Active Ricin Research Articles

Rapid detection of ricin at trace levels in complex matrices by asialofetuin-coated beads and bottom-up proteomics using high-resolution mass spectrometry

Ricin is a toxic protein regarded as a potential chemical weapon for bioterrorism or criminal use. In the event of a ricin incident, rapid analytical methods are essential for ricin confirmation in a diversity of matrices, from environmental to human or food samples. Mass spectrometry–based methods provide specific toxin identification but require prior enrichment by antibodies to reach trace-level detection in matrices. Here, we describe a novel assay using the glycoprotein asialofetuin as an alternative to antibodies for ricin enrichment, combined with the specific detection of signature peptides by high-resolution mass spectrometry. Additionally, optimizations made to the assay reduced the sample preparation time from 5 h to 80 min only. Method evaluation confirmed the detection of ricin at trace levels over a wide range of pH and in protein-rich samples, illustrating challenging matrices. This new method constitutes a relevant antibody-free solution for the fast and specific mass spectrometry detection of ricin in the situation of a suspected toxin incident, complementary to active ricin determination by adenine release assays.Graphical

Sensitive Detection and Differentiation of Biologically Active Ricin and Abrin in Complex Matrices via Specific Neutralizing Antibody-Based Cytotoxicity Assay.

Ricin and abrin are highly potent plant-derived toxins, categorized as type II ribosome-inactivating proteins. High toxicity, accessibility, and the lack of effective countermeasures make them potential agents in bioterrorism and biowarfare, posing significant threats to public safety. Despite the existence of many effective analytical strategies for detecting these two lethal toxins, current methods are often hindered by limitations such as insufficient sensitivity, complex sample preparation, and most importantly, the inability to distinguish between biologically active and inactive toxin. In this study, a cytotoxicity assay was developed to detect active ricin and abrin based on their potent cell-killing capability. Among nine human cell lines derived from various organs, HeLa cells exhibited exceptional sensitivity, with limits of detection reaching 0.3 ng/mL and 0.03 ng/mL for ricin and abrin, respectively. Subsequently, toxin-specific neutralizing monoclonal antibodies MIL50 and 10D8 were used to facilitate the precise identification and differentiation of ricin and abrin. The method provides straightforward and sensitive detection in complex matrices including milk, plasma, coffee, orange juice, and tea via a simple serial-dilution procedure without any complex purification and enrichment steps. Furthermore, this assay was successfully applied in the unambiguous identification of active ricin and abrin in samples from OPCW biotoxin exercises.

SCX-tip-aided LC-MS detection of active ricin via oligonucleotide substrates for depurination kinetics.

The accurate and sensitive detection of active biotoxin proteins and the determination of their kinetics are vital for the upsurge of chemical attacks but still limited. Herein, we report a liquid chromatography-tunable ultraviolet spectroscopic-quadrupole mass spectrometric detection (LC-TUV-QDa) method of active ricin. This method has the advantage of the accurate quantification of active ricin in decreased oligonucleotide (oligo) substrates as well as the produced adenine, in which the QDa detection offers the confirmative evidence of oligo and adenine products. We invented a strong cation exchange (SCX)-tip sample pretreatment way to facilitate the requirement of clean product injection without any fouling proteins. After full-method validation, a wide linear range of 1-5000 ng mL-1 was obtained with a high sensitivity of 1 ng mL-1 active ricin based on the most preferable deoxynucleobase-hybrid RNA (Rd) substrate, Rd12, and without any enrichment. We also fully depicted the kinetic parameters of ricin and its six Rd or RNA substrates and evaluated 11 nucleobase-modified oligos as substrates based on Rd12. Further, we fulfilled an improved molecular docking analysis and revealed that the binding of Rd12 to ricin was more likely to occur at pH 7.4 (typical in vitro and in vivo conditions) than at pH 4.0 (typical ex vitro conditions). With the aid of SCX-tip as a microenzymatic reactor, we can exert the catalytic activity of ricin as N-glycosidase in pH 7.4 toward its Rd12 substrate, with a comparable catalytic efficiency at pH 4.0. This is the first successful implementation of an ex vitro experiment toward oligo substrates at neutral pH, standing on the shoulder of plenty of previously reported efforts all performed under acidic conditions. This method will provide a new and powerful way to detect active ricin when tackling relevant problems in public safety and security.

Rapid detection of whole active ricin using a surface‐enhanced Raman scattering‐based sandwich immunoassay

AbstractRicin is one of the most toxic proteins known, which has been listed as both chemical and biological warfare agent due to its unique properties. Rapid and sensitive method capable of detecting and distinguishing activity of the whole ricin has very important realistic significance in view of national security and defense. This study reports a reliable surface‐enhanced Raman scattering (SERS)‐based sandwich immunoassay for the detection and differentiation of active ricin from other homologous toxins. In the immunoassay design, magnetic bead conjugated with a monoclonal antibody (mAb) MIL50 that can recognize the ricin in its holotoxin form was used as a capture probe to separate ricin from complex matrix, whereas both Raman reporter molecule Nile blue A (NBA) and ricin‐specific mAb 14C12 were modified on gold nanoparticles (AuNPs) to form SERS nanoprobes. In the presence of ricin, SERS nanoprobes bound to the antigen‐captured magnetic beads and formed a sandwich immunocomplex to produce specific hot spots and then generated highly sensitive signals. As a result, the whole ricin could be detected as low as 1 and 5 ng/ml in phosphate‐buffered saline (PBS) and in plasma, respectively. Not only active and inactive ricin can be discriminated distinctly but also ricin from other type II ribosome‐inactivating proteins and their agglutinins. Furthermore, the proposed method made an application to practical samples from the Organization for the Prohibition of Chemical Weapons (OPCW) biotoxin exercise test, proving this SERS‐based sandwich immunoassay a promising assay for timely response of ricin‐related risk assessment in terrorist event and human intoxication.

A Simple, Fast and Portable Method for Electrochemical Detection of Adenine Released by Ricin Enzymatic Activity.

International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin in various sample matrices are considered necessary actions for risk assessment during a suspected exposure. This study reports a portable electrochemical assay for detecting active ricin based on the adenine electro-oxidation released from herring sperm DNA substrate by its catalytic action. Also, kinetic parameters were calculated, and the values were Km of 3.14 µM and Kcat 2107 min−1. A linear response was found in optimized experimental conditions for ricin concentrations ranging from 8 to 120 ng/mL, and with a detection limit of 5.14 ng/mL. This proposed detection strategy emphasizes the possibility of field detection of active ricin in food matrices and can be applied to other endonucleolytic activities.

Highly sensitive MALDI-MS measurement of active ricin: insight from more potential deoxynucleobase-hybrid oligonucleotide substrates.

Herein, we report an improved MALDI-MS method for active ricin to contribute toward countermeasures against its real threat to the public. Compared with commonly used DNA or RNA substrates, the deoxynucleobase-hybrid oligonucleotide (RNA_dA, Rd) substrate containing functional Gd[combining low line]A[combining low line]GA loop was revealed as a substrate with more potential and used for the first time in ricin measurement via MALDI-MS. The Rd sequence greatly prompted ricin to exhibit its catalytic activity as rRNA N-glycosylase in ex vitro condition, which was supported by molecular docking simulation and enzymatic parameters depicted in MALDI-MS. Furthermore, we discovered that a highly pure matrix was the most crucial parameter for enhancing the sensitivity, which addressed the major obstacle encountered in the oligo(deoxy)nucleotide measurement, i.e., the interfering alkali metal ion-adducted signals in MALDI-MS. After the optimization of pH and enzymatic reaction buffer composition in this ex vitro condition, this method can provide a wide linearity of up to three orders of magnitude, i.e., 1-5000 ng mL-1, and a high sensitivity of 1 ng mL-1 without any enrichment. Denatured and active ricin could be distinctly differentiated, and the application to practical samples from one international exercise and a soft drink proved the feasibility of this new method. We believe this MALDI-MS method can contribute to the first response to ricin occurrence events in public safety and security, as well as pave a new way for a deep understanding of ricin and other type II ribosome inactivating proteins involved toxicology.

Examining The Forensic Toxicity of Ricin Using Lc/Ms Techniques

As one the naturally occurring toxins, Ricin is classified among the deadliest poisons available. Ricin and
 Ricinus communis agglutinin (RCA120) are derived from the castor bean plant. Ricin and its related activities
 can be detected using either mass spectrometric (MS) assay or Liquid Chromatography (LC). Further, the
 two methods can be to differentiate ricin from RCA120 because it is less toxic of the two. The sequence of
 Amino acid is identified by monitoring the active ricin using mass spectrometric. This study discusses how
 LC and MS methods can be applied to quantify, detect, and differentiate ricin from RCA120 in test samples.
 On the overall, the two methods were applied to tell aside ricin samples from RCA120. The study concluded
 that mass spectrometry is the most efficient approach in identifying ricin from RCA120.

Ultrasensitive On-Site Detection of Biological Active Ricin in Complex Food Matrices Based on Immunomagnetic Enrichment and Fluorescence Switch-On Nanoprobe.

Ricin is a highly toxic protein largely existing in castor beans, which could be used as a warfare agent due to its unique properties. As a deadenylase, inactivation of ricin means a loss of its toxic threat. Therefore, developing simple, accurate, and sensitive on-site detection of biologically active ricin in wide types of complex matrices is most valuable. Here, antifouling polymer brush modified magnetic beads were prepared first and post modified with ricin monoclonal antibody (the MB@P(C-H)-mAbricin) to efficiently capture ricin from various foods and biological matrices. Active ricin obtained in this manner were sequentially determined by a new designed AuNP/QDs nanoassembly. In this double strand oligodeoxynucleotides (dsODN) linked core-satellite nanoprobe, the fluorescence of satellite QDs was extensively quenched by AuNPs due to the dipole-metal interaction. Active ricin can react with its specific depurination substrates which had been inserted in the dsODN linkers. This reaction would trigger the separation of QDs from Au cores by cutting multiple adenines, and then result in the restoration of QDs fluorescence. By coupling with the magnetic enrichment, this AuNP/QDs nanoprobe provided a qualitative result for active ricin in the range from 10.0 to 100.0 ng mL-1 with the limit of detection as low as 7.46 ng mL-1. Compared with previously proposed methods, this on-site detection strategy offered an easy to handle on-site test for trace amounts of active ricin in a wide range of complex matrices.

Identifying exposures to ribosome-inactivating proteins in blood samples: amplification of ricin-induced ribosomal damage products enables sensitive detection of active toxin and circulating depurinated 28S rRNA

Ricin, a highly potent plant-derived toxin, operates by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. Recently, we presented the TRISOL [Truncated RNA Identification by Synthetic Oligonucleotide Ligation] method for the highly sensitive detection of exposures to type II ribosome-inactivating proteins such as ricin in clinical samples, based on the specific amplification of truncated cDNA molecules formed on ricin-dependent depurinated 28S rRNA templates. In the present study, we demonstrate the application of the TRISOL method to detect ricin exposure in blood samples. Sera collected from ricin-intoxicated animals was tested for the presence of either active ricin or ricin-induced depurinated 28S rRNA. Active ricin in serum samples from mice intranasally or intraperitoneally exposed to ricin was detected by the TRISOL method following incubation with a ribosomal-rich reticulocyte lysate preparation. For the detection of depurinated 28S rRNA in the serum, cell-free RNAs were isolated from the sera of mice and pigs at different time points following ricin intoxication by different exposure routes and analyzed for the presence of depurinated 28S rRNA. The TRISOL method allowed sensitive detection of both active ricin and host ribosomal damage in blood samples. Active ricin was detected at both early and late time points after ricin intoxication by all routes of exposure tested. Depurinated 28S rRNA, detected at later time points after systemic and oral intoxications, enables the determination of ricin-induced damage to cells of the exposed subject.

Fast on-Site Visual Detection of Active Ricin Using a Combination of Highly Efficient Dual-Recognition Affinity Magnetic Enrichment and a Specific Gold Nanoparticle Probe.

Ricin, a highly toxic protein, is a controlled substance by both the Chemical Weapons Convention (CWC) and the Biological Weapons Convention (BWC). Therefore, fast precaution of potential ricin toxin plays an important role in national security and public safety. Herein, a simple, sensitive, and accurate visual detection of active ricin in complex samples is presented by combining magnetic affinity enrichment with a specific gold nanoparticle (AuNP) probe. In the first step, a dual-recognition magnetic absorbent was fabricated by simultaneously incorporating two different affinity ligands (concanavalin A and galactosamine) on low-foul polymer brushes grafted magnetic beads, which showed remarkable multivalent synergy binding capacity for ricin even under complex interfering environments. Subsequently, a homoadenine-constituted oligodeoxynucleotide named poly(21dA) was conjugated to AuNPs (the poly(21dA)-AuNPs), which served as a specific depurination substrate of active ricin. Coralyne can trigger the intact poly(21dA)-AuNPs aggregate by forming a non-Watson-Crick homoadenine/coralyne complex, but the poly(21dA)-AuNPs after reacting with active ricin failed to form this complex due to the loss of adenines. Based on these facts, active ricin can be detected as low as 12.5 ng mL-1 with the naked eyes. This detection strategy could be well-applied in various ricin-spiked complex matrices. The features such as ready operation, facile readout, and easy accessibility make the assay a better choice for fast on-site active ricin detection.

Improved Sensitivity for the Qualitative and Quantitative Analysis of Active Ricin by MALDI-TOF Mass Spectrometry.

Ricin is a highly toxic protein which causes cell death by blocking protein synthesis and is considered a potential bioterrorism agent. Rapid and sensitive detection of ricin toxin in various types of sample matrices is needed as an emergency requirement for public health and antibioterrorism response. An in vitro MALDI TOF MS-based activity assay that detects ricin mediated depurination of synthetic substrates was improved through optimization of the substrate, reaction conditions, and sample preparation. In this method, the ricin is captured by a specific polycolonal antibody followed by hydrolysis reaction. The ricin activity is determined by detecting the unique cleavage product of synthetic oligomer substrates. The detection of a depurinated substrate was enhanced by using a more efficient RNA substrate and optimizing buffer components, pH, and reaction temperature. In addition, the factors involved in mass spectrometry analysis, such as MALDI matrix, plate, and sample preparation, were also investigated to improve the ionization of the depurinated product and assay reproducibility. With optimized parameters, the limit of detection of 0.2 ng/mL of ricin spiked in buffer and milk was accomplished, representing more than 2 orders of magnitude enhancement in assay sensitivity. Improving assay's ruggeddness or reproducibility also made it possible to quantitatively detect active ricin with 3 orders of magnitude dynamic range.

New Surface-Enhanced Raman Sensing Chip Designed for On-Site Detection of Active Ricin in Complex Matrices Based on Specific Depurination.

Quick and accurate on-site detection of active ricin has very important realistic significance in view of national security and defense. In this paper, optimized single-stranded oligodeoxynucleotides named poly(21dA), which function as a depurination substrate of active ricin, were screened and chemically attached on gold nanoparticles (AuNPs, ∼100 nm) via the Au-S bond [poly(21dA)-AuNPs]. Subsequently, poly(21dA)-AuNPs were assembled on a dihydrogen lipoic-acid-modified Si wafer (SH-Si), thus forming the specific surface-enhanced Raman spectroscopy (SERS) chip [poly(21dA)-AuNPs@SH-Si] for depurination of active ricin. Under optimized conditions, active ricin could specifically hydrolyze multiple adenines from poly(21dA) on the chip. This depurination-induced composition change could be conveniently monitored by measuring the distinct attenuation of the SERS signature corresponding to adenine. To improve sensitivity of this method, a silver nanoshell was deposited on post-reacted poly(21dA)-AuNPs, which lowered the limit of detection to 8.9 ng mL(-1). The utility of this well-controlled SERS chip was successfully demonstrated in food and biological matrices spiked with different concentrations of active ricin, thus showing to be very promising assay for reliable and rapid on-site detection of active ricin.

Recommended Mass Spectrometry-Based Strategies to Identify Ricin-Containing Samples.

Ricin is a protein toxin produced by the castor bean plant (Ricinus communis) together with a related protein known as R. communis agglutinin (RCA120). Mass spectrometric (MS) assays have the capacity to unambiguously identify ricin and to detect ricin’s activity in samples with complex matrices. These qualitative and quantitative assays enable detection and differentiation of ricin from the less toxic RCA120 through determination of the amino acid sequence of the protein in question, and active ricin can be monitored by MS as the release of adenine from the depurination of a nucleic acid substrate. In this work, we describe the application of MS-based methods to detect, differentiate and quantify ricin and RCA120 in nine blinded samples supplied as part of the EQuATox proficiency test. Overall, MS-based assays successfully identified all samples containing ricin or RCA120 with the exception of the sample spiked with the lowest concentration (0.414 ng/mL). In fact, mass spectrometry was the most successful method for differentiation of ricin and RCA120 based on amino acid determination. Mass spectrometric methods were also successful at ranking the functional activities of the samples, successfully yielding semi-quantitative results. These results indicate that MS-based assays are excellent techniques to detect, differentiate, and quantify ricin and RCA120 in complex matrices.

Engineering a switch-on peptide to ricin A chain for increasing its specificity towards HIV-infected cells

BackgroundRicin is a type II ribosome-inactivating protein (RIP) that potently inactivates eukaryotic ribosomes by removing a specific adenine residue at the conserved α-sarcin/ricin loop of 28S ribosomal RNA (rRNA). Here, we try to increase the specificity of the enzymatically active ricin A chain (RTA) towards human immunodeficiency virus type 1 (HIV-1) by adding a loop with HIV protease recognition site to RTA. MethodsHIV-specific RTA variants were constructed by inserting a peptide with HIV-protease recognition site either internally or at the C-terminal region of wild type RTA. Cleavability of variants by viral protease was tested in vitro and in HIV-infected cells. The production of viral p24 antigen and syncytium in the presence of C-terminal variants was measured to examine the anti-HIV activities of the variants. ResultsC-terminal RTA variants were specifically cleaved by HIV-1 protease both in vitro and in HIV-infected cells. Upon proteolysis, the processed variants showed enhanced antiviral effect with low cytotoxicity towards uninfected cells. ConclusionsRTA variants with HIV protease recognition sequence engineered at the C-terminus were cleaved and the products mediated specific inhibitory effect towards HIV replication. General significanceCurrent cocktail treatment of HIV infection fails to eradicate the virus from patients. Here we illustrate the feasibility of targeting an RIP towards HIV-infected cells by incorporation of HIV protease cleavage sequence. This approach may be generalized to other RIPs and is promising in drug design for combating HIV.

Immunosorbent analysis of ricin contamination in milk using colorimetric, chemiluminescent and electrochemiluminescent detection

Analytical methodology to detect active ricin in food matrices is important because of the potential use of foodborne ricin as a terrorist weapon. Monoclonal antibodies (mAbs) that bind ricin were used as both capture and detection ligands in sandwich enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescent (ECL) immunosorbent assays. ELISA employed two types of substrate for colorimetric or chemiluminescent detection. Although both fat content and protein content of samples influenced the recovery of ricin, the lower limit of detection (LOD) in ELISA and ECL systems permitted detection of 0.1 ng/mL for milk samples containing 0–4% fat. The assay systems detect pure ricin- or crude ricin-containing castor extract, but do not significantly respond to isolated ricin chains, heat-denatured ricin or the related agglutinin, Ricinus communis agglutinin 1 (RCA-1). Using the standard 96-well-plate formats, the assays detect less than 0.01% of an adult human lethal dose in a typical serving of milk.

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.

Analysis of active ricin and castor bean proteins in a ricin preparation, castor bean extract, and surface swabs from a public health investigation

In late February 2008, law enforcement officials in Las Vegas, Nevada, discovered in a hotel room, a copy of The Anarchist Cookbook, suspected castor beans and a "white powder" thought to be a preparation of ricin. Ricin is a deadly toxin from the seed of the castor bean plant (Ricinus communis). The United States regulates the possession, use, and transfer of ricin and it is the only substance considered a warfare agent in both the Chemical and the Biological Weapons Conventions. Six samples obtained from the hotel room were analyzed by laboratories at the Centers for Disease Control and Prevention using a panel of biological and mass spectrometric assays. The biological assays (real time-PCR, time resolved fluorescence and cytotoxicity) provided presumptive evidence of active ricin in each of the samples. This initial screen was followed by an in-depth analysis using a novel, state-of-the-art mass spectrometry-based ricin functional assay and high sensitivity tandem mass spectrometry for protein identification. Mass spectrometric analysis positively identified ricin and confirmed that in each of the samples it was enzymatically active. The tandem mass spectrometry analysis used here is the most selective method available to detect ricin toxin. In each sample, ricin was unequivocally identified along with other R. communis plant proteins, including the highly homologous protein RCA120. Although database searches using tandem mass spectra acquired from the samples indicated that additional controlled substances were not present in these samples, the mass spectrometric results did provide extensive detail about the sample contents. To the best of our knowledge following a review of the available literature, this report describes the most detailed analysis of a white powder for a public health or forensic investigation involving ricin.

Thermal Stability of Ricin in Orange and Apple Juices

Ricin is a potent protein toxin that could be exploited for bioterrorism. Although ricin may be detoxified using heat, inactivation conditions in foods are not well characterized. Two brands of pulp-free orange juice and 2 brands of single-strength apple juice (one clarified and the other unclarified) containing 100 microg/mL added ricin were heated at 60 to 90 degrees C for up to 2 h. With increasing heating times and temperatures the heat-treated juices exhibited decreasing detectability of ricin by enzyme-linked immunosorbent assay (ELISA) and cytotoxicity to cultured cells. Z-values for ricin inactivation in orange juices were 14.4 +/- 0.8 degrees C and 17 +/- 4 degrees C using cytotoxicity assays, compared to 13.4 +/- 1.5 degrees C and 14 +/- 2 degrees C determined by ELISA. Although insignificant differences were apparent for z-values measured for the 2 orange juice brands, significant differences were found in the z-values for the 2 brands of apple juice. The z-values for ricin inactivation in the clarified and unclarified apple juices were 21 +/- 4 degrees C and 9.5 +/- 1.1 degrees C, determined by cytotoxicity assays, and 20 +/- 2 degrees C and 11.6 +/- 0.7 degrees C, respectively, using ELISA. Overall, there were no significant differences between results measured with ELISA and cytotoxicity assays. Ricin stability in orange juice and buffer was evaluated at 25 degrees C. Half-lives of 10 +/- 3 d and 4.9 +/- 0.4 d, respectively, indicated that active ricin in juice could reach consumers. These results indicate that ricin in apple and orange juices can remain toxic under some processing and product storage conditions. Ricin is a potent toxin that is abundant in castor beans and is present in the castor bean mash by-product after cold-press extraction of castor oil. U.S. Health and Human Services recognizes that ricin could be used for bioterrorism. This study reports the stability of ricin in apple and orange fruit juices at temperatures ranging from 60 to 90 degrees C (140 to 194 degrees F).

Effect of Food Matrices on the Biological Activity of Ricin

A cell-free translation assay was applied for the quick detection of ricin in food samples. Three economically important foods—ground beef, low-fat milk, and liquid chicken egg—were tested. The results indicated that ground beef had very little matrix effect on the assay, whereas low-fat milk and liquid chicken egg showed clear interference on the protein translation. A simple dilution in phosphate-buffered saline (PBS) effectively eliminated the translational inhibition from these foods. The concentrations inhibiting 50% of luciferase translation derived from the current study were 0.01 nM for the pure ricin A chain, 0.02 nM for pure ricin, and 0.087 nM for crude ricin in PBS. In most cases, the half inhibitory concentration values for ricin in food matrices were significantly lower than for those in PBS buffer, suggesting that some components in these food matrices might potentiate the activity of ricin. Thermal stability tests indicated that the ricin A chain was the least stable among the three forms of ricin in all matrices measured. The thermal stability of pure and crude ricins varied depending on the matrices. The specific activities of ricin in PBS buffer were confirmed by a neutralization test with ricin-specific and nonspecific antibodies. This study demonstrates that the cell-free translation assay is a rapid and sensitive method for detection of biologically active ricin toxin in ground beef, low-fat milk, and liquid chicken egg and that food matrices can greatly affect the thermal stability of ricin.