An optical biosensor assay for rapid dual detection of botulinum neurotoxins a and e

ABSTRACT The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of

crucial importance. A chip assay based on Surface Plasmon Resonance monitoring of the cleavage products is a simple method that we have previously established to detect BoNT/A activity. We

have now developed a similar format assay to measure BoNT/E activity. A monoclonal antibody specifically recognizing SNAP25 cleaved by BoNT/E was generated and used to measure the appearance

of the neo-epitope following injection of BoNT/E over SNAP-25 immobilized on a chip. This assay detects BoNT/E activity at 1 LD50/ml within minutes and linear dose-responses curves were

obtained using a multiplexed biosensor. A threshold of 0.01 LD50/ml was achieved after 5 h of cleavage. This assay is 10-fold more sensitive than the _in vivo_ assay for direct detection of

BoNT/E in serum samples. The SNAP25 chip assay is able to discriminate in an automated manner the presence of BoNT/E, BoNT/A or a combination of both toxins. SIMILAR CONTENT BEING VIEWED BY

OTHERS DE NOVO DESIGN OF MODULAR AND TUNABLE PROTEIN BIOSENSORS Article 27 January 2021 A PLUG-AND-PLAY PLATFORM OF RATIOMETRIC BIOLUMINESCENT SENSORS FOR HOMOGENEOUS IMMUNOASSAYS Article

Open access 28 July 2021 IMPROVED IMMUNOASSAY SENSITIVITY AND SPECIFICITY USING SINGLE-MOLECULE COLOCALIZATION Article Open access 12 September 2022 INTRODUCTION Botulinum neurotoxins

(BoNTs) constitute a family of 7 serologically distinct neurotoxins (from A to G) produced by _Clostridium botulinum_ and a few other species of _Clostridium_. BoNT/ A, B and E are the

principal causes of human botulism. Toxicity occurs mainly after oral ingestion of contaminated food. BoNT’s escape the gastro-intestinal tract and a small percentage enters the bloodstream.

Although BoNT’s found in human sera are in the low fmolar concentration range, specific receptors localized in the peripheral nervous system concentrate the toxin, mediate its neuronal

internalization and the consequent disabling of synaptic transmission1. These potent neurotoxins are also considered as one of the most toxic bioweapons constituting a threat for the

public2. Botulism can be effectively treated immunologically using an equine trivalent antitoxin3. However, rapidity is a crucial issue in BoNT diagnosis since anti-toxin antibodies become

ineffective when the toxin penetrates into neurons and no antidote is available. The decision to use serotherapy is often taken on the basis of clinical symptoms and the most direct way to

subsequently confirm diagnosis is to identify the BoNT type in the patient’s serum and/or stool using the standard mouse median lethal dose LD50 method4. The specificity of this _in vivo_

assay lies in the absence of symptoms when specific anti-BoNT antibodies are co-injected with the experimental sample. However, the _in vivo_ method is not really adapted for rapid molecular

diagnosis since several hours or days (range 6–96 h) are necessary for toxin type identification, especially for BoNT concentrations that cause symptoms but not death5. Moreover this

procedure is cumbersome and ethically questionable. Hence _in vitro_ methods allowing rapid and sensitive detection of BoNTs have been developed. Type E botulism affects humans and animals6.

Intoxication is particularly associated with ingestion of marine products as strains producing type E toxin are frequently found in fish and aquatic environments mainly from the Northern

countries of North hemisphere7,8. BoNT/E intoxication is more rarely associated with other foods such as ham9. BoNT/E is synthetized as a single chain molecule of 150 kDa associated with

non-toxic proteins. It acquires higher toxicity upon nicking with an exogenous protease, such as trypsin, at about one third of the length from the N-terminus10,11. The resulting activated

protein consists of a light (50 kDa) and heavy chain (100 kDa) linked by a disulfide bond and non-covalent interactions. The BoNT/E heavy chain controls cellular internalization via binding

to the synaptic vesicle protein SV2 through receptor-mediated endocytosis and translocation12,13. After translocation into the cytoplasm, the light chain of BoNT/E targets

Synaptosomal-associated protein of 25 kDa (SNAP-25), a protein associated with the inner leaflet of the plasma membrane which is involved in the formation of a multi-protein complex that

generates a driving force for exocytosis. BoNT/E cleaves off a C-terminal 26 amino acid fragment of the SNAP-25 inducing a persistent but reversible inhibition of neurotransmission. BoNT/A

and BoNT/C also target SNAP-25, but cut the protein at distinct cleavage sites1. Measurement of BoNT/E enzymatic activity constitutes the most sensitive approach to specifically detect this

toxin. A variety of _in vitro_ activity assays have already been published including methods using capillary gel electrophoresis14, ELISA15, mass spectrometry16,17 or fluorescent and

electrochemical readouts8,18,19. Since the 1990s, Surface Plasmon Resonance (SPR) has proven to be one of the most versatile biophysical methods for biosensor applications in the field of

biology, biomedicine and biochemistry as well as food safety. Optical SPR biosensors operate in a label-free manner by measuring, as molecules bind in real time, the change in the refractive

index within the evanescent field on the apparatus surface. We have recently developed a new SPR-based method to rapidly assay BoNT/A enzymatic activity with very high sensitivity20,21. In

this paper, we describe, using this integrated optical “on-chip” method, the development of an assay able to detect simultaneously the presence of BoNT/A and BoNT/E. RESULTS PRODUCTION OF A

MONOCLONAL ANTIBODY RECOGNIZING THE NEO-EPITOPE GENERATED BY BONT/E Mice were immunized using an eight residue peptide (amino acid 173–180) corresponding to the new C-terminal domain of

SNAP-25 produced by the proteolytic activity of BoNT/E (Fig. 1a). Hybridomas were generated and several clones were selected by ELISA for recognition of the immunization peptide. One clone

(mAb 11C3) was selected and characterized as follows: upon injection in the mobile phase, strong binding to immobilized His-SNAP25 previously cleaved by BoNT/E was measured, whereas no

binding was detected either to an irrelevant control protein (GST) or to uncleaved His-SNAP25 (Fig. 1b). The signal was abolished if mAb11C3 was injected with an excess of the cognate

peptide (Fig. 1b). In order to verify the specificity of mAb11C3 for recognition of a native substrate cleaved in a cellular context, we intoxicated neuronal cultures with BoNT/E.

Immunofluorescence staining using BoNT/E-treated versus untreated neuronal cultures showed that mAb11C3 recognized only BoNT/E treated neurons (Fig. S1). ON-CHIP SUBSTRATE CLEAVAGE BY

BONT/E: ASSAY PRINCIPLE This assay is based on the measurement of the catalytic activity of BoNT/E using an SPR biosensor, integrating all steps on a chip. Figure 2 shows the basic two-step

principle of our approach. The first step consists in injecting BoNT/E at a given concentration for a controlled period of time over a sensor surface coated with His-SNAP25. BoNT/E cleaves

the peptide bond between residues 180–181, thus generating a new SNAP-25 C-terminus. In the second step, mAb11C3 antibody is injected and the extent of cleavage is quantified by monitoring

the increase in Resonance Units (RU), reflecting antibody binding. We found that mAb11C3 binding was reproducible when the antibody injection was repeated after regeneration by acidic pH (CV

 = 3–4%, n = 14). ASSAY SPECIFICITY In order to address the BoNT serotype specificity of the assay, BoNT/A, B, C or E were injected over independent sensor chip flow cells coated with

His-SNAP25. BoNT/A and C cleave SNAP-25 at different sites than BoNT/E (Fig. 1a) and therefore generate different C-termini. Consequently their proteolytic products should not be recognized

by mAb11C3. BoNT/B cleaves the protein VAMP2 and has no proteolytic activity on SNAP-251. As shown in Fig. 3, mAb11C3 bound neither to SNAP-25 cleaved by high concentrations of BoNT/A and C

(1 nM) nor to BoNT/B (1 nM) -treated flow cells. In contrast mAb11C3 strongly bound to BoNT/E-treated SNAP-25 (0.1 nM). This result established that mAb11C3 can specifically detect BoNT/E

catalytic activity using an “on-chip” SPR-based assay. DOSE AND TIME DEPENDENCE OF SNAP-25 BONT/E CLEAVAGE We then explored the time-and dose-dependence of the SPR-based assay. We used a

multiplexed biosensor (ProteOnXPR36) which incorporates six parallel flow channels22. In order to evaluate how these _in vitro_ results compare to the mouse bioassay, BoNT/E concentrations

were expressed in mouse lethal dose units (LD50/ml). Experiments were carried out to measure mAb11C3 binding after repeated injections, for a limited contact time (15 min) of a BoNT/E

concentration range (0.25–4 LD50/ml). Although no signal was measured before BoNT/E injection (Fig. 4, 0 min), after 15 min of cleavage, specific mAb11C3 binding was detected only at the

highest BoNT/E concentrations used (4, 2 and 1 LD50/ml) (Fig. 4, 15 min). Additional rounds of BoNT/E injections were further performed (15 min each) separated by an acidic regeneration of

the chip. As early as the second BoNT/E injection round (cumulated contact time 30 min), a specific mAb11C3 binding signal was observed even with the lowest (0.25 LD50/ml) toxin

concentration (Fig. 4, 30, 45 and 60 min). Binding responses at plateau values were plotted and found to be dependent on cleavage-time (Fig. 4 last panel). For all BoNT/E concentrations in

this time scale, curves were linear, indicating that the reaction is not substrate-limited. In addition, the regeneration step did not affect subsequent cleavage steps on the same

functionalized surface (Fig. 4). Thus, a specific feature of this SPR procedure is that the His-SNAP25 chip can be reused for several assays provided that low BoNT/E concentrations are used,

in order to avoid substrate depletion. However, we noticed that prolonged cleavage times and/or higher BoNT/E concentrations can induce a loss in linearity due to substrate depletion (Fig.

S2). RAPID DETECTION OF BONT/E ACTIVITY AT 1 LD50/ML Using a ProteOn biosensor, BoNT/E proteolytic activity was easily detectable by mAb 11C3 at 1 LD50/ml (22 pM) after only 15 min of

cleavage (Fig. 4 and Table 1). The rapidity of detection at 1 LD50/ml was confirmed using a Biacore T200 biosensor, on which experiments can be implemented more quickly and which has a

slightly higher sensitivity to ProteOn biosensor due to a lower background. In this case, BoNT/E activity at 1 LD50/ml was detected after 5 minutes of contact time (Table 1). It has been

reported that trypsinization of BoNT/E induces an increase in its toxicity in the mouse _in vivo_ bioassay10. In fact, we found that trypsinization of BoNT/E led to a 60 fold increase in

potency in the mouse bioassay (data not shown). However, based on their respective LD50 determinations, the enzymatic activity of trypsinized BoNT/E at 1 LD50/ml (5.8 ± 1 RU, n = 5) was

found to be a third of that of the non trypsinized form (16.3 ± 2.7 RU, n = 5, Table 1), as illustrated in Fig. 5 (mAb11C3). In order to increase SPR responses, a secondary anti-mouse IgG

antibody was used to amplify the mAb11C3 signal via the co-inject mode of the SPR instrument. This automated procedure allows consecutive loading of both mAb11C3 and the secondary antibody

in the injection loops. As shown in Fig. 5a, the secondary antibody binding response took place immediately after the end of mAb11C3 injection. This amplification mode provided a 3–4 fold

increase in RU responses (Table 1 and Fig. 5). Moreover the secondary antibody stabilized the binding of the primary one, as the dissociation step was significantly slowed (compare Fig. 4

and Fig. 5a). This amplification step provided thus an efficient means to amplify binding signals and to detect trypsinized or non trypsinized BoNT/E activity at 1 LD50/ml in 15 minutes.

LIMIT OF SENSITIVITY IN ABSENCE AND PRESENCE OF SERUM As the sensitivity is a function of cleavage time, we explored the lowest BoNT/E concentration that allowed detectable signals after 5 h

of contact with the substrate. Five BoNT/E concentrations (0.03 to 0.25 LD50/ml) were injected and the cleavage products detected using mAb11C3 in tandem with secondary antibody to maximize

sensitivity. The dose-response relationship at these very low BoNT/E concentrations was linear (Fig. 5b). The SPR-based assay exhibited a limit of detection of 0.01 LD50/ml for BoNT/E and

0.03 LD50/ml for trypsinized BoNT/E (Fig. 5b and Table 1). We then evaluated the performance of the SPR assay with trypsinized BoNT/E spiked in serum. In preliminary experiments with a 15 

min cleavage time, we had found that diluting BoNT/E in cleavage buffer containing 10% serum did not affect assay sensitivity (11.4 +/− 2 RU (n = 3) without serum versus 9.8 +/− 3 RU (n = 4)

at 2 LD50/ml with serum). This result indicated that BoNT/E could be directly detected in serum, provided that the specimen is diluted 10-fold before injection. To investigate the _in

vitro_ assay sensitivity in presence of serum and to ensure the robustness of the SPR method using another source of toxin, crude BoNT/E complex was produced and healthy serum samples spiked

with different amounts of toxin. We then compared in parallel the sensitivity of the mouse bioassay and the SPR assay. Aliquots were either administrated in mice without dilution (1 

ml/mouse) or injected for 5 hours on the biosensor after 10 fold dilution in cleavage buffer. As indicated in Table 2, no mouse lethality was observed when using toxin concentrations below 1

LD50/ml. The activity of BoNT/E concentrations as low as 0.125 LD50/ml was still robustly detected using SPR. Thus, using BoNT/E samples in undiluted serum, the _in vitro_ test was nearly

10 times more sensitive than the _in vivo_ assay. Altogether these results indicate that the SPR assay is about 100 fold more sensitive than the _in vivo_ assay in the absence or in the

presence of 10% serum detecting as low as 0.01 LD50/ml. CO-DETECTION OF BONT/E AND BONT/A ACTIVITY We have previously reported the use of a SNAP25 chip to assay BoNT/A activity20 using a

monoclonal antibody (mAb10F12), that specifically recognizes the epitope generated on SNAP25 by BoNT/A activity (Fig. 1a). In an attempt to establish a dual SPR assay, we used both mab10F12

and mAb11C3 to verify whether our SPR method could discriminate between the activities of the two toxins and if it can detect the presence of both toxins in the same sample. BoNT/E and

BoNT/A cleave SNAP-25 at different sites, separated by 17 residues, in the C-terminal domain of the protein (Fig. 1a). BoNT/E activity removes the BoNT/A cleavage site (Fig. 1a) and

therefore could in principle hinder the development of a multiplexed assay. Consequently, BoNT/E activity should be measured on a substrate chip after treatment with BoNT/A, but the opposite

should theoretically not be possible. In order to overcome this problem, we used low concentrations of toxins (1 LD50/ml), at which a single substrate molecule has little probability of

being cleaved by both toxins. We set up a protocol where BoNT/E, BoNT/A or a mixture of both toxins were injected for 15 minutes on different flow cells of a ProteOn apparatus. The cleavage

activity of each toxin was then followed by consecutive injection of mAb11C3 and mAb10F12 for detection of BoNT/E and BoNT/A activity respectively. As shown in Fig. 6, SNAP-25 cleavage

mediated by BoNT/E or BoNT/A separately induced a single positive signal with the corresponding mAb probes and neither of the monoclonal antibodies showed any binding in the absence of

neurotoxins. The signal level for BoNT/A activity detection was also in agreement with the previously published sensitivity20. As shown in the last panel of Fig. 6, the use of moderate

concentrations of BoNT/A and E allowed simultaneous cleavage and sequential detection, with similar signal levels, of both neurotoxins. Thus, these results indicate that our assay provides

an efficient tool for consecutive and specific detection of BoNT/A and E activities on the same biosensor chip. DISCUSSION Highly sensitive, target-specific sensors are needed for

identifying biological threats in health care, security and food safety industries. We have developed an optical SPR biochip to detect and quantify BoNT/E, one of the most potent toxins

affecting humans. Although optical biosensors are widely used to measure biomolecular interactions, the protocol presented in this work does not detect directly the presence of BoNT/E but a

consequence of its interaction with the immobilized substrate. Indeed we have taken advantage of the enzymatic activity driven by the light chain of BoNT/E and its molecular selectivity for

SNAP25 to generate a unique cleavage product on the chip. The method provides a high degree of specificity since positivity is a consequence of three distinct consecutive reactions:

substrate recognition, endoproteolytic cleavage occurring at a single defined peptide bond and finally specific antibody recognition of the cleavage product. The SPR-assay is very simple,

composed of two automated steps consisting in BoNT/E injection and mAb detection. The assay format with one interactant captured on a solid support mimics physiological conditions where

SNAP25 is associated with the inner leaflet of the plasma membrane and cleaved by BoNT/E. We first generated a monoclonal antibody that recognizes specifically the new C-terminal end of

SNAP25 resulting from BoNT/E enzymatic activity but not cross reacting with cleavage products generated by other BoNT subtypes. This mAb provides in a minute timescale a positive gain of

signal to monitor functional BoNT/E activity and constitutes a key specificity determinant of this assay. Compared to polyclonal antibodies with similar specificity15, our mAb should be a

more robust tool, less prone to batch to batch variability. As cleavage product detection by mAb11C3 is performed in controlled buffer conditions after surface washing, the SPR assay avoids

matrix interference23 and retains highly specific detection even when analyzing complex samples (i.e serum). The method has been developed using biosensors equipped with either a single

injection port (Biacore) or multi-ports (ProteOn). The multi-channel apparatus provides the advantage of managing several samples in parallel and especially establishing linear plots across

a range of BoNT/E concentrations. Linear calibration curves were produced in short timescales (15–30 min) for relatively high BoNT/E concentrations (1–20 LD50/ml) or long timescales (5h) for

low concentration ranges (0.01–1 LD50/ml). Sensitivity is thus directly proportional to the cleavage time in a time-window between 0.25 and 5 h, but could be potentially increased providing

that BoNT/E activity remains stable during an extended cleavage time. The SPR assay offers the advantage of rapidity as BoNT/E activity at 1 LD50/ml can be easily detected in less than 15 

minutes. Extension of the cleavage period to 5h increased detection sensitivity for BoNT/E to 0.01 LD50/ml, a value lower than those previously reported for BoNT/E activity-based

assays15,17,19. As LD50 data can depend on assay conditions24 we confirmed our assay sensitivity using two different sources of BoNT/E. We found that BoNT/E trypsinization enhances enzymatic

activity as well as the toxicity in mouse. However, the activity of the trypsinized BoNT/E detected on-chip was slightly lower than what could be expected compared to data obtained from _in

vivo_ experiments. This observation may stem from the fact that trypsin and trypsin inhibitor present in the tryspsinized BoNT/E preparation could slightly interfere with the assay.

Alternatively trypsin activation could increase not only enzymatic activity but also others factors as biodisponibility and/or cell penetration of BoNT/E, factors that are not measured in

the SPR assay. This could account for the greater factor of activation with trypsinized BoNT/E determined using the mouse bioassay. We have shown that BoNT/E could be directly detected in

diluted (1:10) serum samples. A sensitivity of 0.1 LD50/ml was achieved in 5 hours, a concentration that was never lethal in mice. This result suggests that the SPR BoNT/E activity assay

could be a faster and more sensitive method than the mouse assay. A key feature of the SPR-based assay is a rapid readout with a surface that can be probed at various times during the

cleavage step. As detection of BoNT intoxication requires urgent diagnosis for timely treatment, BoNT/E activity could be evaluated using a short cleavage time (i.e. 1 hour) to detect BoNT/E

concentrations ≥1 LD50/ml and then using more prolonged cleavage times to measure lower concentrations. Other _in vitro_ methods have been developed to detect BoNT/E in serum samples.

However, these tests are less convenient than the direct and automated detection method presented in this paper. Indeed, they are based on a two-step approach involving toxin

concentration/purification prior to assaying toxin activity8,16,25,26. Additionally, we have shown that this “on chip” assay can be used to detect and quantify simultaneously both BoNT/A and

BoNT/E activity. This biosensor, which has similar sensitivity for both toxins21, could be connected to drinking water supply systems for continuous monitoring of their presence. This assay

has also the potential to detect the BoNT/C activity that cleaves SNAP-25 and affects animals. Finally, this method should provide a rapid and useful tool to screen for BoNT/A and E

activity inhibitors. Provided that these drugs are membrane permeable, they could be used when toxins enter the nerve cells. MATERIALS AND METHODS CHEMICAL AND REAGENTS His6-SNAP-25 and

mAb10F12 were prepared as described20. Control human sera were obtained from the “Etablissement Francais du Sang” (Marseille, France). Glutathione-S-Transferase (GST) was produced as

described27. Amine coupling kits were from GE Healthcare and Bio-Rad. ZnCl2, fatty acid-free bovine serum albumin (BSA), Tween20, dithiothreitol (DTT) and Fc specific goat anti-mouse IgG

were from Sigma. BoNT/A (MW 500 000 Da, 3.5 × 107 LD50/mg), BoNT/B (MW 550 000 Da, 1.1 × 107 LD50/mg) and BoNT/E (MW 300 000 Da, 1.5 × 105 mLD50/mg) complex toxins at a concentration of 1 

mg/ml were purchased from Metabiologics (Madison, WI). BoNT/C was provided by Prof. S. Kozaki, (Osaka Prefecture University). BoNT/E complex was prepared from culture of _C. botulinum_ E

strain HV in Trypticase-glucose-yeast extract broth culture at 37 °C for 4 days in anaerobic conditions28. BONT/E TRYPSINIZATION In some cases, BoNT/E from Metabiologics was

trypsin-activated by adding one part (by protein weight) of TPCK-treated trypsin (Sigma T1426) to 10 parts of toxin in 0.1 M sodium phosphate buffer pH 6.0 and incubating for 30 min at 37 

°C. The reaction was stopped by adding a 10 fold excess of soybean trypsin inhibitor (Sigma T9003). The activated toxin was aliquoted and frozen. Serum samples spiked with various BoNT/E

concentrations were divided in 2 parts for _in vitro_ and _in vivo_ assays and frozen. MONOCLONAL ANTIBODY PRODUCTION Mice were immunized with a synthetic peptide corresponding to the amino

acid sequence of residues 173–180 of rat SNAP-25 (GeneCust, Europe). This sequence is located immediately N-terminal to the peptide bond cleaved by BoNT/E (R180-I181). A cysteine residue was

added to the N-terminus of the peptide to allow coupling to Keyhole Limpet Hemocyanin (KLH) for immunization. Hybridomas were selected by ELISA for their ability to recognize the

immunization peptide and by SPR for their ability to capture SNAP-25 in plasma membrane vesicles treated with BoNT/E29. The selected monoclonal antibodies (mAb11C3) were affinity purified

using protein A-Sepharose and stored at −20 °C. PREPARATION OF SNAP-25 CHIPS Purified His-SNAP25 was amine coupled at pH 4 to ProteOn GLC (Bio-Rad) sensor chips or at pH 4.5 to Biacore CM5

(GE Healthcare) according to manufacturer’s instructions and phosphate-buffered saline (PBS) as running buffer. The final density was approximately 5 000 RU on GLC chips and 10 000 RU on CM5

chips. All chips were conditioned by injection of cleavage buffer (50 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid)-NaOH pH 7.4, 0.1% BSA, 5 mM DTT, 1% Tween 20, 100 μM

ZnCl2). To determine the specificity of mAb11C3, His-SNAP25 (0.5 mg/ml) was pre-incubated for 2 h at 37 °C in cleavage buffer without BSA in presence or absence of 10 nM BoNT/E. Intact

His-SNAP25, His-SNAP25 cleaved by BoNT/E and GST were coupled (500 RU) on GLC sensor chip as mentioned before. SPR ANALYSIS OF BONT/E ENDOPROTEASE ACTIVITY SPR measurements were performed at

34 °C using ProteOn XPR36 (Bio-Rad) or Biacore T200 (GE Healthcare) apparatus and TBS (10 mM Tris-HCl pH 7.4, 150 mM NaCl) as running buffer. Flow rates were 5 and 25 μl/min, with the

Biacore T200 and ProteonXPR36 apparatus respectively. The ProteOn XPR36 has six parallel flow channels that can be used to uniformly immobilize strips of six ligands on the sensor surface.

The fluidic system can automatically rotate 90° so that up to six different analytes can be injected, allowing simultaneous monitoring of up to 36 individual molecular interactions in a

single run on a single chip (Bravman _et al_., 2006). Data were analyzed using Biaevaluation 4.2 software (GEHealthcare) or ProteOn Manager v. 3.1.0.6 software (Bio-Rad). BoNT/A, B, C and E

(trypsinized or not) were thawed and serially diluted in cleavage buffer. Serially diluted BoNT/E samples and mAb11C3 (10 μg/ml) were placed in the sample rack at 4 °C before injection over

the His-SNAP25 coated chip. BoNT/E samples were injected using different contact times (cleavage step) whereas mAb11C3 was injected for 2 min over all flow cells to measure cleaved SNAP-25

(detection step). In experiments with signal amplification, anti-mouse Fc antibodies were diluted at 10 μg/ml and injected (for 4 minutes) immediately after mAb11C3 (co-inject protocol).

Antibody binding was measured in triplicate, 10 sec after the end of injection(s) and the chip was regenerated with a 8 sec pulse (100 μl/min) of 10 mM glycine-HCl pH 2.0. In all

experiments, non-specific signals were measured by injecting cleavage buffer without BoNT onto a flow cell coated with His-SNAP25. Data obtained from control flow cells were automatically

subtracted from experimental measurements to yield the specific signal. Limits of detection (LODs) were calculated by determining the minimal BoNT concentration producing a signal 3 standard

deviations (SDs) higher than the control values in the absence of BoNT (_n_ = 6). For BoNT/A and BoNT/E co-detection experiments, consecutive co-inject protocols using mAb11C3 (10 

μg/ml)/anti-mouse Fc antibody and mAb10F12/anti-mouse Fc antibody injections were separated by a pH 2 regeneration step. _IN VIVO_ ASSAY Mice were injected intraperitoneally with 1 ml of

serially diluted BoNT/E serum as described 30. Symptoms of botulism were monitored for 4 days. Experiments were performed in accordance with French and European community guidelines for

handling laboratory animals. The protocols of experiments were approved by Pasteur Institute CETEA (Comité d’Ethique en Expérimentation Animale) with the agreement of laboratory animal use

(n° 2013-0118). ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Lévêque, C. _et al_. An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E. _Sci. Rep_. 5,

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ACKNOWLEDGEMENTS This work was supported by INSERM, CNRS and a grant from the DGA (REI N° 0634040). We thank Dr Raymond Miquelis for valuable discussions and careful reading of the

manuscript. AUTHOR INFORMATION Author notes * Lévêque Christian and Ferracci Géraldine contributed equally to this work. AUTHORS AND AFFILIATIONS * INSERM, UMR_S 1072, Marseille, 13015,

France Christian Lévêque, Yves Maulet, Michael Seagar & Oussama El Far * CNRS, UMR 7286, Plate-Forme de Recherche en Neurosciences PFRN, Marseille, 13015, France Géraldine Ferracci &

 Marie-Pierre Blanchard * Aix-Marseille Université, Marseille, 13015, France Christian Lévêque, Géraldine Ferracci, Yves Maulet, Marie-Pierre Blanchard, Michael Seagar & Oussama El Far *

CNR Bactéries Anaérobies et botulisme, Unité des Bactéries anaérobies et toxines. Institut Pasteur, 28 rue du Dr Roux, Paris, Cedex 15, 75724, France Christelle Mazuet & Michel R.

Popoff Authors * Christian Lévêque View author publications You can also search for this author inPubMed Google Scholar * Géraldine Ferracci View author publications You can also search for

this author inPubMed Google Scholar * Yves Maulet View author publications You can also search for this author inPubMed Google Scholar * Christelle Mazuet View author publications You can

also search for this author inPubMed Google Scholar * Michel R. Popoff View author publications You can also search for this author inPubMed Google Scholar * Marie-Pierre Blanchard View

author publications You can also search for this author inPubMed Google Scholar * Michael Seagar View author publications You can also search for this author inPubMed Google Scholar *

Oussama El Far View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS C.L. and G.F. performed SPR experiments, Y.M. prepared recombinant proteins,

C.M. and M.P. prepared BoNT/E and performed experiments on animals. M.-P.B. performed immunocytochemical stainings. C.L., M.S. and O.EF. supervised the study. C.L., G.F., M.S. and O.EF

wrote the manuscript. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY INFORMATION RIGHTS AND

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Lévêque, C., Ferracci, G., Maulet, Y. _et al._ An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E. _Sci Rep_ 5, 17953 (2016).

https://doi.org/10.1038/srep17953 Download citation * Received: 24 September 2015 * Accepted: 02 November 2015 * Published: 09 December 2015 * DOI: https://doi.org/10.1038/srep17953 SHARE

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