Development of sensitive colorimetric capture ELISAs for Clostridium botulinum neurotoxin serotypes E and F.

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed to detect Clostridium botulinum neurotoxin serotypes E (BoNT E) and F (BoNT F) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kD C-fragments of each toxin. Standard curves were linear over 0.5-10 ng/ml (BoNT E) or 2-20 ng/ml (BoNT F). Accurate measurements were achieved at 0.5 ng/ml (BoNT E) or 2 ng/ml (BoNT F) in assay buffer and 10% human serum. Variation between triplicates was typically 5-10%. Less than 1% cross-reactivity occurred between other serotypes A, B, E or F). When tested against toxins complexed to their neurotoxin-associated proteins, interference was absent for BoNT F. However, pure BoNT E and that complexed to associated proteins demonstrated significant quantitative differences. We believe these differences arise from trypsin activation of the toxin. These assays demonstrated sensitivities close to that of the mouse bioassay, without the use of animals, in a much simpler format than other reported assays of similar sensitivity.     

Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

Botulinum neurotoxins (BoNT) are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.     

Sensitive and specific colorimetric ELISAs for Staphylococcus aureus enterotoxins A and B in urine and buffer

We present here simple, sensitive and accurate colorimetric capture ELISAs for staphylococcal enterotoxins A and B. Standard curves were linear over the range 0.5–1 ng/mL, and toxins could be accurately measured at 0.5 ng/mL in assay buffer or 0.1 ng/mL in human urine. Cross-reactivity between serotypes was negligible. Detection in serum was complicated by the presence of specific antibodies to SE’s in most normal sera. These assays offer a viable, cost-effective method for analysis of these ubiquitous toxins. Further, their sensitivity in undiluted urine makes them ideal candidates for evaluating human exposure.     

Sensitive and specific colorimetric ELISAs for Staphylococcus aureus enterotoxins A and B in urine and buffer.

We present here simple, sensitive and accurate colorimetric capture ELISAs for staphylococcal enterotoxins A and B. Standard curves were linear over the range 0.5–1 ng/mL, and toxins could be accurately measured at 0.5 ng/mL in assay buffer or 0.1 ng/mL in human urine. Cross-reactivity between serotypes was negligible. Detection in serum was complicated by the presence of specific antibodies to SE’s in most normal sera. These assays offer a viable, cost-effective method for analysis of these ubiquitous toxins. Further, their sensitivity in undiluted urine makes them ideal candidates for evaluating human exposure.     

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay.     

A highly sensitive immuno-polymerase chain reaction assay for Clostridium botulinum neurotoxin type A.

Our goal was to develop a sensitive method for detecting Clostridium botulinum neurotoxin type A (BoNT/A). We were able to detect BoNT/A in the femtogram (10(-15)g) range using an indirect immuno-polymerase chain reaction (immuno-PCR) assay and an indirect sandwich immuno-PCR assay. For the indirect immuno-PCR assay, enzyme-linked immunosorbent assay (ELISA) plates were coated with BoNT/A that was recognized by anti-BoNT/A monoclonal antibody. For the indirect sandwich immuno-PCR assay, the monoclonal antibody was immobilized on ELISA plates for detecting BoNT/A that was recognized by its polyclonal antibodies. Reporter DNA was prepared by PCR amplification using biotinylated 5'-primers, and it was coupled with biotinylated antibodies through streptavidin. In order to increase sensitivity and reduce background noise, the amounts of reporter DNA (ranging from 50 fg to 50 ng) and streptavidin (ranging from 0.125 ng to 8 ng) were optimized. Using the optimized concentration of reporter DNA and streptavidin, both indirect and indirect sandwich immuno-PCR assays detected BoNT/A as low as 50 fg. These results are a 10(5)-fold improvement over conventional indirect ELISA and indirect sandwich ELISA methods. The assays we developed are currently the most sensitive methods for detecting BoNT/A.     

A cyclic peptide–polymer probe for the detection of Clostridium botulinum neurotoxin serotype A

A Botulinum neurotoxin serotype A (BoNT/A) ELISA detection system was developed based upon an 11-mer cyclic peptide, termed C11-019, that was identified through peptide phage display technology. The assay employs a sandwich format using the C11-019 cyclic peptide attached to a PEMA (poly(ethylene maleic anhydride)) matrix as the capture phase and anti-BoNT/A polyclonal antibodies as the detection phase. Results reported demonstrate that the C11-019 peptide–polymer can specifically bind to BoNT/A with no cross-reactivity to other serotypes examined in assay buffers and a variety of body fluids and foodstuffs. When a highly sensitive chemiluminescent substrate was engaged, the detection of 1 pg/mL could be readily achieved within 3 h with a linear range of 0.1–1 ng/mL. These results demonstrate that an inexpensive peptide–polymer-based capture ELISA system can be used for rapid, sensitive and highly specific BoNT detection.     

Avian eyelid assay, a new diagnostic method for detecting botulinum neurotoxin serotypes A, B and E.

Based upon botulinum neurotoxins' (BoNT) mechanism of action, a novel, rapid, and sensitive avian eyelid assay was developed to detect Clostridium botulinum neurotoxin serotypes A, B and E in assay buffer and mimic samples. It showed that chick was the most optimal model of 20-selected laboratory, non-laboratory animals. The eyelid closure of chick was the indicator symptom for positive results. The detection limits achieved range from 5 to 250 mouse LD(50) for toxin types A, B, and E in a buffer system and mimic samples. No cross reactivity occurred when using staphylococcal enterotoxin B, diphtheria toxin and nerve agent sarin, but cross reactivity was obtained in more than 6h for using high dose of tetanus toxin. This cross reactivity can be differentiated by BoNT neutralization tests with a serotype-specific antiserum in parallel. The avian eyelid assay can be performed within as short a time as 0.4-6 h. We report here the development of avian eyelid assay is the second animal bioassay for the detection of toxin types A, B, and E which approaches the sensitivity of the mouse bioassay, and is simple to perform as well as rapid to yield results.     

Evaluation of Roche Diagnostics ONLINE DAT II, a new generation of assays for the detection of drugs of abuse

A new generation of ONLINE assays has been developed that offers improved performance and enhanced ease of use. This family of assays is being applied to both the COBAS INTEGRA and Roche/Hitachi line of analyzers. The four ONLINE DAT II assays that were evaluated included cocaine (benzoylecgonine) (BE), methadone (MDN), opiates (OP), and tetrahydrocannabinol (THC). The BE assay has a dual cutoff (150/300 ng/mL) with a dynamic range from 0 to 5000 ng/mL. The MDN assay has a cutoff of 300 ng/mL with a dynamic range from 0 to 2000 ng/mL. The opiates assay has a 300 ng/mL cutoff with a 0 to 2000 ng/mL range and a 2000 ng/mL cutoff with a 0 to 8000 ng/mL range. The THC assay has 20, 50, and 100 ng/mL cutoffs with 0 to 100, 0 to 300, and 0 to 300 ng/mL dynamic ranges, respectively. The ranges of the intra-assay precision (coefficients of variation for n = 20) run in the semiquantitative mode are 2.3-7.5% for BE, 2.0-3.8% for MDN, 1.9-4.2% for OP, and 3.9-5.2% for THC. The intra-assay qualitative precision for all of the assays as calculated from absorbance values is generally higher than that of the intra-assay semiquantitative precision at the cutoff. The qualitative precision ranges between 0.4% and 3.1%. The standard curve stability defined for the COBAS INTEGRA systems for these reagents ranges from 35 to 68 days. The clinical sensitivity and specificity were compared to the OnLine generation I and CEDIA immunoassays, as well as gas chromatography-mass spectrometry (GC-MS). The results indicate that for each assay, the sensitivity and specificity were the same or greater when compared to the other two immunoassay technologies. The results of each assay also correlated very well (> 99%) when compared with GC-MS.     

A protein chip membrane-capture assay for botulinum neurotoxin activity

Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC₅₀s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC₅₀ of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.     

A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model

Botulinum neurotoxins (BoNT) are extremely potent and can induce respiratory failure, requiring long-term intensive care to prevent death. Recombinant monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics. In contrast, equine antitoxin cannot be used prophylactically and has a short half-life. Two three-mAb combinations are in development that specifically neutralize BoNT serotype A (BoNT/A) and B (BoNT/B). The three-mAb combinations addressing a single serotype provided pre-exposure prophylaxis in the guinea pig inhalation model. A lyophilized co-formulation of six mAbs, designated G03-52-01, that addresses both A and B serotypes is in development. Here, we investigated the efficacy of G03-52-01 to protect guinea pigs against an aerosol exposure challenge of BoNT/A1 or BoNT/B1. Previously, it was found that each antibody demonstrated a dose-dependent exposure and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intravenous (IV) injection. Here we show that G03-52-01, in a single IM injection of G03-52-01 administered 48 h pre-exposure, protected guinea pigs against an aerosol challenge of up to 238 LD₅₀s of BoNT/A1 and 191 LD₅₀s of BoNT/B1. These data suggest that a single IM administration of G03-52-01 provides pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A1 or BoNT/B1.     

Discrimination of the Activity of Low-Affinity Wild-Type and High-Affinity Mutant Recombinant BoNT/B by a SIMA Cell-Based Reporter Release Assay

Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma–based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.     

Detection of fumonisin b1 and ochratoxin a in grain products using microsphere-based fluid array immunoassays

Grain products are a staple of diets worldwide and therefore, the ability to accurately and efficiently detect foodborne contaminants such as mycotoxins is of importance to everyone. Here we describe an indirect competitive fluid array fluoroimmunoassay to quantify the mycotoxins, fumonisin B1 and ochratoxin A. Both toxins were immobilized to the surface of microspheres using a variety of intermediate molecules and binding of biotinylated "tracer" antibody tracers determined through flow cytometry using streptavidin-phycoerythrin conjugates and the Luminex100 flow cytometer. Competitive assays were developed where the binding of biotinylated monoclonal antibodies to fumonisin B and ochratoxin A was competitively inhibited by different concentrations of those toxins in solution. Concentrations of fumonisin giving 50% inhibition were 300 pg/mL in buffer, 100 ng/g in spiked oats, and 1 μg/g in spiked cornmeal; analogous concentrations for ochratoxin A were 30 ng/mL in buffer, 30 ng/g in spiked oats, and 10 ng/g in spiked corn. The future challenge will be to expand the number of mycotoxins tested both individually and in multiplexed format using this platform.     

A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food

Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A – H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs) capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture) ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s) for individual antibodies ranging from 10 to 48 × 10⁻¹¹ M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D.), ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule) and readily detects toxin in those food samples tested.     

Brotizolam radioimmunoassay: development, evaluation, and application to human plasma samples

A radioimmunoassay for the determination of the hypnotic agent brotizolam (11) was developed. With this procedure, an antiserum was used which was obtained from rabbits immunized with the hapten 10 (We 934) covalently bound to bovine serum albumin and tritium-labeled brotizolam as the radioligand. Compound 10 represents a structural analogue of brotizolam: the bromine was replaced by a carboxyethyl group. By such manipulation high assay specificity against the primary human metabolites was achieved. The sensitivity limit of the assay was about 100 pg of brotizolam per mL of plasma when 0.1-mL samples were used. The assay showed good accuracy and high precision. Repeated assays after keeping plasma samples frozen for various periods again indicated high precision as well as the stability of the brotizolam molecule under these conditions. Application of the assay to plasma samples of eight subjects who received single oral 0.25-mg doses of brotizolam showed a mean maximum plasma concentration of 4.6 ng of unchanged drug per mL at 0.9 h after administration. The brotizolam plasma concentration declined with a mean elimination half-life of 5.1 h. The pharmacokinetic parameters estimated by RIA agree well with those obtained with other specific brotizolam determination procedures.     

Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1

The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing.     

Performance of a microtiter plate ELISA for screening of postmortem blood for cocaine and metabolites

The object of this study was to evaluate the suitability of the Neogen Corporation microtiter plate enzyme-linked immunoassay (ELISA) for cocaine and metabolites for screening of postmortem blood. Sixty-five postmortem whole blood specimens were obtained from drug-involved deaths that had been screened and confirmed positive for cocaine and/or benzoylecgonine (BE). Fifty-eight negative specimens were obtained from noncocaine-involved deaths. Specimens were tested using the Neogen Cocaine/BE microtiter plate ELISA assay. No matrix effects were found for whole blood in this assay. The effect of dilutions of the whole blood specimens of 1:5 through 1:50 was studied. A dilution of 1:5 was chosen to correspond to that used for other Neogen microtiter plate assays for drugs in whole blood. True positives, true negatives, false positives, and false negatives were determined and graphed for the ELISA results against gas chromatography-mass spectrometry (GC-MS), GC-nitrogen-phosphorus detection, and case histories. From these graphs and the receiver operating characteristic curves, the optimal cutoff for the Neogen Cocaine/BE ELISA was found to be 5 ng/mL BE equivalents at a 1:5 dilution. The optimum cutoff for a 1:50 dilution was 50 ng/mL BE equivalents. The Neogen Cocaine/BE ELISA had a sensitivity of 93.8% +/- 2.9% and a specificity of 96.6% +/- 2.4% versus GC-MS at a cutoff of 5 ng/mL BE equivalents (1:5 dilution) and a sensitivity of 100% +/- 0.5% and specificity of 98.3% +/- 1.7% versus GC-MS at a 50 ng/mL BE equivalents cutoff (1:50 dilution).     

In vitro potency determination of botulinum neurotoxin B based on its receptor-binding and proteolytic characteristics

Botulinum neurotoxins (BoNTs) are the most potent toxins known. However, the paralytic effect caused by BoNT serotypes A and B is taken advantage of to treat different forms of dystonia and in cosmetic procedures. Due to the increasing areas of application, the demand for BoNTs A and B is rising steadily. Because of the high toxicity, it is mandatory to precisely determine the potency of every produced BoNT batch, which is usually accomplished by performing toxicity testing (LD₅₀ test) in mice. Here we describe an alternative in vitro assay for the potency determination of the BoNT serotype B. In this assay, the toxin is first bound to its specific receptor molecules. After the proteolytic subunit of the toxin has been released and activated by chemical reduction, it is exposed to synaptobrevin, its substrate protein. Finally the proteolytic cleavage is quantified by an antibody-mediated detection of the neoepitope, reaching a detection limit below 0.1 mouse LD₅₀/ml. Thus, the assay, named BoNT/B binding and cleavage assay (BoNT/B BINACLE), takes into account the binding as well as the protease function of the toxin, thereby measuring its biological activity.     

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 Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.