Machine learning powered detection of biological toxins in association with confined lateral flow immunoassay (c-LFA).

Biological weapons, primarily dispersed as aerosols, can spread not only to the targeted area but also to adjacent regions following the movement of air driven by wind. Thus, there is a growing demand for toxin analysis because biological weapons are among the most influential and destructive. Specifically, such a technique should be hand-held, rapid, and easy to use because current methods require more time and well-trained personnel. Our study demonstrates the use of a novel lateral flow immunoassay, which has a confined structure like a double barbell in the detection area (so called c-LFA) for toxin detection such as staphylococcal enterotoxin B (SEB), ricinus communis (Ricin), and botulinum neurotoxin type A (BoNT-A). Additionally, we have explored the integration of machine learning (ML), specifically, a toxin chip boosting (TOCBoost) hybrid algorithm for improved sensitivity and specificity. Consequently, the ML powered c-LFA concurrently categorized three biological toxin types with an average accuracy as high as 95.5%. To our knowledge, the sensor proposed in this study is the first attempt to utilize ML for the assessment of toxins. The advent of the c-LFA orchestrated a paradigm shift by furnishing a versatile and robust platform for the rapid, on-site detection of various toxins, including SEB, Ricin, and BoNT-A. Our platform enables accessible and on-site toxin monitoring for non-experts and can potentially be applied to biosecurity.

New approach for the rational selection of markers to identify botulinum toxins.

The application of mass spectrometry (MS) to detect unique peptide markers has been widely employed as a means of identifying bacterial proteins. Botulinum neurotoxins (BoNTs) are bacterial proteins that cause the life-threatening disease botulism. BoNTs are divided into several antigenically distinct serotypes and several dozen subtypes. The toxins' molecular heterogeneity makes their detection highly challenging. In this study, we describe a new LC-MS/MS-based platform for the direct identification of proteins derived from various species and subspecies in a single assay, as exemplified by BoNTs. The platform employs a rational down-selection process through several steps based on a combination of bioinformatics, tryptic digestion, and LC-MS, each leads to the final panel of markers. This approach has been demonstrated for all 8 subtypes of botulinum serotype A (BoNT/A). Ab-independent and Ab-dependent assays were developed based on the identification of 4 rationally selected markers or a combination of some of them, which enables full selectivity coverage. The Ab-independent assay, which is highly simple and rapid, has a sample-to-result turnaround time of approximately 40 min and enables the identification of 500 MsLD50/mL (5 ng/mL) BoNT/A in complex environmental matrices. The Ab-dependent assay, which is based on toxin's specific enrichment, has a turnaround time of 100 min, but enables improved sensitivity (50 MsLD50/mL, 0.5 ng/mL). Both assays were verified and validated using various environmental samples. This approach can easily be expanded to other botulinum serotypes and exhibits the potential for even further extension as a highly multiplexed assay for protein-based toxins, viruses, and organisms.

In vitro quantification of botulinum neurotoxin type A1 using immobilized nerve cell-mimicking nanoreactors in a microfluidic platform.

The bacterial toxin botulinum neurotoxin A (BoNT/A) is not only an extremely toxic substance but also a potent pharmaceutical compound that is used in a wide spectrum of neurological disorders and cosmetic applications. The quantification of the toxin is extremely challenging due to its extraordinary high physiological potency and is further complicated by the toxin's three key functionalities that are necessary for its activity: receptor binding, internalization-translocation, and catalytic activity. So far, the industrial standard to measure the active toxin has been the mouse bioassay (MBA) that is considered today as outdated due to ethical issues. Therefore, recent introductions of cell-based assays were highly anticipated; their impact however remains limited due to their labor-intensive implementation. This report describes a new in vitro approach that combines a nanosensor based on the use of nerve cell-mimicking nanoreactors (NMN) with microfluidic technology. The nanosensor was able to measure all three key functionalities, and therefore suitable to quantify the amount of physiologically active BoNT/A. The integration of such a sensor in a microfluidic device allowed the detection and quantification of BoNT/A amounts in a much shorter time than the MBA (<10 h vs. 2-4 days). Lastly, the system was also able to reliably quantify physiologically active BoNT/A within a simple final pharmaceutical formulation. This complete in vitro testing system and its unique combination of a highly sensitive nanosensor and microfluidic technology represent a significant ethical advancement over in vivo measures and a possible alternative to cell-based in vitro detection methods.

Optimization of SNAP-25-derived peptide substrate for improved detection of botulinum A in the Endopep-MS assay.

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Endopeptidase-mass-spectrometry (Endopep-MS) is used as a specific and rapid in-vitro assay to detect BoNTs. In this assay, immunocaptured toxin cleaves a serotype-specific-peptide-substrate, and the cleavage products are then detected by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type A (BoNT/A). Our strategy was based on reported BoNT/A-SNAP-25 interactions integrated with analysis method efficiency considerations. Integration of the newly designed substrate led to a 10-fold increase in the assay sensitivity both in buffer and in clinically relevant samples.

Highly Stable Lyophilized Homogeneous Bead-Based Immunoassays for On-Site Detection of Bio Warfare Agents from Complex Matrices.

This study shows the development of dry, highly stable immunoassays for the detection of bio warfare agents in complex matrices. Thermal stability was achieved by the lyophilization of the complete, homogeneous, bead-based immunoassay in a special stabilizing buffer, resulting in a ready-to-use, simple assay, which exhibited long shelf and high-temperature endurance (up to 1 week at 100 °C). The developed methodology was successfully implemented for the preservation of time-resolved fluorescence, Alexa-fluorophores, and horse radish peroxidase-based bead assays, enabling multiplexed detection. The multiplexed assay was successfully implemented for the detection of Bacillus anthracis, botulinum B, and tularemia in complex matrices.

Multiplex Biosensing Based on Highly Sensitive Magnetic Nanolabel Quantification: Rapid Detection of Botulinum Neurotoxins A, B, and E in Liquids.

We present a multiplex quantitative lateral flow (LF) assay for simultaneous on-site detection of botulinum neurotoxin (BoNT) types A, B, and E in complex matrixes, which is innovative by virtually no sacrifice in performance while transition from the single-plex assays and by characteristics on the level of laboratory quantitative methods. The novel approach to easy multiplexing is realized via joining an on-demand set of single-plex LF strips, which employ magnetic nanolabels, into a miniature cylinder cartridge that mimics LF strip during all assay stages. The cartridge is read out by an original portable multichannel reader based on the magnetic particle quantification technique. The developed reader offers the unmatched 60 zmol detection limit and 7-order linear dynamic range for volumetric registration of magnetic labels inside a cartridge of several millimeters in diameter regardless of its optical transparency. Each of the test strips, developed here as building blocks for the multiplex assay, can be used "as is" for autonomous quantitative single-plex detection with the same measuring setup, exhibiting the limits of detection (LOD) of 0.22, 0.11, and 0.32 ng/mL for BoNT-A, -B, and -E, respectively. The proposed multiplex assay has demonstrated the remarkably similar LOD values of 0.20, 0.12, 0.35 ng/mL under the same conditions. The multiplex assay performance was successfully validated by BoNT detection in milk and apple and orange juices. The developed methods can be extended to other proteins and used for rapid multianalyte tests for point-of-care in vitro diagnostics, food analysis, biosafety and environmental monitoring, forensics, and security, etc.

A chip-based assay for botulinum neurotoxin A activity in pharmaceutical preparations.

The production of botulinum neurotoxin A (BoNT/A) for therapeutic and cosmetic applications requires precise determination of batch potency, and the enzymatic activity of BoNT/A light chain is a crucial index that can be measured in vitro. We previously established a SNAP-25 chip-based assay using surface plasmon resonance (SPR) that is more sensitive than the standard mouse bioassay for the quantification of BoNT/A activity. We have now adapted this procedure for pharmaceutical preparations. The optimized SPR assay allowed multiple measurements on a single chip, including the kinetics of substrate cleavage. The activity of five different batches of a pharmaceutical BoNT/A preparation was determined in a blind study by SPR and found to be in agreement with data from the in vivo mouse lethality assay. Biosensor detection of specific proteolytic products has the potential to accurately monitor the activity of pharmaceutical BoNT/A preparations, and a single chip can be used to assay more than 100 samples.

Infant botulism with prolonged faecal excretion of botulinum neurotoxin and Clostridium botulinum for 7 months.

In Finland in April 2010, a 3-month old baby was diagnosed with type A infant botulism. He excreted botulinum neurotoxin and/or Clostridium botulinum in his faeces until November 2010. Five months of excretion was after clinical recovery and discharge from hospital. C. botulinum isolates recovered from the household dust in the patient's home were genetically identical to those found in the infant's stool samples. Long-term faecal excretion of C. botulinum may pose a possible health risk for the parents and others in close contact with the infant.

An electrochemiluminescence assay for the detection of bio threat agents in selected food matrices and in the screening of Clostridium botulinum outbreak strains associated with type A botulism.

BACKGROUND: Specific screening methods for complex food matrices are needed that enable unambiguous and sensitive detection of bio threat agents (BTAs) such as Bacillus anthracis spores and microbial toxins (e.g. staphylococcal enterotoxin B (SEB) and clostridial botulinum neurotoxins (BoNTs)). The present study describes an image-based 96-well Meso Scale Discovery (MSD) electrochemiluminescence (ECL) assay for simultaneous detection of BTAs in dairy milk products. RESULTS: The limit of detection of this ECL assay is 40 pg mL⁻¹ for BoNT/A complex, 10 pg mL⁻¹ for SEB and 40000 CFU mL⁻¹ for Bacillus anthracis spores in dairy milk products. The ECL assay was successfully applied to screen type A Clostridium botulinum outbreak strains. CONCLUSION: The results of the study indicate that this ECL assay is very sensitive, rapid (<6 h) and multiplex in nature. The ECL assay has potential for use as an in vitro screening method for BTAs over other comparable immunoassays.

Improved detection of botulinum neurotoxin serotype A by Endopep-MS through peptide substrate modification.

Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to humans. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype-specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep-MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype-specific antibodies and detecting the unique and serotype-specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep-MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity 5-fold with toxin spiked into buffer solution or different biological matrices.

Bioluminescence assay for the highly sensitive detection of botulinum neurotoxin A activity.

This article describes a novel bioluminescence assay for detecting the proteolytic activity of Botulinum NeuroToxins (BoNT) in complex matrices. The assay is capable of detecting traces of BoNT in blood samples as well as in food drinks. The assay was responsive to BoNT/A subtypes 1 to 5, and serotype E3 in buffered solutions. It was responsive to filtered Clostridium botulinum supernatants and BoNT/A1 in complex with neurotoxin associated proteins in bouillon and milk (3.8% fat) down to 400 fM after 4 h RT incubation and in bouillon at concentrations down to 120 fM after 21 h RT incubation. In combination with an immunocapture/enrichment step it could detect BoNT/A1 in citrated plasma at concentrations down to 30 fM (1.2 mouse LD50 per mL). The simplicity of the assay, combined with a demonstrated ability to lyophilize the reagents, demonstrates its usefulness for detection of BoNT in non-specialised analytical laboratories.

Development of xMAP assay for detection of six protein toxins.

xMAP technology was used for simultaneous identification of six protein toxins (staphylococcal enterotoxins A and B, cholera toxin, ricin, botulinum toxin A, and heat labile toxin of E. coli). Monoclonal antibody-conjugated xMAP microspheres and biotinilated monoclonal antibodies were used to detect the toxins in a sandwich immunoassay format. The detection limits were found to be 0.01 ng/mL for staphylococcal enterotoxin A, cholera toxin, botulinum toxin A, and ricin in model buffer (PBS-BSA) and 0.1 ng/mL for staphylococcal enterotoxin B and LT. In a complex matrix, such as cow milk, the limits of detection for staphylococcal enterotoxins A and B, cholera toxin, botulinum toxin A, and ricin increased 2- to 5-fold, while for LT the detection limit increased 30-fold in comparison with the same analysis in PBS-BSA. In the both PBS-BSA and milk samples, the xMAP test system was 3-200 times (depending on the toxin) more sensitive than ELISA systems with the same pairs of monoclonal antibodies used. The time required for a simultaneous analysis of six toxins using the xMAP system did not exceed the time required for ELISA to analyze one toxin. In the future, the assay may be used in clinical diagnostics and for food and environmental monitoring.

Development of a fluorescence internal quenching correction factor to correct botulinum neurotoxin type A endopeptidase kinetics using SNAPtide.

Botulinum neurotoxins (BoNTs), which are highly toxic proteins responsible for botulism, are produced by different strains of Clostridium botulinum. These various strains of bacteria produce seven distinct serotypes, labeled A-G. Once inside cells, the zinc-dependent proteolytic light chain (LC) degrades specific proteins involved in acetylcholine release at neuromuscular junctions causing flaccid paralysis, specifically synaptosomal-associated protein 25 (SNAP-25) for botulinum neurotoxin type A (BoNT/A). BoNT endopeptidase assays using short substrate homologues have been widely used and developed because of their ease of synthesis, detection limits, and cost. SNAPtide, a 13-amino acid fluorescence resonance energy transfer (FRET) peptide, was used in this study as a SNAP-25 homologue for the endopeptidase kinetics study of BoNT/A LC. SNAPtide uses a fluorescein isothiocyanate/4-((4-(dimethylamino)phenyl)azo) benzoic acid (FITC/DABCYL) FRET pair to produce a signal upon substrate cleavage. Signal quenching can become an issue after cleavage since quencher molecules can quench cleaved fluorophore molecules in close proximity, reducing the apparent signal. This reduction in apparent signal provides an inherent error as SNAPtide concentrations are increased. In this study, fluorescence internal quenching (FIQ) correction factors were derived using an unquenched SNAPtide peptide to quantify the signal quenching over a range of SNAPtide concentrations and temperatures. The BoNT/A LC endopeptidase kinetics at the optimally active temperature (37 °C) using SNAPtide were studied and used to demonstrate the FIQ correction factors in this study. The FIQ correction factors developed provide a convenient method to allow for improved accuracy in determining and comparing BoNT/A LC activity and kinetics using SNAPtide over a broad range of concentrations and temperatures.

Detection of botulinum neurotoxin serotype A, B, and F proteolytic activity in complex matrices with picomolar to femtomolar sensitivity.

Rapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and precision. In this study, we present three biochemical assays for the detection and quantification of BoNT serotype A, B, and F proteolytic activities in complex matrices that offer picomolar to femtomolar sensitivity with small assay volumes and total assay times of less than 24 h. These assays consist of magnetic beads conjugated with BoNT serotype-specific antibodies that are used to purify BoNT from complex matrices before the quantification of bound BoNT proteolytic activity using the previously described BoTest reporter substrates. The matrices tested include human serum, whole milk, carrot juice, and baby food, as well as buffers containing common pharmaceutical excipients. The limits of detection were below 1 pM for BoNT/A and BoNT/F and below 10 pM for BoNT/B in most tested matrices using 200-µl samples and as low as 10 fM for BoNT/A with an increased sample volume. Together, these data describe rapid, robust, and high-throughput assays for BoNT detection that are compatible with a wide range of matrices.

A functional dual-coated (FDC) microtiter plate method to replace the botulinum toxin LD50 test.

Conventional capture ("Sandwich") ELISAs equally detect denatured inactive and native active botulinum type A toxin. Light chain endoprotease activity assays also fail to distinguish between various inactive molecules including partially denatured and fragmented material still retaining this protease activity. By co-coating microtiter plates with SNAP25 substrate and a monoclonal antibody specific for a conformational epitope of the toxin's Hc domain, it was possible to develop a highly sensitive (130 aM LoD), precise (1.4% GCV) new assay specific for the biologically active toxin molecule. Capture was performed in phosphate buffer with a fixed optimal concentration of chaotropic agent (e.g., 1.2 M urea) to differentially isolate functional toxin molecules. Addition of enzymatically favorable buffer containing zinc and DTT reduced the interchain disulfide bond releasing and activating the captured L-chain with subsequent specific cleavage of the SNAP25(1-206) substrate. A neoepitope antibody specific for the newly exposed Q(197) epitope was used to quantify the cleaved SNAP25(1-197). The assay's requirement for the intact toxin molecule was demonstrated with pre-reduced toxin (heavy and light chains), recombinant LHn fragments, and stressed samples containing partially or fully denatured material. This is the first known immunobiochemical assay that correlates with in vivo potency and provides a realistic alternative.

[Development and optimization of immunoassays for the detection of botulinum toxins].

Monoparametric immunoassay tests for detecting botulinum toxins types A and B and multiparametric assays for simultaneous detection of botulinum toxins type A and B have been developed. It is shown that the sensitivity of assays is affected by the size of nanoparticles of colloidal gold used as a marker of antibodies, load intensity of antibodies of colloidal gold in conjugates, the type of analytical membranes, as well as the chemical composition of buffer solutions used for the storage of conjugates and immunoassay analysis. The detection limit of monoparametric immunoassay tests is 0.5 ng/ml; that of multiparametric assays, 5.0 ng/ml. The developed immunoassay can be used for rapid assay of product quality, for grade control of botulinum toxins in pharmaceuticals, and environmental monitoring.

Sensitive and quantitative detection of botulinum neurotoxin in neurons derived from mouse embryonic stem cells.

Botulinum neurotoxins (BoNTs), the most poisonous protein toxins known, represent a serious bioterrorism threat but are also used as a unique and important bio-pharmaceutical to treat an increasing myriad of neurological disorders. The only currently accepted detection method by the United States Food and Drug Administration for biological activity of BoNTs and for potency determination of pharmaceutical preparations is the mouse bioassay (MBA). Recent advances have indicated that cell-based assays using primary neuronal cells can provide an equally sensitive and robust detection platform as the MBA to reliably and quantitatively detect biologically active BoNTs. This study reports for the first time a BoNT detection assay using mouse embryonic stem cells to produce a neuronal cell culture. The data presented indicate that this assay can reliably detect BoNT/A with a similar sensitivity as the MBA.

Improved detection of botulinum neurotoxin type A in stool by mass spectrometry.

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humankind. Rapid and sensitive detection of BoNTs is necessary for timely clinical confirmation of the disease state in botulism. BoNTs cleave proteins and peptide mimics at specific sites. A mass spectrometry (MS)-based method, Endopep-MS, can detect these cleavages and has detection limits of 0.05-0.5 mouse LD(50) (U) in serum, depending on the BoNT serotypes. In this method, the products generated from cleavage of peptide substrates using antibody affinity-purified toxins are detected by MS. Nonspecific bound endogenous proteases or peptidases in stool can coextract with the toxin, cleaving the peptide substrates and reducing the sensitivity of the method. Here we report a method to reduce nonspecific substrate cleavage by reducing stool protease coextraction in the Endopep-MS assay.

[Monoclonal antibodies to type A, B, E and F botulinum neurotoxins].

Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.

Multilocus outbreak of foodborne botulism linked to contaminated sausage in Hebei province, China.

In 2007, an outbreak of foodborne botulism occurred in Hebei province, China. An epidemiological investigation and laboratory detection studies showed that sausage contaminated by type A Clostridium botulinum caused this outbreak of food poisoning. Its clinical and epidemiological features were different from previous reports of food poisoning.