve ELISA performance was examined using ELISAs for the detection of the plant toxin ricin and gliadin. The ricin ELISA normally requires several one hour incubations at 37 °C, a total assay time of approximately five hours, and employs a complex buffer containing PBS, Tween-20®, and non-fat milk. Different energy levels and pulse designs were compared to the use of abbreviated incubation times at 37 °C for the detection of ricin in food. The use of microwave irradiation had no significant advantage over the application of heat using an oven incubator and performed worse with some foods. In contrast, a gliadin ELISA that relied on 30 min incubation steps at room temperature and a salt-based buffer performed better upon irradiation but also displayed improvement upon incubating the microtiter plate at 37 °C. Whether microwave irradiation was advantageous compared to incubation in an oven was inconclusive. However, by abbreviating the incubation time of the ricin ELISA, it was possible to cut the assay time to less than 2 hours and still display LOD values < 10 ppb and recoveries of 78%–98%.

Piezoresistive microcantilever aptasensor for ricin detection and kinetic analysis

Up to now, there has been no report on target molecules detection by a piezoresistive microcantilever aptasensor. In order to evaluate the test performance and investigate the response dynamic characteristics of a piezoresistive microcantilever aptasensor, a novel method for ricin detection and kinetic analysis based on a piezoresistive microcantilever aptasensor was proposed, where ricin aptamer was immobilised on the microcantilever surface by biotin-avidin binding system. Results showed that the detection limit of ricin was 0.04μg L−1 (S/N ≥ 3). A linear relationship between the response voltage and the concentration of ricin in the range of 0.2μg L−1-40μg L−1 was obtained, with the linear regression equation of ΔUe = 0.904C + 5.852 (n = 5, R = 0.991, p &amp;lt; 0.001). The sensor showed no response for abrin, BSA, and could overcome the influence of complex environmental disruptors, indicating high specificity and good selectivity. Recovery and reproducibility in the result of simulated samples (simulated water, soil, and flour sample) determination met the analysis requirements, which was 90.5∼95.5% and 7.85%∼9.39%, respectively. On this basis, a reaction kinetic model based on ligand-receptor binding and the relationship with response voltage was established. The model could well reflect the dynamic response of the sensor. The correlation coefficient (R) was greater than or equal to 0.9456 (p &amp;lt; 0.001). Response voltage (ΔUe) and response time (t0) obtained from the fitting equation on different concentrations of ricin fitted well with the measured values.

Paper diagnostic device for quantitative electrochemical detection of ricin at picomolar levels.

We report a paper-based assay platform for detection of ricin a chain. The paper platform is assembled by simple origami paper folding. The sensor is based on quantitative, electrochemical detection of silver nanoparticle labels linked to a magnetic microbead support via a ricin immunosandwich. Importantly, ricin was detected at concentrations as low as 34 pM. Additionally, the assay is robust, even in the presence of 100-fold excess hoax materials. Finally, the device is easily remediated after use by incineration. The cost of the device, not including reagents, is just $0.30. The total assay time, including formation of the immunosandwich, is 9.5 min.

Fabrication of a SERS based aptasensor for detection of ricin B toxin.

In this report, we have developed a novel surface-enhanced Raman scattering (SERS) aptasensor for ricin B toxin recognition based on Ag nanoparticles labeled with 4,4'-bipyridyl (Bpy, Raman reporter) and ricin B aptamer. The hybrid silicon substrate was first prepared via surface-mediated RAFT polymerization of N-acryoyl-l-valine in the presence of 2-(butylthio-carbonothioylthio)-2-methylpropionic acid-modified silicon wafer as a RAFT agent and then biofunctionalized with ricin B aptamer. In this novel system, the ricin B aptamer functionalized silicon substrate was used as a scavenger for target ricin B molecules. After ricin B molecules were separated from the matrix, the sandwich assay procedure was applied using Ag nanoparticles labeled with Bpy and ricin B aptamer which act as SERS probes. Meanwhile, to enhance the SERS signal, silver deposition on the sandwich complex was also performed. The correlation between the ricin B concentration and SERS signal was found to be linear within the range of 1.0 fM to 50 pM. The limit of detection for the SERS aptasensor was determined as 0.32 fM. Furthermore, the SERS aptasensor was also evaluated for detecting ricin B in artificially contaminated orange juice, milk, blood and urine. Finally, this method has the potential to be used for the detection of other protein toxins in a complex matrix if a specific aptamer for that protein toxin can be designed.

Homogeneous time-resolved fluorescence assay for the detection of ricin using an aptamer immobilized on europium-doped KGdF4 nanoparticles and graphene oxide as a quencher

We have developed an aptamer-based assay for the food toxin ricin. It is based on a competitive quenching strategy along with time-resolved fluorescence detection. The assay involves the following steps: (a) The aptamer is immobilized on the europium-doped KGdF4 nanoparticles (NPs); (b) these NPs are added to the sample where ricin binds to the aptamer; (c) graphene oxide (GO) is added and competitively binds to the aptamer on the NPs that are not blocked by ricin; this causes the quenching of the fluorescence of the NPs; (d) fluorescence is detected at 593 nm in a microplate reader in the time-resolved mode at an excitation wavelength of 273 nm, a delay time of 100 μs, and a gating time of 1 s. Under optimal conditions, the calibration plot is linearly related to the concentration of ricin in the 50 pg·mL−1 to 50 ng·mL−1 range (R2 = 0.9975), and the limit of detection is 8 pg·mL−1. The method was compared to a standard ELISA, and correlation was excellent. The assay presented here provides a sensitive, dependable and convenient platform that is expected to have promising applications for the homogeneous assay of various other target analytes.

Toxin Content of Commercial Castor Cultivars

The castor plant Ricinus communis L. is the source of castor oil which has numerous uses and is a key chemical feedstock for an array of products from polymers to cosmetics. Although castor was once widely grown throughout the world, the presence of the toxic protein ricin has deterred the re-introduction of this useful crop. In order to develop a low-ricin or no-ricin castor crop, a robust, accurate method for screening castor seed for ricin content is essential. In this study, we validated a sandwich enzyme-linked immunosorbent assay for the detection of ricin and the homologous Ricinus communis agglutinin (RCA) in castor beans and screened a collection of castor lines including commercial varieties using this assay. Although the average content derived by extracting several seeds provided a fair representation of the ricin content for a given selection, there was enough seed-to-seed variation to justify determining the ricin/RCA content of individual seeds. Ricin levels observed ranged from 1.16 to 6.25 % by weight.

Highly sensitive and rapid visual detection of ricin using unmodified gold nanoparticle probes

A sensitive aptamer-based colorimetric biosensor for the detection of ricin using unmodified gold nanoparticles as probe was developed.

Rapid and simultaneous detection of ricin, staphylococcal enterotoxin B and saxitoxin by chemiluminescence-based microarray immunoassay.

Simultaneous detection of small and large molecules on microarray immunoassays is a challenge that limits some applications in multiplex analysis. This is the case for biosecurity, where fast, cheap and reliable simultaneous detection of proteotoxins and small toxins is needed. Two highly relevant proteotoxins, ricin (60 kDa) and bacterial toxin staphylococcal enterotoxin B (SEB, 30 kDa) and the small phycotoxin saxitoxin (STX, 0.3 kDa) are potential biological warfare agents and require an analytical tool for simultaneous detection. Proteotoxins are successfully detected by sandwich immunoassays, whereas competitive immunoassays are more suitable for small toxins (<1 kDa). Based on this need, this work provides a novel and efficient solution based on anti-idiotypic antibodies for small molecules to combine both assay principles on one microarray. The biotoxin measurements are performed on a flow-through chemiluminescence microarray platform MCR3 in 18 minutes. The chemiluminescence signal was amplified by using a poly-horseradish peroxidase complex (polyHRP), resulting in low detection limits: 2.9 ± 3.1 μg L(-1) for ricin, 0.1 ± 0.1 μg L(-1) for SEB and 2.3 ± 1.7 μg L(-1) for STX. The developed multiplex system for the three biotoxins is completely novel, relevant in the context of biosecurity and establishes the basis for research on anti-idiotypic antibodies for microarray immunoassays.

Comprehensive Laboratory Evaluation of a Highly Specific Lateral Flow Assay for the Presumptive Identification of Ricin in Suspicious White Powders and Environmental Samples

Ricin, a heterodimeric toxin that is present in the seeds of the Ricinus communis plant, is the biothreat agent most frequently encountered by law enforcement agencies in the United States. Even in untrained hands, the easily obtainable seeds can yield a highly toxic product that has been used in various types of threats, including "white-powder" letters. Although the vast majority of these threats are hoaxes, an impediment to accurate hazard assessments by first responders is the unreliability of rapid detection assays for ricin, such as lateral flow assays (LFAs). One of the complicating factors associated with LFAs is the incorporation of antibodies of poor specificity that cross-react with near-neighbors or with plant lectins that are capable of nonspecifically cross-linking the capture and detector antibodies. Because of the compelling and critical need to promote the interests of public safety and public health, the Department of Homeland Security conducted a comprehensive laboratory evaluation study of a commercial LFA for the rapid detection of ricin. This study was conducted using comprehensive inclusivity and exclusivity panels of ricin and near-neighbor plant materials, along with panels of lectins and "white-powders," to determine the specificity, sensitivity, limits of detection, dynamic range, and repeatability of the assay for the specific intended use of evaluating suspicious white powders and environmental samples in the field.

Evaluation of a novel ultra-sensitive nanoparticle probe-based assay for ricin detection

A gold nanoparticle (GNP) probe-based assay (GNPA) modified from the bio-barcode assay (BCA) was developed for ultrasensitive detection of ricin, a potential biothreat agent. In the GNPA, a chain of ricin was captured by a GNP probe coated with polyclonal antibodies and single-stranded signal DNA. A magnetic microparticle (MMP) probe coated with ricin A chain monoclonal antibody was then added to form an immuno-complex. After being magnetically separated, the immuno-complex containing the single-stranded signal DNA was characterized by PCR and real-time PCR. A detection limit of 10−2 fg/ml was determined for the ricin A chain; this is eight orders of magnitude more sensitive than that achieved with an ELISA and two orders more sensitive than that obtained with the BCA. The coefficients of variation (CV) of the intra- and inter-assay values ranged from 3.82–6.46%. The results here show that this novel assay is an ultrasensitive method for detection of ricin proteins and may be suitable for the ultrasensitive detection of other proteins.

Single domain antibody–quantum dot conjugates for ricin detection by both fluoroimmunoassay and surface plasmon resonance

The combination of stable biorecognition elements and robust quantum dots (QDs) has the potential to yield highly effective reporters for bioanalyses. Llama-derived single domain antibodies (sdAb) provide small thermostable recognition elements that can be easily manipulated using standard DNA methods. The sdAb was self-assembled on dihydrolipoic acid (DHLA) ligand-capped CdSe–ZnS core–shell QDs made in our laboratory through the polyhistidine tail of the protein, which coordinated to zinc ions on the QD surface. The sdAb–QD bioconjugates were then applied in both fluorometric and surface plasmon resonance (SPR) immunoassays for the detection of ricin, a potential biothreat agent. The sdAb–QD conjugates functioned in fluoroimmunoassays for the detection of ricin, providing equivalent limits of detection when compared to the same anti-ricin sdAb labeled with a conventional fluorophore. In addition, the DHLA-QD–sdAb conjugates were very effective reporter elements in SPR sandwich assays, providing more sensitive detection with a signal enhancement of ∼10-fold over sdAb reporters and 2–4 fold over full sized antibody reporters. Commercially prepared streptavidin-modified polymer-coated QDs also amplified the SPR signal for the detection of ricin when applied to locations where biotinylated anti-ricin sdAb was bound to target; however, we observed a 4-fold greater amplification when using the DHLA-QD–sdAb conjugates in this format.

Localized Surface Plasmon Resonance Detection of Biological Toxins Using Cell Surface Oligosaccharides on Glyco Chips

We have detected biological toxins using localized surface plasmon resonance (LSPR) and synthetic glycosyl ceramides (β-lactoside, globosyl trisaccharide (Gb3), or GM1 pentasaccharide) attached to gold (Au) nanoparticles. The particle diameters ranged from 5-100 nm. The detection sensitivity for three toxins (ricin, Shiga toxin, and cholera toxin) was found to depend not only on the attached glycoside but also on the diameter of the Au nanoparticles. For the detection of ricin, the 20-nm β-lactoside-coated Au nanoparticle exhibited the highest LSPR response, whereas 40-nm Gb3- and GM1-coated Au nanoparticles gave the best results for Shiga toxin and cholera toxin, respectively. In addition, a blocking process on the nanoparticle surface greatly improved the detection sensitivity for cholera toxin. The LSPR system enabled us to detect ricin at 30 ng/mL, Shiga toxin at 10 ng/mL, and the cholera toxin at 20 ng/mL.

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

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.

A Pilot Project to Develop in Vivo Detection of Ricin through Fluorescence Sandwich ELISA Technique

Aims & Objective: In the present study our main objective was to develop a fluorescence sandwich ELISA assay for in vivo detection of ricin from plasma of mice after intoxication. Ricin (also called RCA-II or RCA60), one of the most potent toxins and documented bioweapons, is derived from castor beans of Ricinus communis. Material & Method: The assay was performed in plasma obtained from ricin treated and control mice after 30 minutes of exposure. The assay utilizes an antiricin rat polyclonal antibody to adsorb ricin from plasma of ricin treated mice. The rabbit polyclonal antiricin antibody is then used to form a sandwich, and anti-rabbit FITC-conjugate allows development of fluorescence. The developed fluorescence was read at an excitation-emission wavelength of 485/528nm. The graph was drawn by plotting the ricin concentration against the fluorescence (rf) values. Result: We were able to detect more than 19.5ng/ml of ricin in assay buffer, 28ng/ml in diluted plasma (1:20480) at the dose of 10mg/kg and 27ng/ml of diluted Plasma (1:80) at the dose of 5mg/ kg body weight. The coefficient of variation ranged from 22% for ricin in assay buffer, 9% for 5mg/ kg body weight dose plasma, 14% for 10mg/kg body weight dose plasma and 4% for non treated control plasma. Conclusions: The detection of ricin from serum/plasma of individuals exposed to ricin will be quite difficult at lethal dose, estimated for humans to be 1 to 10μg per kg body weight following inhalation or injection. Hence, more efforts are needed for the detection of ricin at its lethal exposure dose.

Mass Spectrometric Detection of Protein-Based Toxins

This review focuses on mass spectrometric detection of protein-based toxins, which are among the most toxic substances known. Special emphasis is given to the bacterial toxins botulinum neurotoxin from Clostridium botulinum and anthrax toxins from Bacillus anthracis as well as the plant toxin ricin produced by Ricinus communis. A common feature, apart from their extreme toxicity, is that they are composed of 2 polypeptide chains, one of which is responsible for cell uptake and another that has enzymatic function with the ability to destroy basic cellular functions. These toxins pose a threat, both regarding natural spread and from a terrorism perspective. In order for public health and emergency response officials to take appropriate action in case of an outbreak, whether natural or intentional, there is a need for fast and reliable detection methods. Traditionally, large molecules like proteins have been detected using immunological techniques. Although sensitive, these methods suffer from some drawbacks, such as the risk of false-positives due to cross-reactions and detection of inactive toxin. This article describes recently developed instrumental methods based on mass spectrometry for the reliable detection of botulinum neurotoxins, anthrax toxins, and ricin. Unequivocal identification of a protein toxin can be carried out by mass spectrometry-based amino acid sequencing. Furthermore, in combination with antibody affinity preconcentration and biochemical tests with mass spectrometric detection demonstrating the toxin's enzymatic activity, very powerful analytical methods have been described. In conclusion, the advent of sensitive, easily operated mass spectrometers provides new possibilities for the detection of protein-based toxins.

Single Domain Antibodies for the Detection of Ricin Using Silicon Photonic Microring Resonator Arrays

Ricin is a lethal protein toxin derived from the castor bean plant. Given its notorious history as a biowarfare agent and homicidal weapon, ricin has been classified as a category B bioterrorism agent. Current ricin detection methods based on immunoassays lack the required sensitivity and specificity for many homeland security surveillance applications. Importantly, many conventional antibody-based methodologies are unable to distinguish ricin from RCA 120, a nontoxic protein also found in the castor bean plant. Single domain antibodies (sdAbs), which are recombinantly derived from immunized llamas, are known to have high affinities for ricin A or B chains and low cross-reactivity with RCA 120. Herein, we demonstrate the use of silicon photonic microring resonators for antibody affinity profiling and one-step ricin detection at concentrations down to 300 pM using a 15 min, label-free assay format. These sdAbs were also simultaneously compared with a commercial anti-RCA IgG antibody in a multicapture agent, single target immunoassay using arrays of microrings, which allowed direct comparison of sensitivity and specificity. A selected sdAb was also found to exhibit outstanding specificity against another biotoxin, saporin, which has mechanism of action similar to ricin. Given the rapidity, scalability, and multiplexing capability of this silicon-based technology, this work represents a step toward using microring resonator arrays for the sensitive and specific detection of biowarfare agents.

Comparison of single domain antibody immobilization strategies evaluated by surface plasmon resonance

The use of single domain antibodies (sdAbs) in place of conventional antibodies for both therapeutic and diagnostic applications continues to grow. SdAbs offer a number of advantages when compared to conventional antibodies such as their small size and low structural complexity which allows them to readily be produced as fusions in a variety formats. In this work we compared the utility of various C-terminal fusions and immobilization strategies for two sdAbs; one which recognizes ricin and the other EA1, an S-layer protein, of Bacillus anthracis. Comparisons were made between direct covalent attachment and affinity immobilization using a biotin-streptavidin interaction for the standard sdAb monomers, randomly and site-specifically biotinylated monomers, and fusion constructs of alkaline phosphatase dimers and streptavidin core tetramers. The sdAb binding and regeneration was evaluated by surface plasmon resonance in a multiplexed format. The construct that provided the highest density of active molecules by at least a factor of two was the sdAb–streptavidin core tetramer, followed by the sdAb-alkaline phosphatase and then the site-specifically biotinylated monomer. The poorest performing immobilization methods were the two most common, direct covalent attachment and the randomly biotinylated sdAb attached via NeutrAvidin. These improvements directly correlated to antigen capture in SPR assays. Similarly, the oriented immobilization method also translated to improvements in limit of detection assays using a bead-based system. The sdAb–streptavidin core provided more than a 100-fold improvement in the limit of detection of EA1, from ~200ng/mL to to 1.6ng/mL, while improvement for ricin detection was less but still a significant 5-fold decrease, going from 1.6ng/mL down to 0.32ng/mL. This demonstrated improvement in limits of detection is an advantage that should be transferable to most assay formats.

Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR)

Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

Sensitive Bioassay for Detection of Biologically Active Ricin in Food

The potential use of ricin as an agent of biological warfare highlights the need to develop fast and effective methods to detect biologically active ricin. The current "gold standard" for ricin detection is an in vivo mouse bioassay; however, this method is not practical to test on a large number of samples and raises ethical concerns with regard to the use of experimental animals. In this work, we generated adenoviral vectors that express the green fluorescent protein gene and used the relative fluorescence units intensity inhibition by transduced cells for quantitative measurement of biologically active ricin. The detection limit of the assay was 200 pg/ml, which is over 500,000 times greater than the adult human lethal oral dose. The inhibition of fluorescence intensity between ricin treatment and control was higher in 72-h posttransduction Vero cells than 24-h human embryonic kidney cells. Therefore, to detect biologically active ricin in food matrices that might influence the assay, we used 72-h posttransduction Vero cells. This simple assay could be used for large-scale screening to detect biologically active ricin in food without added substrates or use of cell fixation methods.

Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices

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.