Detection Of Botulinum Neurotoxin Type Research Articles

ELISA Based Detection of Botulinum Neurotoxin Type F in Red Meat and Canned Fish

Botulinum neurotoxin (BoNT) is the most toxic protein molecule so far known to animals and humans, due to its extreme toxicity, CDC has been listed as category ‘A' Biological warfare agents. In India, there is no commercial detection system available for the detection of botulism. The present study was aimed to develop an ELISA based detection system for botulinum neurotoxin serotype F in red meat and canned fish. Polyclonal antibodies were generated against the recombinant BoNT/F LC protein and IgG was purified. Sandwich ELISA system was successfully optimized with LOD of ~ 7.8 ng/ml in PBS. Similarly, different concentrations of BoNT/F were spiked in red meat and canned fish and extracted the toxin and the detection LOD was achieved for red meat ~ 62 ng/ml and for canned fish ~ 31 ng/ml. The developed detection system is highly specific. The developed assay will be useful for the screening of botulinum toxins in a large number of food samples.

A sensitive thrombin-linked sandwich immunoassay for protein targets using high affinity phosphorodithioate modified aptamer for thrombin labeling

Thrombin and its aptamers have been well studied and widely used as models in aptamer based assays and sensors. Here we reported a thrombin-linked sandwich immunoassay for proteins to demonstrate new applications of thrombin and the aptamers, converting protein detection to analysis of thrombin label. In this assay, target protein was sandwiched by the capture antibody on a microplate and the biotinylated detection antibody. Thrombin bound to one biotinylated aptamer, and then the thrombin-labeled aptamer was attached on the sandwich complex through streptavidin-biotin interaction by using streptavidin as a linker. Thrombin catalyzed cleavage of fluorogenic peptide substrates, generating fluorescence signals for target detection. Among a few different anti-thrombin aptamers, the use of one nuclease resistant RNA aptamer having phosphorodithioate (PS2) modification on a specific backbone position enabled higher assay sensitivity due to its much higher affinity. This thrombin-linked sandwich immunoassay allowed detection of prostate-specific antigen (PSA) at 2 pM, an important protein related cancer disease, with high sensitivity and specificity. The strategy was general, and also enabled sensitive detection of botulinum neurotoxin type A (BoNTA) light chain, one toxin protein causing risk to human health. This assay combines advantages of antibody recognition, aptamer affinity labeling, high affinity of aptamers, and enzyme activity of thrombin. Labeling thrombin on the immunosandwich complex through simple affinity binding overcomes limitations of covalent conjugating enzyme on antibody in conventional immunoassay. This assay is promising in applications for protein detection.

A monoclonal enzyme-linked immunoassay for the detection of botulinum neurotoxin type E

Background and Aim: Botulinum neurotoxin Type E (BoNT/E), one of the most lethal toxin known, is the common contamination in fishery products or fish consumption that causes foodborne botulism. It is necessary to establish a sensitive and specific method for the detection of BoNT/E because of its extremely low lethal dose. Methods: In this study, a practical enzyme-linked immunosorbent assay for BoNT/E detection was developed. The assay is based on the sandwich format using monoclonal antibodies of two distinct specificities. An affinity-purified anti-BoNT/E light chain Mab (1E1) is utilized to adsorb BoNT/E from solution, and the second anti-BoNT/E heavy chain C terminus Mab (5E1) conjugated with peroxidase is then used to form sandwich complexes, and peroxidase allows color development and measurement of optical density at 450 nm. Results: Standard curves were linear over the range of 5–100 ng/ml BoNT/E. The limit of detection was below 10 ng/ml in phosphate-buffered saline buffer. The developed BoNT/E assay also showed no cross-reaction to Type A neurotoxin (BoNT/A) and Type B neurotoxin (BoNT/B). Conclusion: Herein, a sensitive and accurate ELISA for BoNT/E detection was presented. It has the potential to utilize in vivo BoNT/E analysis and contamination monitoring.

An enzymatic reporter cell line for detection of botulinum neurotoxin type B

An enzymatic reporter cell line for detection of botulinum neurotoxin type B

Commentary: A Cell Line for Detection of Botulinum Neurotoxin Type B.

Commentary: A Cell Line for Detection of Botulinum Neurotoxin Type B.

A Cell Line for Detection of Botulinum Neurotoxin Type B.

Botulinum neurotoxins (BoNTs) type A and type B are commonly used as biopharmaceutics for neurological diseases, uniquely allowing months-long paralysis of target muscles. Their exquisite neuronal specificity is conferred by a multistep process of binding, internalization, cytosolic escape and cleavage of the neuron-specific proteins, SNAP-25 and vesicle-associated membrane proteins (VAMPs), ultimately to inhibit secretion of neurotransmitters. Currently the mouse lethality bioassay is the only available method for quality control testing of VAMP-cleaving botulinum products. Refined assays for botulinum product testing are urgently needed. Specifically, in vitro replacement assays which can account for all steps of BoNT intoxication are in high demand. Here, we describe a novel SiMa cell-based approach where re-engineering of the VAMP molecule allows detection of all BoNT/B intoxication steps using a luminescent enzymatic reaction with sensitivity comparable to mouse LD50 bioassay. The presented one-step enzyme-linked immunosorbent assay meets 3Rs (replacement, reduction, and refinement of the use of animals) objectives, is user-friendly and will accelerate development of new botulinum drugs. The sensitive enzymatic reporter cell line could also be adapted for the detection of toxin activity during the manufacture of botulinum and tetanus vaccines.

Label-free electrochemical detection of botulinum neurotoxin type E based on its enzymatic activity using interdigitated electrodes

We report a simple label-free electrochemical method of detecting low concentrations of botulinum neurotoxin type E light chain (BoNT/E LC) based on its peptide cleavage activity. Dual-mode cyclic voltammetry was employed to observe changes in the redox signal of ferri-/ferro-cyanide on interdigitated microelectrodes, whose surfaces were covered by peptides designed from synaptosomal-associated protein 25 to be cleaved by BoNT/E LC. With the introduction of BoNT/E LC, the redox signal showed a time-dependent increase due to cleavage of the immobilized peptide molecules. In addition to the increased redox signal intensity, its time-dependence can be considered as a strong evidence of BoNT/E sensing, since the time-dependent increase can only result from the enzymatic activity of BoNT/E LC. Using this method, BoNT/E LC, at concentrations as low as 5 pg/ml, was readily measurable with only an hour of incubation.

Improved detection of botulinum type E by rational design of a new peptide substrate for endopeptidase–mass spectrometry assay

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humans. 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. To further improve the sensitivity of the assay, we report here the rational design of a new substrate peptide for the detection of botulinum neurotoxin type E (BoNT/E). Our strategy was based on previously reported structural interactions integrated with analysis method efficiency considerations. Integration of the newly designed substrate has led to a more than one order of magnitude increased sensitivity of the assay.

A Bioanalytical Platform for Simultaneous Detection and Quantification of Biological Toxins

Prevalent incidents support the notion that toxins, produced by bacteria, fungi, plants or animals are increasingly responsible for food poisoning or intoxication. Owing to their high toxicity some toxins are also regarded as potential biological warfare agents. Accordingly, control, detection and neutralization of toxic substances are a considerable economic burden to food safety, health care and military biodefense. The present contribution describes a new versatile instrument and related procedures for array-based simultaneous detection of bacterial and plant toxins using a bioanalytical platform which combines the specificity of covalently immobilized capture probes with a dedicated instrumentation and immuno-based microarray analytics. The bioanalytical platform consists of a microstructured polymer slide serving both as support of printed arrays and as incubation chamber. The platform further includes an easy-to-operate instrument for simultaneous slide processing at selectable assay temperature. Cy5 coupled streptavidin is used as unifying fluorescent tracer. Fluorescence image analysis and signal quantitation allow determination of the toxin’s identity and concentration. The system’s performance has been investigated by immunological detection of Botulinum Neurotoxin type A (BoNT/A), Staphylococcal enterotoxin B (SEB), and the plant toxin ricin. Toxins were detectable at levels as low as 0.5–1 ng·mL−1 in buffer or in raw milk.

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.

Detection of Botulinum Neurotoxin Type A by In Vitro Bioassay Based on Endopeptidase Activity

Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that cleaves SNAP-25 (synaptosome-associated protein of 25 kDa), a specific cellular protein essential for neurotransmitter release. As well as mouse bioassay to detect BoNT/A, various assay methods based on its endopeptidase activity have been developed. In this study, we tried to develop a BoNT/A assay system using recombinant SNAP-25 with glutathione S-transferase (GST) tags at both termini as substrate. The recombinant GST-SNAP-25-GST with 70 kDa was expressed and purified in E. coli and synthesized N-terminal 50 kDa and C-terminal 25 kDa fragment after cleavage at the Gln197-Arg198 bond by BoNT/A. To detect both fragments, we obtained rabbit antisera against peptides corresponding to the cleaved ends of each fragment. In the western blotting, the N-terminal fragment was detected by the antibody specifically recognizing the newly exposed C-terminus (corresponding to amino acid residue 191-197). This assay system was able to detect until 3.125 ng of BoNT/A, which corresponded to about 90 fold LD50 in mice. These results suggest that the in vitro endopeptidase assay developed in this study would replace others to detect BoNT/A.

Micellar electrokinetic chromatography and laser induced fluorescence detection of botulinum neurotoxin type A activity using a dual-labelled substrate

The activity of Botulinum neurotoxin type A (BoNT A) can be measured by monitoring the toxin's endopeptidase reaction with its peptide substrate. In this report, a sensitive and simple capillary electrophoresis (CE) method for analysing BoNT A activity was developed using a peptide substrate labelled with Fluorescein isothiocynate (FITC) at the N-terminal and biotin at the C-terminal. This dual labelling enables not only highly sensitive laser induced fluorescence (LIF) detection of the reaction product, but also good analytical separation of the product from the peptide substrate by Micellar Electrokinetic Chromatography (MEKC). The separation between the product peak and the substrate peak was approximately 5 min using the dual-labelled substrate, while just about 1 min using the FITC-labelled substrate without biotinylation. Using the current assay method, BoNT A with concentration as low as 0.1 ng ml−1 (3.6 U mL−1 in mouse LD50) in water was detected with a S:N ratio of 3 (RSD <19%) and a linear range of four orders of magnitude. With CE's advantages of very small sample volume needed, this method may find particular applications as in assays of BoNT A activity in water samples and kinetic analyses of toxin activity.

Development of Capture ELISA Using a Biotinylated Monoclonal Antibody for Detection of Botulinum Neurotoxin Type A

A capture enzyme-linked immunosorbent assay (capture ELISA) was developed to detect Clostridium botulinum neurotoxin type A (BoNT/A) in assay buffer and human serum. The assay is based upon affinity-purified rabbit polyclonal and biotinylated monoclonal antibodies directed against the BoNT/A complex purified from C. botulinum ATCC19397. For the capture ELISA, the optimized amount (2 μg/ml) of rabbit polyclonal antibody was immobilized on ELISA plates to detect BoNT/A (ranging from 0 to 500 ng/ml), which was recognized by 2 μg/ml of the monoclonal antibody. From three independent repeated experiments, standard curves were linear over the range of 0~31.25 ng/ml BoNT/A and the coefficients (r2) ranged from 0.9951~0.9999 for all assays. The inter-variations were typically 0.50~6.93% and the specificity was confirmed by showing no cross-reactivity against BoNT/B and /E. The detection limit of capture ELISA was 0.488 ng/ml, which was close to mouse LD50. In addition, application with BoNT/A-spiking human sera showed a possibility to detect BoNT/A with capture ELISA from the contaminated human sera. Taken together, the newly developed capture ELISA could serve as a rapid and sensitive screening tool for detecting BoNT/A simultaneously from massive specimens.

Substrate-Modified Hydrogels for Autonomous Sensing of Botulinum Neurotoxin Type A

We report the development of substrate-cross-linked hydrogels for the specific detection of botulinum neurotoxin type A (BoNT/A), the most poisonous toxin known. Peptide-cross-linked BoNT/A-sensitive hydrogels were photopolymerized within microfluidic channels to create autonomous biosensors capable of sensing toxin enzymatic activity with a visual readout.

Development and comparison of two immunoassay formats for rapid detection of botulinum neurotoxin type A

We have evaluated two formats of immunoassays for the rapid detection of Clostridium botulinum neurotoxin type A (BoNT/A), in assay buffer and various matrices (human serum and nasal swabs, fresh milk, sugar, flour and talcum). The two formats, a vertical-flow strip immunochromatography (ICT) and a small disposable immunoaffinity column (IAC), were selected because they are both rapid and readily usable in the field without sophisticated equipment. We utilised the same critical reagents to develop and optimise both assays, making it possible to compare the corresponding technologies on the same toxin preparations, without interference due to the properties of the antibodies. Results were interpreted using a standard statistical test (ANOVA) and showed little difference of sensitivity between matrices. Though both assays were completed in 40 min, the sensitivity of the IAC, evaluated at 0.45 pM (5 mouse LD50 units/ml), was 40 to 80 times better than that of the ICT. Furthermore, the sensitivity of the IAC assay was improved to 0.09 pM (1 mouse LD50 unit/ml) when performed on a 5-ml volume of human serum. Thus, the IAC appears to be one of the most sensitive and rapid assays for the detection of BoNT/A reported to date and, because it is also highly transportable, it is amongst the best suited for field diagnosis of BoNT/A poisoning.

Evaluation of lateral flow assays for the detection of botulinum neurotoxin type A and their application in laboratory diagnosis of botulism

Four lateral flow assays (LFAs) were evaluated for the detection of purified botulinum neurotoxin A, toxin complex, and unpurified culture supernatant. They included the BioThreat (Tetracore, Rockville, MD), SMART (New Horizons Diagnostics, Columbia, MD), BADD (ADVNT Biotechnologies, Phoenix, AZ), and RAMP (Response Biomedical, Burnaby, BC, Canada) assays. BioThreat and SMART did not detect the purified toxin. The best sensitivity was achieved with the RAMP test (50 ng mL −1). BioThreat and SMART measured as low as 10 ng mL −1 of the toxin complex. Specificity data differed among the tests. BADD gave false-positive signals with uninoculated bacterial culture medium. BioThreat and RAMP were further evaluated with clinical sample matrices (serum, gastric, and rectum contents from pigs). Because of matrix effects and a generally low positive response, the assays are unsuitable for the direct detection of the toxin. However, the LFAs can be a helpful tool in screening bacterial cultures for toxigenic Clostridium botulinum, if further validated according to the laboratory needs.