Bio-barcode triggered isothermal amplification in a fluorometric competitive immunoassay for the phytotoxin abrin.

Abrin is one of the most toxic phytotoxins to date, and is a potential biological warfare agent. A bio-barcode triggered isothermal amplification for fluorometric determination of abrin is described. Free abrin competes with abrin-coated magnetic microparticles (MMP) probes to bind to gold nanoparticle (AuNP) probes modified with abrin antibody and bio-barcoded DNA. Abundant barcodes are released from the MMP-AuNP complex via dithiothreitol treatment. This triggers an exponential amplification reaction (EXPAR) that is monitored by real-time fluorometry, at typical excitation/emission wavelengths of 495/520 nm. The EXPAR assay is easily operated, highly sensitive and specific. It was used to quantify abrin in spiked commercial samples. The detection limit (at S/N = 3; for n = 6) is 5.6 pg·mL-1 which is considerably lower than previous reports. This assay provides a universal sensing platform and has great potential for determination of various analytes, including small molecules, proteins, DNA, and cells. Graphical abstract Schematic representation of the bio-barcode triggered exponential amplification reaction (EXPAR) for a fluorometric competitive immunoassay for abrin. The limit of detection is 5.6 pg mL-1 with a large dynamic range from 10 pg mL-1 to 1 µg mL-1.

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.

A Novel Humanized Anti-Abrin A Chain Antibody Inhibits Abrin Toxicity In Vitro and In Vivo.

Abrin, a type-II ribosome inactivating protein from the seed of Abrus precatorius, is classified as a Category B bioterrorism warfare agent. Due to its high toxicity, ingestion by animals or humans will lead to death from multiple organ failure. Currently, no effective agents have been reported to treat abrin poisoning. In this study, a novel anti-abrin neutralizing antibody (S008) was humanized using computer-aided design, which possessed lower immunogenicity. Similar to the parent antibody, a mouse anti-abrin monoclonal antibody, S008 possessed high affinity and showed a protective effect against abrin both in vitro and in vivo, and protected mice that S008 was administered 6 hours after abrin. S008 was found that it did not inhibit entry of abrin into cells, suggesting an intracellular blockade capacity against the toxin. In conclusion, this work demonstrates that S008 is a high affinity anti-abrin antibody with both a neutralizing and protective effect and may be an excellent candidate for clinical treatment of abrin poisoning.

A recombinant mutant abrin A chain expressed in Escherichia coli can be used as an effective vaccine candidate.

We used site-directed mutagenesis to mutate two key amino acid residues, Glu164 and Arg167, of abrin A chain (ABRA), creating a mutant ABRA(E164AR167L). The mutant ABRA(mABRA) encoded by mABRA(E164AR167L) was expressed in the cytoplasm of Escherichia coli, and used to develop an effective vaccine to protect mice against native abrin intoxication. The cytotoxicity of mABRA was dramatically reduced as compared to that of recombinant ABRA(rABRA) and native abrin, but the antigenicity and immunogenicity remained the same. Balb/c mice were vaccinated with purified mABRA, and survival was evaluated after challenge with native abrin. Mice that were given three vaccinations developed a protective immune response that was 100% protective against an intraperitoneal (i.p.) administration of 10×LD50 of native abrin. Furthermore, the sera from immunized mice provided complete passive protection for naive mice. This study describes the generation of a substantial amount of mABRA from E. coli and the potential application of mABRA as an effective vaccine candidate for humans, to protect against a high-dose of native abrin.

Differentiation, Quantification and Identification of Abrin and Abrus precatorius Agglutinin.

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.

Structural basis for neutralization of cytotoxic abrin by monoclonal antibody D6F10.

Abrin, an extremely cytotoxic Type II ribosome-inactivating protein (RIP), is a potential bio-warfare agent. Abrin A-chain (ABA) depurinates an adenosine of sarcin-ricin loop (SRL) from eukaryotic 28S rRNA, thereby arresting protein synthesis and leading to cell death. Monoclonal antibody (mAb) D6F10 is the only known antibody that neutralizes ABA's activity in cell-free systems as well as abrin's toxicity in vitro and in vivo. However, how binding of mAb D6F10 to abrin interferes with abrin's catalytic activity at ribosome is still poorly understood. To provide structural basis for mAb D6F10-mediated rescue of ribosome inactivation by abrin, we determined crystal structures of ABA with and without substrate analogs. The structures of ABA-substrate analogs and ribosome were used in an experiment-guided computational protocol, to construct the ABA-Ribosome complex. A homology model of the variable region (Fv ) of mAb D6F10 was generated and docked with the apo-ABA structure to construct the ABA-D6F10 Fv complex. Structural superposition of ABA common to ABA-D6F10 Fv and ABA-Ribosome complexes reveals steric hindrance as the primary mechanism by which mAb D6F10 neutralizes abrin. In contrast to ABA alone, ABA bound to mAb D6F10 is unable to access the SRL on the ribosome owing to steric clashes of mAb D6F10 with the ribosome. Crystal structures of ABA also reveal a catalytic water molecule implicated in hydrolyzing N-glycosidic bond of the susceptible adenosine by RIPs. Furthermore, our strategy provides structural details of steric hindrance important for neutralization of ricin, another RIP, by mAb 6C2 and hence is of wide applicability. ENZYME: EC3.2.2.22. DATABASE: Structural data have been deposited in the Protein Data Bank (PDB) under the accession numbers 5Z37, 5Z3I, and 5Z3J.

Novel Phage Display-Derived Anti-Abrin Antibodies Confer Post-Exposure Protection against Abrin Intoxication.

Abrin toxin is a type 2 ribosome inactivating glycoprotein isolated from the seeds of Abrus precatorius (jequirity pea). Owing to its high toxicity, relative ease of purification and accessibility, it is considered a biological threat agent. To date, there is no effective post-exposure treatment for abrin poisoning and passive immunization remains the most effective therapy. However, the effectiveness of anti-abrin monoclonal antibodies for post-exposure therapy following abrin intoxication has not been demonstrated. The aim of this study was to isolate high affinity anti-abrin antibodies that possess potent toxin-neutralization capabilities. An immune scFv phage-display library was constructed from an abrin-immunized rabbit and a panel of antibodies (six directed against the A subunit of abrin and four against the B subunit) was isolated and expressed as scFv-Fc antibodies. By pair-wise analysis, we found that these antibodies target five distinct epitopes on the surface of abrin and that antibodies against all these sites can bind the toxin simultaneously. Several of these antibodies (namely, RB9, RB10, RB28 and RB30) conferred high protection against pulmonary intoxication of mice, when administered six hours post exposure to a lethal dose of abrin. The data presented in this study demonstrate for the first time the efficacy of monoclonal antibodies in treatment of mice after pulmonary intoxication with abrin and promote the use of these antibodies, one or several, for post-exposure treatment of abrin intoxication.

A Monoclonal-Monoclonal Antibody Based Capture ELISA for Abrin.

Abrin, one of the most highly potent toxins in the world, is derived from the plant, Abrus precatorius. Because of its high toxicity, it poses potential bioterror risks. Therefore, a need exists for new reagents and technologies that would be able to rapidly detect abrin contamination as well as lead to new therapeutics. We report here a group of abrin-specific monoclonal antibodies (mAbs) that recognize abrin A-chain, intact A-B chain toxin, and agglutinin by Western blot. Additionally, these mAbs were evaluated for their ability to serve as capture antibodies for a sandwich (capture) ELISA. All possible capture-detector pairs were evaluated and the best antibody pair identified and optimized for a capture ELISA. The capture ELISA based on this capture-detector mAb pair had a limit of detection (L.O.D) of ≈1 ng/mL measured using three independent experiments. The assay did not reveal any false positives with extracts containing other potential ribosome-inactivating proteins (RIPs). Thus, this new capture ELISA uses mAbs for both capture and detection; has no cross-reactivity against other plant RIPs; and has a sensitivity comparable to other reported capture ELISAs using polyclonal antibodies as either capture or detector.

Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies.

Abrin, a member of the ribosome-inactivating protein family, is produced by the Abrus precatorius plant. Having the potential to pose a severe threat to both human and animal health, abrin is classified as a Select Agent by the U.S. Department of Health and Human Services. However, an immunoassay that is specific for intact abrin holotoxin has not yet been reported. In this study, seven new monoclonal antibodies (mAbs), designated as Abrin-1 through Abrin-7 have been developed. Isotyping analyses indicate these mAbs have IgG1, IgG2a, or IgG2b heavy-chains and kappa light-chains. Western blot analyses identified two abrin A-chain specific mAbs, Abrin-1 and Abrin-2, and four B-chain specific mAbs (Abrin-3, -5, -6, and -7). A sandwich enzyme-linked immunosorbent assay (ELISA), capable of detecting a mixture of abrin isoforms and agglutinins was developed using B-chain specific Abrin-3 for capture and A-chain specific Abrin-2 as detector. The ELISA is highly sensitive and detects 1 ng/mL of the abrin holotoxin in phosphate-buffered saline, nonfat milk, and whole milk, significantly below concentrations that would pose a health concern for consumers. This ELISA also detects native abrin in plant extracts with a very low background signal. The new abrin mAbs and ELISA should be useful for detecting this potent toxin in the milk supply chain and other complex matrices.

Immunotoxins to a human melanoma-associated antigen: comparison of gelonin with ricin and other A chain conjugates.

Gelonin, a ribosome-inactivating protein from the seeds of Gelonium multiflorum, has been conjugated to antibodies. Previous reports have indicated variable potency of such immunotoxins. The lack of toxicity of gelonin, however, makes it attractive for immunoconjugate production. The ribosome-inactivating protein was covalently linked (using N-succinimidyl-3-(2-pyridyldithio)propionate) to monoclonal antibody, 9.2.27, directed to a human melanoma-associated glycoprotein/proteoglycan. The immunoconjugate showed high selectivity with dose-dependent cytotoxic activity to cultured human melanoma cells (50% inhibitory dose; 1-3 X 10(-11) M versus antigen-positive cells; 1-3 X 10(-7) M versus antigen-negative cells). Specificity and immunoreactivity of the conjugate were similar to those of unconjugated antibody. Biodistribution studies with iodine trace-labeled conjugate in nude mice indicated that tumor localization of the gelonin conjugate was decreased compared to unconjugated antibody. However, a significant therapeutic effect of the conjugate was found with multiple but not single dose i.v. treatment in nude mice bearing established palpable melanoma. These in vivo experiments showed that gelonin conjugates are not toxic up to 2 mg total dose/mouse and significantly retarded the growth of established s.c. tumor. Comparison of gelonin conjugates in vitro and in vivo with other A-chain conjugates of 9.2.27 (abrin and ricin) indicated that gelonin had similar potency, better selectivity, better tumor localization, and more significant therapeutic effects.

A monoclonal antibody to an abrin chimera recognizing a unique epitope on abrin A chain confers protection from abrin-induced lethality.

Abrin, obtained from the seeds of Abrus precatorius plant, is a potent toxin belonging to the family of type II ribosome-inactivating proteins. Recently, a recombinant vaccine consisting of the A subunits of abrin and its homolog Abrus precatorius agglutinin (APA) was demonstrated to protect mice from abrin lethality. Toward identifying neutralizing epitopes recognized during this response, we generated monoclonal antibodies against the proposed vaccine candidate. One antibody, namely A7C4, the corresponding epitope of which was found to be distal to the active site of the enzymatic A chain, prevented abrin-mediated toxicity on cells and abrin-induced lethality in mice but did not inhibit the catalytic activity of the A chain. The in vivo protection conferred by monoclonal antibody A7C4 highlights the potential use of this antibody as a promising immunotherapeutic.

Rapid detection of abrin in foods with an up-converting phosphor technology-based lateral flow assay.

Abrin is a natural plant toxin found in the seeds of Abrus precatorius. It may be used for food poisoning or bioterrorism, seriously endangering public health. In this study, a reliable method for the rapid detection of abrin in foods was developed, based on an up-converting phosphor technology-based lateral flow assay (abrin-UPT-LFA). Nine high-affinity monoclonal antibodies (mAbs) against abrin were prepared, and the optimum mAbs (mAb-6F4 and mAb-10E11) were selected for use in the assay in double-antibody-sandwich mode. The assay was confirmed to be specific for abrin, with a detection sensitivity of 0.1 ng mL-1 for standard abrin solutions. Good linearity was observed for abrin quantitation from 0.1 to 1000 ng mL-1 (r = 0.9983). During the analysis of various abrin-spiked food samples, the assay showed strong sample tolerance and a satisfactory limit of detection for abrin (0.5-10 ng g-1 for solid and powdered samples; 0.30-0.43 ng mL-1 for liquid samples). The analysis of suspected food samples, from sample treatment to result feed-back, could be completed by non-professionals within 20 min. Therefore, the abrin-UPT-LFA is a rapid, sensitive, and reliable method for the on-site detection of abrin in foods.

A comparison of the in vitro and in vivo activities of conjugates of anti-mouse lymphocyte globulin and abrin.

Anti-mouse lymphocyte globulin and normal immunoglobulin have been conjugated to abrin using two procedures, one involving linkage through an amide bond and a piperazine ring and the other the introduction of two amide bonds flanking a disulphide bridge. The four conjugates produced were equipotent as inhibitors of protein synthesis in rabbit reticulocyte lysates. Each antibody-containing conjugate was a more effective inhibitor of protein synthesis in cultured cells than the equivalent normal immunoglobulin-containing conjugate. In addition the conjugates with disulphide linkage groups were ten times more potent than their counterparts. The disulphide conjugates were also twice as toxic to mice in an acute toxicity test but when used to suppress their immune responses to sheep red blood cells it was the non-disulphide-linked conjugates that were superior. In all instances antibody-containing conjugates were more powerful immunosuppressants than those containing normal IgG. The results are taken to indicate a relative lack of stability of the disulphide conjugates in the tissues.

Primary structure of three distinct isoabrins determined by cDNA sequencing. Conservation and significance.

A family of toxic proteins, the isoabrins, which possess N-glycosylase activity toward eukaryotic 28 S r-RNA, may have potential use in cancer chemotherapy. By polymerase chain reaction techniques, cDNA clones of three isoabrins, carrying A and B-chain sequences, were isolated and their nucleotide sequences were determined. The isoabrins consist of an A-chain comprised of 250 of 251 amino acids, followed by a 10 amino acid linker and a B-chain of 267 amino acids. There is substantial conservation in the B-chain of the three isoabrins, with less than six amino acid substitutions, whereas as many as 46 amino acid substitutions occur in the A-chains. Based on the relationships between the biological activities and the putative amino acid sequences of the isoabrins, three isoabrins, abrin-a, -b and -d, could be identified and the potential epitope of immunological response of these isoabrins could be assigned.

Development of ELISA and Colloidal Gold-PAb Conjugate-Based Immunochromatographic Assay for Detection of Abrin-a.

When abrin-a was combined with several polyclonal antibodies (PAb), the detection limit could be increased. In this way, a monoclonal antibody (capture) and polyclonal antibody (detection) sandwich enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-PAb conjugate-based immunochromatographic assay for detection of abrin-a were developed. The ELISA had a detection limit of 3.9 ng/mL for abrin-a in standard solution and 7.8 ng/mL in soybean milk, and was more sensitive than polyclonal antibody (capture) and monoclonal antibody (detection) ELISA, which had a detection limit of 15.6 ng/mL. The test strip had a detection range of 50 to 500 ng/mL for abrin-a and a detection limit in standard solution or soybean milk samples of 50 ng/mL. However, the test strip had a reduced detection capability compared with a colloidal gold-monoclonal antibody conjugate-based immunochromatographic assay test strip, which had a lower detection limit of 10 ng/mL. The developed ELISAs and test strip show the specificity towards abrin-a and have no cross-reactivity towards abrin-b, -c, -d, ricin, or the agglutinins from either castor beans or rosary peas.

Antibody treatment against pulmonary exposure to abrin confers significantly higher levels of protection than treatment against ricin intoxication.

Abrin, a potent plant-derived toxin bearing strong resemblance to ricin, irreversibly inactivates ribosomes by site-specific depurination, thereby precipitating cessation of protein synthesis in cells. Due to its high availability and ease of preparation, abrin is considered a biological threat, especially in context of bioterror warfare. To date, there is no established therapeutic countermeasure against abrin intoxication. In the present study, we examined the progress of pulmonary abrin intoxication in mice, evaluated the protective effect of antibody-based post-exposure therapy, and compared these findings to those observed for ricin intoxication and therapy. Salient features of abrin intoxication were found to be similar to those of ricin and include massive recruitment of neutrophils to the lungs, high levels of pro-inflammatory markers in the bronchoalveolar lavage fluid and damage of the alveolar-capillary barrier. In contrast, the protective effect of anti-abrin antibody treatment was found to differ significantly from that of anti-ricin treatment. While anti-ricin treatment efficiency was quite limited even at 24h post-exposure (34% protection), administration of polyclonal anti-abrin antibodies even as late as 72h post-exposure, conferred exceedingly high-level protection (>70%). While both anti-toxin antibody treatments caused neutrophil and macrophage levels in the lungs to revert to normal, only anti-abrin treatment brought about a significant decline in the pulmonary levels of the pro-inflammatory cytokine IL-6. The differential ability of the anti-toxin treatments to dampen inflammation caused by the two similar toxins, abrin and ricin, could explain the radically different levels of protection achieved following antibody treatment.

A novel recombinant vaccine protecting mice against abrin intoxication.

Abrin toxin (AT) consisting of an A chain and a B chain is a potential agent for bioterrorism and an effective vaccine against AT poisoning is urgently required. In this study, AT B chain (ATB) was successfully expressed in the Escherichia coli (E. coli) and assessed the protection capacity against AT intoxication. The recombinant ATB (rATB) subunit induces a good immune response after 4 immunizations. All BALB/c mice immunized intraperitoneally (i.p.) with the purified rATB protein survived after challenged with 5 × LD50 of AT. Transfusion of sera from immunized mice provided passive protection in naive mice. Furthermore, histological findings showed that immunization with rATB decreased the severity of toxin-related tissue damage. This work indicates that the rATB protein may be a promising vaccine candidate against human exposure to AT.

The low uptake of an abrin A-chain immunotoxin by rat hepatic cells in vivo and in vitro.

The therapeutic value of antibody-ricin A-chain conjugates (immunotoxins) as antineoplastic agents is limited by their rapid removal from the circulation, primarily by cells of the liver which take up the immunotoxin through receptor mediated recognition of mannose-containing oligosaccharides in the toxin A-chain. We have therefore examined the uptake by rat hepatic cells of a monoclonal antibody (LICR-LOND Fib 75) conjugate assembled with the ricin related, but carbohydrate free, A-chain of the plant toxin abrin. The abrin A-chain immunotoxin was very poorly taken up in vivo and in vitro by both hepatic parenchymal and non parenchymal cells whereas a comparable conjugate assembled with ricin A-chain was actively accumulated by liver cells particularly the hepatic non-parenchymal cells. Furthermore, the abrin A-chain immunotoxin uptake by non-parenchymal cells in vitro was unaffected by mannose and the immunotoxin bound less readily to liver cells than did the ricin A-chain conjugate, consistent with a proposal that its accumulation by hepatic cells is brought about by endocytosis following non-specific binding or by fluid phase pinocytosis. These results suggest abrin A-chain immunotoxins might be further explored as anti-cancer agents since in some cases they could have an improved therapeutic efficacy over immunotoxins constructed with ricin A-chains.

[The establishment of a magnetic nanoparticle-based immunocapturing method for the detection of abrin].

OBJECTIVE: To develop a high sensitive and specific method for the detection of abrin. METHODS: The abrin monoclonal antibody (mAb) 7D1 coated with Fe3O4 magnetic nanoparticles (MNPs) and abrin mAb labeled with horseradish peroxidase (HRP-mAb) were used to establish the immunocapturing method for abrin detection. The results were compared with the traditional double antibody sandwich ELISA. RESULTS: The detecting linear of immunocapture for abrin was 2.5-60 ng/mL, and the linear regression equation was y=0.012x-0.015 with the detection limit of 2.5 ng/mL. Ricin at different concentrations did not interfere the abrin detection results, which demonstrated that the method had a good specificity . This approach showed good reproducibility with relative standard deviation ranging from 5.18%-8.67%. It could be used for analyzing abrin-contaminated specimens such as water, beverage, and milk, etc. The results of comparison with the conventional double antibody sandwich ELISA indicated that the immunocapture have a broader linear scale, higher sensitivity, and a shorter detection time. CONCLUSION: The developed immunocapturing method can be used for detecting traces of abrin.

A recombinant chimeric protein containing B chains of ricin and abrin is an effective vaccine candidate.

Both ricin toxin (RT) and abrin toxin (AT) are 2 important toxin agents as potantial bioweapons. A dual subunit vaccine against RT and AT exposure is a promising option for developing prophylactic vaccination. In this study, we constructed a dual vaccine with RT B chain and AT B chain named RTB-ATB. The RTB-ATB chimeric protein was expressed in Escherichia coli (E. coli), and the purified protein was used to evaluate the immune response by a 2 × 2 × 2 × 2 factorial design. The main effects included dose of RTB-ATB, route of immunization injection, immunization time interval, and dose of native toxins challenge. For 2 × LD(50) challenge of RT or AT, 100% of the RTB-ATB immunized mice survived and regained or exceeded their initial weights within 10 days. For 4 × LD(50) challenge, different routes of immunization injection caused significant difference (P < 0.05), intraperitoneal (i.p.) administration of immunogen protected mice better than the subcutaneous (s.c.) administration. In conclusion, when administered i.p. to mice with 25 µg per mouse and immunization time interval Π in the absence of adjuvant, the chimeric protein elicited a stronger immune response and protected the animals from a dose of native toxins which was 4 times higher than their LD(50) in unvaccinated mice. Besides, the RTB-ATB chimeric protein could induce specific neutralizing antibodies against these 2 toxins. We anticipate that this study will open new possibilities in the preparation of RTB-ATB dual subunit vaccine against the exposure to deadly RT and AT.