Search Results (35)

Abrin and ricin, both type II ribosome-inactivating proteins, are toxins of significant concern and are under international restriction by the Chemical Weapons Convention and the Biological and Toxin Weapons Convention. The development of a rapid and sensitive detection method for these toxins is of the utmost importance for the first emergency response. Emerging rapid detection techniques, such as surface-enhanced Raman spectroscopy (SERS) and lateral flow assay (LFA), have garnered attention due to their high sensitivity, good selectivity, ease of operation, low cost, and disposability. In this work, we generated stable and high-affinity nanotags, via an efficient freezing method, to serve as the capture module for SERS-LFA. We then constructed a sandwich-style lateral flow test strip using a pair of glycoproteins, asialofetuin and concanavalin A, as the core affinity recognition molecules, capable of trace measurement for both abrin and ricin. The limit of detection for abrin and ricin was 0.1 and 0.3 ng/mL, respectively. This method was applied to analyze eight spiked white powder samples, one juice sample, and three actual botanic samples, aligning well with cytotoxicity assay outcomes. It demonstrated good inter-batch and intra-batch reproducibility among the test strips, and the detection could be completed within 15 min, indicating the suitability of this SERS-LFA method for the on-site rapid detection of abrin and ricin toxins. Full article

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. Full article

Shiga-toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology. Five potential biosensor cell lines were evaluated for their ability to detect Stx2. The results using the best biosensor cell line (T5) indicated that this biosensor was stable after reconstitution with assay buffer covered in foil at 4 °C for up to 10 days with an estimated limit of detection (LOD) of ≈0.1–0.2 ng/mL for days up to day 5 and ≈0.4 ng/mL on day 10. The assay detected a broad range of Stx2 subtypes, including Stx2a, Stx2b, Stx2c, Stx2d, and Stx2g but did not cross-react with closely related Stx1, abrin, or ricin. Additionally, this assay was able to detect Stx2 in culture supernatants of STEC grown in media with mitomycin C at 8 and 24 h post-inoculation. These results indicate that the STEC CANARY biosensor developed in this study is sensitive, reproducible, specific, rapid (≈3 min), and may be applicable for surveillance of the environment and food to protect public health. Full article

Rapid and accurate detection of protein toxins is crucial for public health. The Raman spectra of several protein toxins, such as abrin, ricin, staphylococcal enterotoxin B (SEB), and bungarotoxin (BGT), have been studied. Multivariate scattering correction (MSC), Savitzky–Golay smoothing (SG), and wavelet transform methods (WT) were applied to preprocess Raman spectra. A principal component analysis (PCA) was used to extract spectral features, and the PCA score plots clustered four toxins with two other proteins. The k-means clustering results show that the spectra processed with MSC and MSC-SG methods have the best classification performance. Then, the two data types were classified using partial least squares discriminant analysis (PLS-DA) with an accuracy of 100%. The prediction results of the PCA and PLS-DA and the partial least squares regression model (PLSR) perform well for the fingerprint region spectra. The PLSR model demonstrates excellent classification and regression ability (accuracy = 100%, Rcv = 0.776). Four toxins were correctly classified with interference from two proteins. Classification models based on spectral feature extraction were established. This strategy shows excellent potential in toxin detection and public health protection. These models provide alternative paths for the development of rapid detection devices. Full article

Jigucao capsules (JGCC) have the effects of soothing the liver and gallbladder and clearing heat and detoxification. It is a good medicine for treating acute and chronic hepatitis cholecystitis with damp heat of the liver and gallbladder. However, the existing quality standard of JGCC does not have content determination items, which is not conducive to quality control. In this study, serum pharmacochemistry technology and UNIFI data processing software were used to identify the blood prototype components and metabolites under the condition of the obvious drug effects of JGCC, and the referenced literature reports and the results from in vitro analysis of JGCC in the early stage revealed a total of 43 prototype blood components and 33 metabolites in JGCC. Quality markers (Q-markers) were discovered, such as abrine, trigonelline, hypaphorine and isoschaftoside. In addition, ultra-high-performance liquid chromatography–triple quadrupole mass spectrometry (UPLC-QQQ-MS) was used to determine the active ingredients in JGCC. The components of quantitative analysis have good correlation in the linear range with R² ≥ 0.9993. The recovery rate is 93.15%~108.92% and the relative standard deviation (RSD) is less than 9.48%. The established UPLC-MS/MS quantitative analysis method has high sensitivity and accuracy, and can be used for the quality evaluation of JGCC. Full article

Pulchellin is a plant biotoxin categorized as a type 2 ribosome-inactivating protein (RIPs) which potentially kills cells at very low concentrations. Biotoxins serve as targeting immunotoxins (IT), consisting of antibodies conjugated to toxins. ITs have two independent protein components, a human antibody and a toxin with a bacterial or plant source; therefore, they pose unique setbacks in immunogenicity. To overcome this issue, the engineering of epitopes is one of the beneficial methods to elicit an immunological response. Here, we predicted the tertiary structure of the pulchellin A-chain (PAC) using five common powerful servers and adopted the best model after refining. Then, predicted structure using four distinct computational approaches identified conformational B-cell epitopes. This approach identified some amino acids as a potential for lowering immunogenicity by point mutation. All mutations were then applied to generate a model of pulchellin containing all mutations (so-called PAM). Mutants’ immunogenicity was assessed and compared to the wild type as well as other mutant characteristics, including stability and compactness, were computationally examined in addition to immunogenicity. The findings revealed a reduction in immunogenicity in all mutants and significantly in N146V and R149A. Furthermore, all mutants demonstrated remarkable stability and validity in Molecular Dynamic (MD) simulations. During docking and simulations, the most homologous toxin to pulchellin, Abrin-A was applied as a control. In addition, the toxin candidate containing all mutations (PAM) disclosed a high level of stability, making it a potential model for experimental deployment. In conclusion, by eliminating B-cell epitopes, our computational approach provides a potential less immunogenic IT based on PAC. Full article

Bladder cancer (BLCA) is a common malignancy of the urinary system. The gut microbiome produces various metabolites that play functional roles in tumorigenesis and tumor progression. However, the integrative analysis of the gut microbiome and metabolome in BLCA has still been lacking. Thus, the aim of this study was to identify microbial and functional characteristics and metabolites in BLCA in a Chinese population. Metagenomics, targeted metabolomics, bioinformatics, and integrative analysis were used in fecal samples of BLCA patients and healthy individuals. We found gut microbiomes were significantly dysregulated in BLCA patients, including Bifidobacterium, Lactobacillus, Streptococcus, Blautia, and Eubacterium. We also found 11Z-eicosenoic acid, 3-methoxytyrosine, abrine, aniline-2-sulfonate, arachidic acid, conjugated linoleic acids, elaidic acid, glycylleucine, glycylproline, leucyl-glycine, linoelaidic acid, linoleic acid, nicotinamide hypoxanthine dinucleotide, oleic acid, petroselinic acid, and ricinoleic acid to be significantly decreased, while cholesterol sulfate was significantly increased in BLCA patients. Integration of metagenomics and metabolomics revealed interactions between gut microbiota and metabolites and the host. We identified the alterations of gut microbiomes and metabolites in BLCA in a Chinese population. Moreover, we preliminarily revealed the associations between specific gut microbiomes and metabolites. These findings determined potential causative links among gut dysbiosis, dysregulated metabolites, and BLCA. Full article

A quantitative structure–activity relationship (QSAR) model for the structure and affinity of abrin aptamers was established. A higher affinity abrin aptamer based on the established QSAR model was screened by site-directed mutagenesis. The fluorescence quenching effect between magnetic microspheres and fluorescent molecules was studied for the first time. A new method for abrin detection based on the interaction between target molecules and fluorescently labeled aptamers on magnetic microspheres was developed, with the detection limit of 5 ng mL⁻¹. This method can overcome the influence of complex environmental interferents in abrin detection and can meet the analysis requirements for simulated samples such as water, soil, and food. Full article

Abrin is a highly toxic protein obtained from the seeds of the rosary pea plant Abrus precatorius, and it is closely related to ricin in terms of its structure and chemical properties. Both toxins inhibit ribosomal function, halt protein synthesis and lead to cellular death. The major clinical manifestations following pulmonary exposure to these toxins consist of severe lung inflammation and consequent respiratory insufficiency. Despite the high similarity between abrin and ricin in terms of disease progression, the ability to protect mice against these toxins by postexposure antibody-mediated treatment differs significantly, with a markedly higher level of protection achieved against abrin intoxication. In this study, we conducted an in-depth comparison between the kinetics of in vivo abrin and ricin intoxication in a murine model. The data demonstrated differential binding of abrin and ricin to the parenchymal cells of the lungs. Accordingly, toxin-mediated injury to the nonhematopoietic compartment was shown to be markedly lower in the case of abrin intoxication. Thus, profiling of alveolar epithelial cells demonstrated that although toxin-induced damage was restricted to alveolar epithelial type II cells following abrin intoxication, as previously reported for ricin, it was less pronounced. Furthermore, unlike following ricin intoxication, no direct damage was detected in the lung endothelial cell population following abrin exposure. Reduced impairment of intercellular junction molecules following abrin intoxication was detected as well. In contrast, similar damage to the endothelial surface glycocalyx layer was observed for the two toxins. We assume that the reduced damage to the lung stroma, which maintains a higher level of tissue integrity following pulmonary exposure to abrin compared to ricin, contributes to the high efficiency of the anti-abrin antibody treatment at late time points after exposure. Full article

The compound EACC (ethyl (2-(5-nitrothiophene-2-carboxamido) thiophene-3-carbonyl) carbamate) was recently reported to inhibit fusion of autophagosomes with lysosomes in a reversible manner by inhibiting recruitment of syntaxin 17 to autophagosomes. We report here that this compound also provides a strong protection against the protein toxin ricin as well as against other plant toxins such as abrin and modeccin. The protection did not seem to be caused by inhibition of endocytosis and retrograde transport, but rather by inhibited release of the enzymatically active A-moiety to the cytosol. The TANK-binding kinase 1 (TBK1) has been reported to phosphorylate syntaxin 17 and be required for initiation of autophagy. The inhibitor of TBK1, MRT68601, induced in itself a strong sensitization to ricin, apparently by increasing transport to the Golgi apparatus. Importantly, MRT68601 increased Golgi transport of ricin even in the presence of EACC, but EACC was still able to inhibit intoxication, supporting the idea that EACC protects at a late step along the retrograde pathway. These results also indicate that phosphorylation of syntaxin 17 is not required for the protection observed. Full article

Abrin is a cytotoxin with strong lethality, which is a serious threat to human health and public safety, and thus, highly sensitive detection methods are urgently needed. The phage display affibody has two major modules, among which, the affibody fragment, with small molecular weight, high affinity and easy preparation, can be used for the specific recognition of the target, and the phage shell, with numerous protein copies, can be used as a carrier for the massive enrichment of signal molecules, and thus is particularly suitable as a sensitive probe for signal amplification in high-sensitivity biosensors. In this study, with antibody-coated magnetic microspheres as capture probes, Ru(bpy)₃²⁺ and biotin dual-labeled phage display affibodies as the specific signal probes and AuNPs@Ru(bpy)₃²⁺ (Ru(bpy)₃²⁺-coated gold nanoparticles) as the signal amplification nanomaterials, a new electrochemiluminescence (ECL) biosensor with a four-level sandwich structure of “magnetic capture probe-abrin-phage display affibody-AuNPs@Ru(bpy)₃²⁺” was constructed for abrin detection. In this detection mode, AuNPs@Ru(bpy)₃²⁺, a gold nanocomposite prepared rapidly via electrical interaction, contained an extremely high density of signal molecules, and the phage display affibodies with powerful loading capacity were not only labeled with Ru(bpy)₃²⁺, but also enriched with AuNPs@Ru(bpy)₃²⁺ in large amounts. These designs greatly improved the detection capability of the sensor, ultimately achieving the ultra-sensitive detection of abrin. The limit of detection (LOD) was 4.1 fg/mL (3δ/S), and the quantification range was from 5 fg/mL to 5 pg/mL. The sensor had good reproducibility and specificity and performed well in the test of simulated samples. This study expanded the application of affibodies in the field of biosensing and also deeply explored the signal amplification potential of phage display technology, which is of high value for the construction of simple and efficient sensors with high sensitivity. Full article

Ricin and abrin are phytotoxins that can be easily used as biowarfare and bioterrorism agents. Therefore, developing a rapid detection method for both toxins is of great significance in the field of biosecurity. In this study, a novel nanoforest silicon microstructure was prepared by the micro-electro-mechanical systems (MEMS) technique; particularly, a novel microfluidic sensor chip with a capillary self-driven function and large surface area was designed. Through binding with the double antibodies sandwich immunoassay, the proposed sensor chip is confirmed to be a candidate for sensing the aforementioned toxins. Compared with conventional immunochromatographic test strips, the proposed sensor demonstrates significantly enhanced sensitivity (≤10 pg/mL for both toxins) and high specificity against the interference derived from juice or milk, while maintaining good linearity in the range of 10–6250 pg/mL. Owing to the silicon nanoforest microstructure and improved homogeneity of the color signal, short detection time (within 15 min) is evidenced for the sensor chip, which would be helpful for the rapid tracking of ricin and abrin for the field of biosecurity. Full article

Abrin is a types II ribosome-inactivating protein (RIP) isolated from Abrus precatorious seeds, which comprises a catalytically active A chain and a lectin-like B chain linked by a disulfide bond. Four isotoxins of abrin have been reported with similar amino-acid composition but different cytotoxicity, of which abrin-a is the most potent toxin. High lethality and easy availability make abrin a potential bioterrorism agent. However, there are no antidotes available for managing abrin poisoning, and treatment is only symptomatic. Currently, neutralizing antibodies remain the most effective therapy against biotoxin poisoning. In this study, we prepared, identified, and acquired a high-affinity neutralizing monoclonal antibody (mAb) 10D8 with a potent pre- and post-exposure protective effect against cytotoxicity and animal toxicity induced by abrin-a or abrin crude extract. The mAb 10D8 could rescue the mouse injected intraperitoneally with a 25 × LD₅₀ dose of abrin-a from lethality and prevent tissue damages. Results indicated that 10D8 does not prevent the binding and internalization of abrin-a to cells but inhibits the enzymatic activity of abrin-a and reduces protein synthesis inhibition of cells. The high affinity, good specificity, and potent antitoxic efficiency of 10D8 make it a promising candidate for therapeutic antibodies against abrin. Full article

The high toxic abrin from the plant Abrus precatorius is a type II ribosome-inactivating protein toxin with a human lethal dose of 0.1–1.0 µg/kg body weight. Due to its high toxicity and the potential misuse as a biothreat agent, it is of great importance to developing fast and reliable methods for the identification and quantification of abrin in complex matrices. Here, we report rapid and efficient acetonitrile (ACN)- and ultrasound-assisted on-bead trypsin digestion method combined with HPLC-MS/MS for the quantification of abrin isoforms in complex matrices. Specific peptides of abrin isoforms were generated by direct ACN-assisted trypsin digestion and analyzed by HPLC-HRMS. Combined with in silico digestion and BLASTp database search, fifteen marker peptides were selected for differential detection of abrin isoforms. The abrin in milk and plasma was enriched by immunomagnetic beads prepared by biotinylated anti-abrin polyclonal antibodies conjugated to streptavidin magnetic beads. The ultrasound-assisted on-bead trypsin digestion method was carried out under the condition of 10% ACN as denaturant solvent, the entire digestion time was further shortened from 90 min to 30 min. The four peptides of T3A^a,b,c,d, T12A^a, T15A^b, and T9A^c,d were chosen as quantification for total abrin, abrin-a, abrin-b, and abrin-c/d, respectively. The absolute quantification of abrin and its isoforms was accomplished by isotope dilution with labeled AQUA peptides and analyzed by HPLC-MS/MS (MRM). The developed method was fully validated in milk and plasma matrices with quantification limits in the range of 1.0-9.4 ng/mL for the isoforms of abrin. Furthermore, the developed approach was applied for the characterization of abrin isoforms from various fractions from gel filtration separation of the seeds, and measurement of abrin in the samples of biotoxin exercises organized by the Organization for the Prohibition of Chemical Weapons (OPCW). This study provided a recommended method for the differential identification of abrin isoforms, which are easily applied in international laboratories to improve the capabilities for the analysis of biotoxin samples. Full article

Abrin, the toxic lectin from the rosary pea plant Abrus precatorius, has gained considerable interest in the recent past due to its potential malevolent use. However, reliable and easy-to-use assays for the detection and discrimination of abrin from related plant proteins such as Abrus precatorius agglutinin or the homologous toxin ricin from Ricinus communis are sparse. To address this gap, a panel of highly specific monoclonal antibodies was generated against abrin and the related Abrus precatorius agglutinin. These antibodies were used to establish two sandwich ELISAs to preferentially detect abrin or A. precatorius agglutinin (limit of detection 22 pg/mL for abrin; 35 pg/mL for A. precatorius agglutinin). Furthermore, an abrin-specific lateral flow assay was developed for rapid on-site detection (limit of detection ~1 ng/mL abrin). Assays were validated for complex food, environmental and clinical matrices illustrating broad applicability in different threat scenarios. Additionally, the antibodies turned out to be suitable for immuno-enrichment strategies in combination with mass spectrometry-based approaches for unambiguous identification. Finally, we were able to demonstrate for the first time how the developed assays can be applied to detect, identify and quantify abrin from a clinical sample derived from an attempted suicide case involving A. precatorius. Full article