Multiplex quantification of protein toxins in human biofluids and food matrices using immunoextraction and high-resolution targeted mass spectrometry.

The development of rapid methods for unambiguous identification and precise quantification of protein toxins in various matrices is essential for public health surveillance. Nowadays, analytical strategies classically rely on sensitive immunological assays, but mass spectrometry constitutes an attractive complementary approach thanks to direct measurement and protein characterization ability. We developed here an innovative multiplex immuno-LC-MS/MS method for the simultaneous and specific quantification of the three potential biological warfare agents, ricin, staphylococcal enterotoxin B, and epsilon toxin, in complex human biofluids and food matrices. At least 7 peptides were targeted for each toxin (43 peptides in total) with a quadrupole-Orbitrap high-resolution instrument for exquisite detection specificity. Quantification was performed using stable isotope-labeled toxin standards spiked early in the sample. Lower limits of quantification were determined at or close to 1 ng·mL(-1). The whole process was successfully applied to the quantitative analysis of toxins in complex samples such as milk, human urine, and plasma. Finally, we report new data on toxin stability with no evidence of toxin degradation in milk in a 48 h time frame, allowing relevant quantitative toxin analysis for samples collected in this time range.

Fast and direct extraction of cell-associated hepatotoxins from toxic cyanobacteria.

Microcystins are an important group of toxins produced by cyanobacteria of different genera. An increasing number of water contaminations by this class of toxins have been reported that are susceptible to generate important public health problems. We designed an efficient method for extracting these toxins on-site for a rapid testing of potentially contaminated water. The extraction parameters have been optimized using Microcystis aeruginosa and the technique successfully applied to different laboratory cultures and field samples. The procedure employs a simple and stable reagent mix of propanol, Tween 20, and trifluoroacetic acid. It is directly active on crude cell suspensions without any pre-treatment. Extraction yields measured by immunological quantification were at least equal to the best values obtained with the most commonly used laboratory techniques. An additional simple concentration/extraction step is also described that allows measurements on samples too dilute for direct detection by immunochromatographic techniques.

A Monoclonal Antibody with a High Affinity for Ricin Isoforms D and E Provides Strong Protection against Ricin Poisoning.

Ricin is a highly potent toxin that has been used in various attempts at bioterrorism worldwide. Although a vaccine for preventing ricin poisoning (RiVax™) is in clinical development, there are currently no commercially available prophylaxis or treatments for ricin intoxication. Numerous studies have highlighted the potential of passive immunotherapy using anti-ricin monoclonal antibodies (mAbs) and have shown promising results in preclinical models. In this article, we describe the neutralizing and protective efficacy of a new generation of high-affinity anti-ricin mAbs, which bind and neutralize very efficiently both ricin isoforms D and E in vitro through cytotoxicity cell assays. In vivo, protection assay revealed that one of these mAbs (RicE5) conferred over 90% survival in a murine model challenged intranasally with a 5 LD50 of ricin and treated by intravenous administration of the mAbs 6 h post-intoxication. Notably, a 35% survival rate was observed even when treatment was administered 24 h post-exposure. Moreover, all surviving mice exhibited long-term immunity to high ricin doses. These findings offer promising results for the clinical development of a therapeutic candidate against ricin intoxication and may also pave the way for novel vaccination strategies against ricin or other toxins.

Fast and sensitive detection of Bacillus anthracis spores by immunoassay.

Bacillus anthracis is one of the most dangerous potential biological weapons, and it is essential to develop a rapid and simple method to detect B. anthracis spores in environmental samples. The immunoassay is a rapid and easy-to-use method for the detection of B. anthracis by means of antibodies directed against surface spore antigens. With this objective in view, we have produced a panel of monoclonal antibodies against B. anthracis and developed colorimetric and electrochemiluminescence (ECL) immunoassays. Using Meso Scale Discovery ECL technology, which is based on electrochemiluminescence (ECL) detection utilizing a sulfo-Tag label that emits light upon electrochemical stimulation (using a dedicated ECL plate reader, an electrical current is placed across the microplate with electrodes integrated into the bottom of the plate, resulting in a series of electrically induced reactions leading to a luminescent signal), a detection limit ranging between 0.3 × 10(3) and 10(3) CFU/ml (i.e., 30 to 100 spores per test), depending on the B. anthracis strain assayed, was achieved. In complex matrices (5 mg/ml of soil or simulated powder), the detection level (without any sample purification or concentration) was never altered more than 3-fold compared with the results obtained in phosphate-buffered saline.

NeuroTorp, a lateral flow test based on toxin-receptor affinity for in-situ early detection of cyclic imine toxins.

The emergent cyclic imine toxins produced by marine dinoflagellates are potent antagonists of nicotinic acetylcholine receptors. Shellfish accumulate cyclic imine toxins following filter-feeding on toxic dinoflagellates vectoring them to humans. Herein is presented a lateral flow test for the detection of cyclic imine toxins based on three new concepts for test strips: i) the immobilization of lipoprotein vesicles in the test-line, ii) the high affinity of neurotoxins for their receptor targets and iii) the use of high porosity glass fiber filter membranes as support for the fabrication of the lateral flow test NeuroTorp (WO2017108115). Purified electrocyte membrane vesicles from Torpedo marmorata were used as a source of receptor and were immobilized in the test-line. Biotin-α-bungarotoxin was used as toxin tracer for the NeuroTorp LFT given its high affinity for nicotinic acetylcholine receptors while neutravidin nanogold particle conjugates enable its visual detection. Herein is reported for the first time the use of GF/C glass fiber membranes as the stationary phase for a lateral flow test. The GF/C filter ensures both: the immobilization of a complex lipoprotein in the test-line and the capillary migration of the mobile phase. Scanning electron microscopy studies shed light into the mechanism by which Torpedo-electrocyte membranes vesicles are immobilized in the GF/C glass microfiber. The electrocyte membrane vesicles anchor in neighboring microfibers randomly disposed in the same plane of the GF/C filter forming stable microfilm structures ensuring the functionality of nicotinic acetylcholine receptors. NeuroTorp is a ready-to-use low-cost early warning device for rapid detection of cyclic imine toxins in shellfish by end-users.

Ricin Antibodies' Neutralizing Capacity against Different Ricin Isoforms and Cultivars.

Ricin, a highly toxic protein from Ricinus communis, is considered a potential biowarfare agent. Despite the many data available, no specific treatment has yet been approved. Due to their ability to provide immediate protection, antibodies (Abs) are an approach of choice. However, their high specificity might compromise their capacity to protect against the different ricin isoforms (D and E) found in the different cultivars. In previous work, we have shown the neutralizing potential of different Abs (43RCA-G1 (anti ricin A-chain) and RB34 and RB37 (anti ricin B-chain)) against ricin D. In this study, we evaluated their protective capacity against both ricin isoforms. We show that: (i) RB34 and RB37 recognize exclusively ricin D, whereas 43RCA-G1 recognizes both isoforms, (ii) their neutralizing capacity in vitro varies depending on the cultivar, and (iii) there is a synergistic effect when combining RB34 and 43RCA-G1. This effect is also demonstrated in vivo in a mouse model of intranasal intoxication with ricin D/E (1:1), where approximately 60% and 40% of mice treated 0 and 6 h after intoxication, respectively, are protected. Our results highlight the importance of evaluating the effectiveness of the Abs against different ricin isoforms to identify the treatment with the broadest spectrum neutralizing effect.

Role of T3SS-1 SipD Protein in Protecting Mice against Non-typhoidal Salmonella Typhimurium.

BACKGROUND: Salmonella enterica species are enteric pathogens that cause severe diseases ranging from self-limiting gastroenteritis to enteric fever and sepsis in humans. These infectious diseases are still the major cause of morbidity and mortality in low-income countries, especially in children younger than 5 years and immunocompromised adults. Vaccines targeting typhoidal diseases are already marketed, but none protect against non-typhoidal Salmonella. The existence of multiple non-typhoidal Salmonella serotypes as well as emerging antibiotic resistance highlight the need for development of a broad-spectrum protective vaccine. All Salmonella spp. utilize two type III Secretion Systems (T3SS 1 and 2) to initiate infection, allow replication in phagocytic cells and induce systemic disease. T3SS-1, which is essential to invade epithelial cells and cross the barrier, forms an extracellular needle and syringe necessary to inject effector proteins into the host cell. PrgI and SipD form, respectively, the T3SS-1 needle and the tip complex at the top of the needle. Because they are common and highly conserved in all virulent Salmonella spp., they might be ideal candidate antigens for a subunit-based, broad-spectrum vaccine. PRINCIPAL FINDINGS: We investigated the immunogenicity and protective efficacy of PrgI and SipD administered by subcutaneous, intranasal and oral routes, alone or combined, in a mouse model of Salmonella intestinal challenge. Robust IgG (in all immunization routes) and IgA (in intranasal and oral immunization routes) antibody responses were induced against both proteins, particularly SipD. Mice orally immunized with SipD alone or SipD combined with PrgI were protected against lethal intestinal challenge with Salmonella Typhimurium (100 Lethal Dose 50%) depending on antigen, route and adjuvant. CONCLUSIONS AND SIGNIFICANCE: Salmonella T3SS SipD is a promising antigen for the development of a protective Salmonella vaccine, and could be developed for vaccination in tropical endemic areas to control infant mortality.

Recommended Immunological Assays to Screen for Ricin-Containing Samples.

Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC) as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories' capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120). Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests). Using these immunological methods "dangerous" samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin.

Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples.

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A-G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as "category A" bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.

A one-step and biocompatible cellulose functionalization for covalent antibody immobilization on immunoassay membranes.

Among bioactive papers, many multiplexed assays implement methods incompatible with the conventional lateral flow immunoassay (LFIA) carrier material, nitrocellulose. Consequently, its replacement by cellulose has to be considered. This technological breakthrough requires a surface chemistry which ensures both the biomolecules covalent grafting to cellulose and the conservation of their biological activity. To comply with these requirements, the process elaborated in this study implements compounds and methods compatible with biological material. While cellulose chemical modification is usually operated under harsh conditions in organic solvents, the diazonium-based functionalization procedure presented here was performed onto cellulose sheets in water and at room temperature. Paper sheets have been successfully modified and bear different chemical functions which enable grafting of biomolecules by common bioconjugate techniques and to perform LFIAs. More generally, the chemical ways developed in this study are suitable for many biomolecules and would be helpful for any sensitive molecule immobilization onto cellulose sheets.

Characterization of botulinum neurotoxin type A neutralizing monoclonal antibodies and influence of their half-lives on therapeutic activity.

Botulinum toxins, i.e. BoNT/A to/G, include the most toxic substances known. Since botulism is a potentially fatal neuroparalytic disease with possible use as a biowarfare weapon (Centers for Disease Control and Prevention category A bioterrorism agent), intensive efforts are being made to develop vaccines or neutralizing antibodies. The use of active fragments from non-human immunoglobulins (F(ab')(2), Fab', scFv), chemically modified or not, may avoid side effects, but also largely modify the in vivo half-life and effectiveness of these reagents. We evaluated the neutralizing activity of several monoclonal anti-BoNT/A antibodies (mAbs). F(ab')(2) fragments, native or treated with polyethyleneglycol (PEG), were prepared from selected mAbs to determine their half-life and neutralizing activity as compared with the initial mAbs. We compared the protective efficiency of the different biochemical forms of anti-toxin mAbs providing the same neutralizing activity. Among fourteen tested mAbs, twelve exhibited neutralizing activity. Fragments from two of the best mAbs (TA12 and TA17), recognizing different epitopes, were produced. These two mAbs neutralized the A1 subtype of the toxin more efficiently than the A2 or A3 subtypes. Since mAb TA12 and its fragments both exhibited the greatest neutralizing activity, they were further evaluated in the therapeutic experiments. These showed that, in a mouse model, a 2- to 4-h interval between toxin and antitoxin injection allows the treatment to remain effective, but also suggested an absence of correlation between the half-life of the antitoxins and the length of time before treatment after botulinum toxin A contamination. These experiments demonstrate that PEG treatment has a strong impact on the half-life of the fragments, without affecting the effectiveness of neutralization, which was maintained after preparation of the fragments. These reagents may be useful for rapid treatment after botulinum toxin A contamination.