Rapid and sensitive identification of ricin in environmental samples based on lactamyl agarose beads using LC‐MS/MS (MRM)

Ricin, a plant‐derived toxin extracted from the seeds of Ricinus communis (castor bean plant), is one of the most toxic proteins known. Ricin's high toxicity, widespread availability, and ease of its extraction make it a potential agent for bioterrorist attacks. Most ricin detection methods are based on immunoassays. These methods may suffer from low efficiency in matrices containing interfering substances, or from false positive results due to antibody cross reactivity, with highly homologous proteins. In this study, we have developed a simple, rapid, sensitive, and selective mass spectrometry assay, for the identification of ricin in complex environmental samples. This assay involves three main stages: (a) Ricin affinity capture by commercial lactamyl‐agarose (LA) beads. (b) Tryptic digestion. (c) LC‐MS/MS (MRM) analysis of tryptic fragments. The assay was validated using 60 diverse environmental samples such as soil, asphalt, and vegetation, taken from various geographic regions. The assay's selectivity was established in the presence of high concentrations of competing lectin interferences. Based on our findings, we have defined strict criteria for unambiguous identification of ricin. Our novel method, which combines affinity capture beads followed by MRM‐based analysis, enabled the identification of 1 ppb ricin spiked into complex environmental matrices. This methodology has the potential to be extended for the identification of ricin in body fluids from individuals exposed (deliberately or accidentally) to the toxin, contaminated food or for the detection of the entire family of RIP‐II toxins, by applying multiplex format.     

Competitive bindings of histones with DNA in formation of plant nucleohistones

An acid form of ricin — ricin T — with a molecular weight of 58 kDa, an isoelectric point, pI, of 7.0–7.1, and a sedimentation coefficient of 4.60 S has been isolated from the seeds of the Central Asian castor-oil plantRicinus communis by gel filtration on Sephadex G-75 followed by ion exchange chromatography on DEAE- and CM-celluloses. The amino acid composition and the N-terminal amino acid residues of ricin T have been determined.Institute of the Chemistry of Plant Substances, Uzbek Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 883–887, November–December, 1987.     

Seed proteins of Ricinus communis. II. Isolation and comparative characterization of the acid form of ricin

An acid form of ricin — ricin T — with a molecular weight of 58 kDa, an isoelectric point, pI, of 7.0–7.1, and a sedimentation coefficient of 4.60 S has been isolated from the seeds of the Central Asian castor-oil plantRicinus communis by gel filtration on Sephadex G-75 followed by ion exchange chromatography on DEAE- and CM-celluloses. The amino acid composition and the N-terminal amino acid residues of ricin T have been determined.     

Detection of ricin in food using electrochemiluminescence-based technology

Ricin is a toxic ribosome inactivating protein (RIP-II) present in beans of the castor plant, Ricinus communis. Its potential as a biodefense threat has made the rapid, sensitive detection of ricin in food important to the U.S. Food and Drug Administration. Samples of juice, dairy products, soda, vegetables, bakery products, chocolate, and condiments were spiked with varying concentrations of ricin and analyzed using a 96-well format, electrochemiluminescence (ECL) immunoassay. Assay configurations included the use of a monoclonal capture antibody coupled with either a polyclonal or monoclonal detector antibody. The samples and detector antibodies were either added sequentially or in combination during the capture step. Using the polyclonal antibody, 0.04 ng/mL ricin was detected in analytical samples prepared from several beverages. By simultaneously incubating the sample with detector antibody, it was possible to decrease the assay time to a single 20 min incubation step with a limit of detection <10 ng/mL. Assays run according to this single incubation step exhibited a hook effect (decrease in signal at high concentrations of ricin), but because of the large signal-to-noise ratio associated with the ECL assay, the response remained above background and detectable. Thus, the ECL assay was uniquely suited for the screening of samples for ricin.     

Development and validation of an ELISA kit for the detection of ricin toxins from biological specimens and environmental samples

Ricin is a toxin that can be easily extracted from seeds of Ricinus communis plants. Ricin is considered to be a major bio-threat as it can be freely and easily acquired in large quantities. A deliberate release of such toxin in civilian populations would very likely overwhelm existing public health systems, resulting in public fear and social unrest. There is currently no commercially available or FDA-approved prophylaxis such as vaccines, or therapeutic antitoxins or antidotes, available for ricin intoxication. Patient treatment is typically supportive care based on symptoms, often designed to reinforce the body’s natural response. This paper describes the development and validation of a robust ELISA test kit, which can be used to screen for ricin in biological specimens such as whole blood and faeces. Faecal specimens are shown in this study to have better diagnostic sensitivity and a wider diagnostic window compared to whole blood. From these results, it is concluded that faeces is the most suitable clinical specimen for diagnosis of ricin poisoning via the oral route. The ELISA test kit can also detect ricin in environmental samples. An advantage of this ELISA kit over other commercial off-the-shelf (COTS) detection kits currently on the market that are developed to screen environmental samples only is its ability to diagnose ricin poisoning from clinical specimens as well as detect ricin from environmental samples.     

Stable Sugar‐Chain‐Immobilized Fluorescent Nanoparticles for Probing Lectin and Cells

Sugar chains are important molecules in cellular recognition and signaling, and quantum dots (QDs) are a very powerful tool for in vitro and in vivo imaging. Herein, we report the preparation of stable sugar‐chain‐immobilized fluorescent nanoparticles (SFNPs) and their application to the analysis of sugar‐chain–protein interactions and cellular imaging. SFNPs were easily prepared by mixing CdTe/CdS core/shell QDs with our previously developed sugar‐chain–ligand conjugates. The obtained SFNPs were very stable and could be stored for several months. In the binding analysis, β‐galactose‐ and α‐glucose‐immobilized SFNPs were specifically interacted with Ricinus communis agglutinin I and concanavalin A, respectively, and made into aggregates. The binding interaction was detected visually, fluorescently, or both. In the experiment for cellular imaging, it was found that SFNPs were predominantly taken up by human hepatocyto carcinoma cells (HepG2), suggesting the possible usage of our designed SFNPs for various biochemical analyses of sugar chains.     

Evaluation of the Effect of Pear Stone Cells on the Recovery of Multi-Class Pesticides

The widespread sample preparation method for the extraction of pesticide residues, the QuEChERS method, sometimes yields different pesticide recovery values for different food matrices. Pear (Pyrus communis L.) is an example of a matrix that can be characterized with somewhat lower pesticide recoveries. In order to investigate the reason for this result, the most characteristic structural feature of pears, the stone cells, possessing a specific surface due to their greater cellulose and lignin contents, were isolated and purified. The isolated stone cells were used for the preparation of a model sample in combination with tomato, a food matrix showing high pesticide recovery values, in a pesticide spiking experiment. Eleven multi-class pesticides were selected for the study where the quantification was carried out with HPLC-ESI-MS/MS. No significant differences were found between the pesticide recoveries obtained from the tomato and the combined tomato-pear stone cells matrices, which indicates the reason behind the lower pesticide recoveries in pear matrices is still to be determined.     

Biosensor-based small molecule fragment screening with biolayer interferometry

Biosensor-based fragment screening is a valuable tool in the drug discovery process. This method is advantageous over many biochemical methods because primary hits can be distinguished from non-specific or non-ideal interactions by examining binding profiles and responses, resulting in reduced false-positive rates. Biolayer interferometry (BLI), a technique that measures changes in an interference pattern generated from visible light reflected from an optical layer and a biolayer containing proteins of interest, is a relatively new method for monitoring small molecule interactions. The BLI format is based on a disposable sensor that is immersed in 96-well or 384-well plates. BLI has been validated for small molecule detection and fragment screening with model systems and well-characterized targets where affinity constants and binding profiles are generally similar to those obtained with surface plasmon resonsance (SPR). Screens with challenging targets involved in protein–protein interactions including BCL-2, JNK1, and eIF4E were performed with a fragment library of 6,500 compounds, and hit rates were compared for these targets. For eIF4E, a protein containing a PPI site and a nucleotide binding site, results from a BLI fragment screen were compared to results obtained in biochemical HTS screens. Overlapping hits were observed for the PPI site, and hits unique to the BLI screen were identified. Hit assessments with SPR and BLI are described.     

A single DNA aptamer functions as a biosensor for ricin

The use of microorganisms or toxins as weapons of death and fear is not a novel concept; however, the modes by which these agents of bioterrorism are deployed are increasingly clever and insidious. One mechanism by which biothreats are readily disseminated is through a nation's food supply. Ricin, a toxin derived from the castor bean plant, displays a strong thermostability and remains active at acidic and alkaline pHs. Therefore, the CDC has assigned ricin as a category B reagent since it may be easily amendable as a deliberate food biocontaminate. Current tools for ricin detection utilize enzymatic activity, immunointeractions and presence of castor bean DNA. Many of these tools are confounded by complex food matrices, display a limited dynamic range of detection and/or lack specificity. Aptamers, short RNA and single stranded DNA sequences, have increased affinity to their selected receptors, experience little cross-reactivity to other homologous compounds and are currently being sought after as biosensors for bacterial contaminants in food. This paper describes the selection and characterization of a single, dominant aptamer, designated as SSRA1, against the B-chain of ricin. SSRA1 displays one folding conformation that is stable across 4-63 °C (ΔG = -5.05). SSRA1 is able to concentrate at least 30 ng mL(-1) of ricin B chain from several liquid food matrices and outcompetes a currently available ELISA kit and ricin aptamer. Furthermore, we show detection of 25 ng mL(-1) of intact ricin A-B complex using SSRA1 combined with surface enhanced Raman scattering technique. Thus, SSRA1 would serve well as pre-analytical tool for processing of ricin from liquid foods to aid current diagnostics as well as a sensor for direct ricin detection.     

Exploratory Analysis for Elemental Characterization of Biomass Residues From Biodiesel Production by Inductively Coupled Plasma Optical Emission Spectrometry

Edible and non-edible seeds with high oil content are used as the main raw material for biodiesel production. The residual biomass (press cake or oil cake) can be used as animal feed, to aggregate value to the biodiesel production cycle. In this work, a microwave-assisted-digestion procedure for elementary determination by inductively coupled plasma optical emission spectrometry (ICP OES) using dilute nitric acid was developed. Inter-laboratorial reference material was used to check the accuracy. A full factorial design in addition to exploratory analysis through principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to classify the samples: press cakes from castor (Ricinus communis), jatropha (Jatropha curcas), radish (Raphanus sativus), sunflower (Heliantus annus), and coffee (Coffe arabica), according to their similarities.     

Development and validation of a sandwich ELISA for the determination of potentially allergenic sesame (Sesamum indicum) in food

This paper presents a sandwich enzyme-linked immunosorbent assay (ELISA) that allows the determination of traces of sesame in food. Chicken anti-sesame antibodies, used as coating antibodies, and rabbit anti-sesame antibodies, used as secondary antibodies, were prepared by immunization with a protein extract of white, peeled sesame. The ELISA did not show any cross-reactivity with 19 food ingredients commonly found in sesame-containing foodstuffs such as seeds, nuts, and cereals. In whole grain bread, crisp toast, and snacks, the limit of detection (S/N = 3) was 0.5, 0.5, and 0.3 μg sesame protein/g, and the limit of quantification (S/N = 10) was 0.6, 0.8, and 1.4 μg sesame protein/g, respectively. The analysis of blank food matrices (whole grain bread, white bread, crisp toast, and snacks) spiked with sesame protein at four spike levels generally resulted in mean recoveries from 72% to 145%. In the case of spiking blank food matrices with sesame seeds, the ELISA proved to be more accurate for whole wheat cookies than for whole wheat bread.     

Synergic effect of multi-walled carbon nanotubes and gold nanoparticles towards immunosensing of ricin with carbon nanotube-gold nanoparticles-chitosan modified screen printed electrode

An amperometric immunosensor for the specific detection of Ricinus communis is reported. Screen printed electrodes (SPEs) were modified with gold nanoparticles (GNPs) loaded multiwalled carbon nanotubes (MWCNTs)-chitosan (Ch) film. The ratio of MWCNT and GNP was optimised to get best electrochemically active electrode. Sandwich immunoassay format was used for the immunosensing of ricin. The revealing antibodies tagged with the enzyme alkaline phosphatase (ALP) converts the substrate 1-naphthyl phosphate into 1-naphthol that was determined with the amperometric technique. The amperometric current obtained was correlated with the concentration of ricin. The prepared GNP-MWCNT-Ch-SPE showed high stability due to the Ch film, short response time with good reproducibility and increased shelf life of the electrodes immobilised with antibodies. The electrochemical activity of the electrode improved because of optimization of composition of CNTs and gold nanoparticles. Under the optimal conditions, the modified electrode showed a wide linear response to the concentration of ricin in the range of 2.5-25 ng mL(-1) with a limit of detection of 2.1 ng mL(-1) and with a relative standard deviation of 5.1% and storage life of 32 days.     

蓖麻内源防御肽的MALDI-MSI方法及溯源应用研究

国际范围内,蓖麻毒素、蓖麻籽粗提物以及蓖麻籽等均为蓖麻毒素信件的潜在来源,屡屡引发公共安全威胁.因此,蓖麻籽产地溯源至关重要.该研究选择高丰度、低毒性的蓖麻内源性防御肽(RCB),系统探讨优化了冷冻切片制备、基质喷雾等条件,首次建立了RCB 的基质辅助激光解吸电离-质谱成像(MALDI-MSI)方法.选择四川、山西为中...     

Separation and detection of multiple pathogens in a food matrix by magnetic SERS nanoprobes

A rapid and sensitive method was developed here for separation and detection of multiple pathogens in food matrix by magnetic surface-enhanced Raman scattering (SERS) nanoprobes. Silica-coated magnetic probes (MNPs@SiO₂) of ∼100 nm in diameter were first prepared via the reverse microemulsion method using cetyltrimethylammonium bromide as a surfactant and tetraethyl orthosilicate as the silica precursor. The as-prepared MNPs@SiO₂ were functionalized with specific pathogen antibodies to first capture threat agents directly from a food matrix followed by detection using an optical approach enabled by SERS. In this scheme, pathogens were first immuno-magnetically captured with MNPs@SiO₂, and pathogen-specific SERS probes (gold nanoparticles integrated with a Raman reporter) were functionalized with corresponding antibodies to allow the formation of a sandwich assay to complete the sensor module for the detection of multiple pathogens in selected food matrices, just changing the kinds of Raman reporters on SERS probes. Here, up to two key pathogens, Salmonella enterica serovar Typhimurium and Staphylococcus aureus, were selected as a model to illustrate the probability of this scheme for multiple pathogens detection. The lowest cell concentration detected in spinach solution was 10³ CFU/mL. A blind test conducted in peanut butter validated the limit of detection as 10³ CFU/mL with high specificity, demonstrating the potential of this approach in complex matrices.     

Acetone powder from dormant seeds of Ricinus communis L

The influence of several factors on the hydrolytic activity of lipase, present in the acetone powder from dormant castor seeds (Ricinus communis) was evaluated. The enzyme showed a marked specificity for short-chain substrates. The best reaction conditions were an acid medium, Triton X-100 as the emulsifying agent and a temperature of 30 degrees C. The lipase activity of the acetone powder of different castor oil genotypes showed great variability and storage stability of up to 90%. The toxicology analysis of the acetone powder from genotype Nordestina BRS 149 showed a higher ricin (toxic component) content, a lower 2S albumin (allergenic compound) content, and similar allergenic potential compared with untreated seeds.     

Fabrication of Highly Stable Glyco‐Gold Nanoparticles and Development of a Glyco‐Gold Nanoparticle‐Based Oriented Immobilized Antibody Microarray for Lectin (GOAL) Assay

The design of high‐affinity lectin ligands is critical for enhancing the inherently weak binding affinities of monomeric carbohydrates to their binding proteins. Glyco‐gold nanoparticles (glyco‐AuNPs) are promising multivalent glycan displays that can confer significantly improved functional affinity of glyco‐AuNPs to proteins. Here, AuNPs are functionalized with several different carbohydrates to profile lectin affinities. We demonstrate that AuNPs functionalized with mixed thiolated ligands comprising glycan (70 mol %) and an amphiphilic linker (30 mol %) provide long‐term stability in solutions containing high concentrations of salts and proteins, with no evidence of nonspecific protein adsorption. These highly stable glyco‐AuNPs enable the detection of model plant lectins such as Concanavalin A, wheat germ agglutinin, and Ricinus communis Agglutinin 120, at subnanomolar and low picomolar levels through UV/Vis spectrophotometry and dynamic light scattering, respectively. Moreover, we develop in situ glyco‐AuNPs‐based agglutination on an oriented immobilized antibody microarray, which permits highly sensitive lectin sensing with the naked eye. In addition, this microarray is capable of detecting lectins presented individually, in other environmental settings, or in a mixture of samples. These results indicate that glyconanoparticles represent a versatile and highly sensitive method for detecting and probing the binding of glycan to proteins, with significant implications for the construction of a variety of platforms for the development of glyconanoparticle‐based biosensors.     

A combinatorial approach to obtain affinity media with binding properties towards the aflatoxins

In our work we performed a combinatorial solid-phase synthesis in aqueous medium to prepare peptide libraries from which to select an amino acid sequence with binding properties towards aflatoxins.We used polystyrene beads, functionalised with carboxylic groups as solid support and eight amino acids as monomers. During the first step 64 different sequences of two amino acids were prepared by exploiting the principles of combinatorial chemistry; then the binding properties of all sequences towards aflatoxin B(1 )were checked. We determined binding constants towards aflatoxin B(1) and towards aflatoxins B(2), G(1) and G(2). Results were promising, so we prepared a new library by using the selected dipeptide as the starting solid phase. After selecting the best tetrapeptide sequences, we determined binding constants towards the quoted aflatoxins. We obtained binding constants ( K>10(4) M(-1)) similar to those shown by human serum albumin for similar compounds. Preliminary studies on an extraction column were promising for the development of an SPE system and for its application in food matrices.     

Conjugates of polyhedral boron compounds with carbohydrates 8. Synthesis and properties of nido-ortho-carborane glycoconjugates containing one to three ��-lactosylamine residues

??-Lactosylamine derivatives containing one to three disaccharide residues with the bromine atom at the terminal position of aglycon were synthesized. Conjugation of these derivatives with 1-mercapto-1,2-dicarba-closo-dodecaborane was carried out in DMSO, which was accompanied by deboronation to form nido-ortho-carborane (7,8-dicarba-nido-undecaborate) glycoconjugates in up to 70% yield. The hydrolytic stability of glycoconjugates and the binding efficiency of their triethylammonium salts to castor-bean galectin (Ricinus communis aggluinin, RCA₁₂₀) were estimated. Glycoconjugates are decomposed in an aqueous solution by ??15% at 37 °C in three days. The glycoconjugate salt with one ??-lactosylamine residue binds to galectin analogously to lactose; after indroduction of the second ??-lactosylamine residue into glycoconjugate, the binding efficiency increases ??6-fold due to the cluster effect.     

Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays

Proteins of the 2S albumin family, such as Ara h2 and Ara h6, are most frequently involved in peanut allergy. We have developed a reverse enzyme allergo-sorbent test (EAST) in which total serum IgE antibodies are first captured by immobilised anti-human IgE monoclonal antibodies, and then the binding of the anti-Ara h2 and anti-Ara h6 specific IgE to the corresponding labelled allergens is measured. This reverse immunoassay was used either as a direct EAST or as an EAST inhibition assay to study the interactions of whole peanut protein extract and purified Ara h2 and Ara h6 with IgE antibodies from peanut-allergic patients. Finally, we identified some IgE-binding epitopes on Ara h6 using a format of EAST in which the protein is immobilised in a particular, well defined, manner through interactions with specific monoclonal antibodies (mAbs) coated on the micro-plates. The fine specificity of those mAbs has been characterised at the epitope level, and their binding to the allergen thus masks a known particular epitope and makes it unavailable for recognition by IgE antibodies. The reverse EAST increased the ratio specific signal/background. It avoids interferences with competitors such as anti-peanut protein IgG antibodies and allows the study of the specificity and/or affinity of the interactions between IgE antibodies and Ara h2 or Ara h6 with a higher sensitivity and accuracy than the conventional EAST. The EAST results obtained when the allergens are presented by specific mAbs suggest that the homologous molecular domain(s) in peanut 2S albumins encompass major IgE epitope(s) and are strongly involved in peanut allergenicity.     

Following aptamer-ricin specific binding by single molecule recognition and force spectroscopy measurements

Single molecule recognition imaging and dynamic force spectroscopy (DFS) analysis showed strong binding affinity between an aptamer and ricin, which was comparable with antibody-ricin interaction. Molecular simulation showed a ricin binding conformation with aptamers and gave different ricin conformations immobilizing on substrates that were consistent with AFM images.