Journal of Immunological Methods 330 (2008) 120–129www.elsevier.com/locate/jim

Research paper

A sensitive sandwich enzyme immunoassay for free or complexedClostridium botulinum neurotoxin type A

Hervé Volland a,⁎, Patricia Lamourette a, Marie-Claire Nevers a, Christelle Mazuet b,Eric Ezan a, Laure-Marie Neuburger a, Michel Popoff b, Christophe Créminon a

a CEA, iBiTecS, Service de Pharmacologie et d'Immunoanalyse, Gif sur Yvette Cedex 91191, Franceb Unité des Bactéries anaérobies et Toxines, Institut Pasteur, 28 rue du Docteur Roux, 75015 Paris, France

Received 30 January 2007; received in revised form 17 October 2007; accepted 14 November 2007Available online 17 December 2007

Abstract

Fourteen monoclonal antibodies (mAbs) against Clostridium botulinum type A neurotoxin were raised in mice using a carboxyterminal fragment of the toxin for immunization. A two-site immunometric assay was developed which allowed detection of 8LD50/ml (40 pg/ml) botulinum neurotoxin A and an accurate quantification close to 25 LD50/ml (150 pg/ml). No cross-reactivitywas observed with other toxinotypes. During the development of this assay, interference induced by associated protein wasobserved. By comparing the effect of different buffers, a buffer composed with Tris–HCl and NaCl salts was demonstrated todissociate protein complexed with the neurotoxin A. Applied to the measurement of the toxin in different matrices, this dissociatingbuffer ensures the correct quantification.© 2007 Elsevier B.V. All rights reserved.

Keywords: Botulinum A toxin; Enzyme immmunometric assay; Monoclonal antibody; Associated non-toxic protein

1. Introduction

Seven serologically different botulinum toxins, knownas BoNT/A to /G, are produced by different strains ofa Gram-positive, spore-forming anaerobic bacteria,Clostridium botulinum. These neurotoxins are the mosttoxic substances known, and, for example, are 100000timesmore toxic than sarin (Broussard, 2001). Threemainforms of botulism have been characterized: (i) food-bornebotulism; (ii) infant botulism, due to toxin production ininfant intestines after consumption of the bacillus spores;

Abbreviations: kDa, kilodalton; EIA, enzyme immunoassay.⁎ Corresponding author. Tel.: +33 1 69 08 13 14.E-mail address: herve.volland@cea.fr (H. Volland).

0022-1759/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jim.2007.11.006

(iii) wound botulism (Liu et al., 2003). Cases of botulismare rare but often life-threatening, since the recoveryperiod can take several months and requires intensivecare. Vaccination against botulism is available and con-sists of a long-lasting inoculation protocol; it has onlybeen used for people at high risk of exposure and not forwhole populations. However, if diagnosed early, botulismcan also be treated by passive immunization using anti-toxin sera.

The high toxicity of BoNTs and the relative simplicityof their production and dissemination make them a verypotent biological weapon, which is why they are listed asone of the most powerful biowarfare threats by theCenters for Disease Control and Prevention in the UnitedStates. Therefore, the development of rapid and sensitive

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detection methods for BoNTs is needed. Currently, theAOAC mouse bioassay method is the only referencemethod for detecting botulinum toxin during botulisminvestigations. Although highly sensitive (5–20 pg/ml),this method is expensive, time-consuming and requiresspecific authorization and facilities such as mousehusbandry to perform the test. To date, several detectionmethods have been developed for BoNTs as an alter-native to the mouse bioassay: PCR-based assays(Lindstrom et al., 2001; Dahlenborg et al., 2001; Kimuraet al., 2001; Fach et al., 2002), enzyme activity-basedassays (Boyer et al., 2005; Schmidt et al., 2001; Liu et al.,2003; Wictome et al., 1999) and immunoassays (Shoneet al., 1985; Ekong et al., 1995; Doellgast et al., 1993;Peruski et al., 2002; Ahn-Yoon et al., 2004; Szilagyiet al., 2000; Poli et al., 2002). Since PCR only detects thebotulinum toxin gene, this assay could not detect abotulism case or a bioterrorist attack where the causalagent is the preformed toxin and not C. botulinum itself.The most recently published immunoassays exhibit asensitivity close to the mouse bioassay but only twopublications (Szilagyi et al., 2000; Poli et al., 2002)reported comparison of the results obtained by assayingeither the neurotoxin alone or associated with non-toxicproteins, further showing that these last can interferewith immunoassays.

Botulinum neurotoxins are produced complexed toother proteins, e.g. hemagglutinins (HA) and non-toxicnon-hemagglutinin (NTNH). These complexes, calledprogenitor toxins, differ in molecular weight dependingon the toxin and the corresponding type of association.BoNT/A complexes exists in three forms, M (300 kDa),L (500 kDa) and LL (900 kDa), BoNT/B, /C and /Dcomplexes have two forms (M and L), and BoNT/Fand /G occur in one form (M and L, respectively). Theneurotoxin alone is named form S (150 kDa). Amongother functions, these toxin-associated proteins protectthe neurotoxin from the effect of the acidic medium andproteases through the gastrointestinal tract (Sugii et al.,1977). It has thus been reported that in rats the oraltoxicity of the LL complex is 360 times that of the150 kDa (S) botulinum neurotoxin alone (Ohishi et al.,1977). Interestingly, the complexed forms of BoNTshave been used effectively as therapeutic reagentsagainst several neuromuscular disorders (Schantz andJohnson, 1992).

Complexing proteins modify the recognition of theneurotoxin by antibodies (Chen et al., 1997; Singh et al.,1996), and their diversity suggests the involvement ofdifferent areas of interaction, potentially leading to sterichindrance. Any immunoassay of the neurotoxin, in what-ever form, must take these observations into account.

The present paper describes the development of avery sensitive conventional two-site immunoassay forBoNT/A using a procedure which allows accurate quan-tification of all the neurotoxin forms, i.e. complexed ornot.

2. Materials and methods

2.1. Reagents

Unless otherwise stated, all reagents were from Sigma(St. Louis, MO). The culture supernatants of differenttoxinotypes of C. botulinumwere prepared from: BoNT/A, strain NCTC 2916; BoNT/B, strain BL8; BoNT/C,strain 468; BoNT/D, strain 1873; and BoNT/E, strainP34). BoNT (either purified or from culture supernatant)requires special handling precautions due to its toxicity.Appropriate laboratory attire should be worn, including alab coat, gloves and safety glasses. BoNT-contaminatedmaterials were inactivated by immersion in bleach solu-tion for 24 h.

A recombinant Fc BoNT/A fragment was prepared asdescribed previously (Tavallaie et al., 2004), corre-sponding to the binding domain of the toxin.

BoNT/Awas purified from NCTC2916 culture uponacidification, toxin extraction and ion exchange chro-matography as described previously (Shone and Tranter,1995).

When performing immunoassays, all reagents werediluted in enzyme immunoassay (EIA) buffer (0.1 Mphosphate buffer, pH 7.4, containing 0.15 M NaCl,0.1% bovine serum albumin (BSA) and 0.01% sodiumazide). Plates were washed with washing buffer (0.01 Mphosphate buffer, pH 7.4, containing 0.05% Tween 20).

2.2. Apparatus

Immunometric assay was performed using Titertekmicrotitration equipment from Labsystem (Helsinki,Finland), including an automatic plate washer (Washer120), an automatic plate reader (Multiskan Bichro-matic). Ninety-six-well microtitre plates (Maxisorp)were obtained from Nunc (Roskilde, Denmark).

2.3. Purification of acetylcholinesterase

Acetylcholinesterase (AChE) (EC 3.1.1.7) wasextracted from the electric organ of the electric eel,Electrophorus electricus, and purified by one-step affinitychromatography, as described by Massoulie and Bon(1976). The tetrameric form of AChE (the polymorphismof AChE is described in detail by Massoulie and Bon,

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1982) used in this study and named ‘G4’ was preparedfrom the purified AChE preparation by treatment withtrypsin (1 µg/ml) for 18 h at 25 °C in 0.1 M phosphatebuffer (pH 7; 1/2000 trypsin/AChE ratio (w/w)).

2.4. Measurement of acetylcholinesterase activity

Acetylcholinesterase (AChE) activity was measuredby the method of Ellman et al. (1961). Ellman's mediumcomprises a mixture of 7.5×10−4 M acetylthiocholineiodide (enzyme substrate) and 2.5×10− 4 M 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (reagent forthiol colorimetric measurement) in 0.1 M phosphatebuffer (pH 7.4). Enzymatic activity was expressed inEllman Units (EU). One EU is defined as the amount ofenzyme producing an absorbance increase of one unitduring 1min in 1ml of medium, for an optical pathlengthof 1 cm; it corresponds to about 8 ng of enzyme.

2.5. Immunization and hybridoma procedure

Fc BoNT/A (50 kDa), was used as immunogen. Anti-Fc BoNT/A antibodies were raised in mice (BALB/c)using the following procedure. On day zero, Fc BoNT/A(10 µg) emulsified in Freund's complete adjuvant wasinjected into the foot pad. The mice were bled 2 weekslater. Murine anti-Fc BoNT/A antibodies in the antiserawere detected by their capacity to bind Fc BoNT/A-biotin(see below). A booster injection of 10 µg of antigen wasgiven on day 21 and on day 35 in the foot pad. Bleedingswere performed 1 week after each booster. The mousewith the highest titre was selected for preparation ofmonoclonal antibodies. Three days before fusion, it wasgiven a final booster injection (10 µg Fc BoNT/A, i.v.injection). Spleen cells from the mouse were fused withNS1 mouse myeloma cells as previously described(Grassi et al., 1988). Anti-Fc BoNT/A antibodies in mye-loma culture supernatants were detected using an immu-noenzymatic test (Frobert and Grassi, 1998) (see below).

2.6. Labelling of Fc BoNT/A with biotin

Fc BoNT/A was labelled with biotin using an acti-vated N-hydroxysuccinimide ester of biotin that reactswith the primary amino group of the protein. Briefly,20 µl of 10 mg/ml biotin–NHS ester in anhydrous DMFwere added to 100 µg of Fc BoNT/A dissolved in 0.1 Mborate buffer (pH 8.5). After 1-h incubation at roomtemperature, 100 µl of 1 M Tris–HCl buffer (pH 8) wereadded for 15 min, before completing with 500 µl of EIAbuffer. This preparation was stored frozen at–20 °C untiluse.

2.7. Purification and labelling of monoclonalantibodies

Monoclonal antibodies (mAbs) from ascitic fluid werepurified using caprylic acid precipitation (Reik et al.,1987), and their purity was assessed by polyacrylamidegel electrophoresis (PAGE) in denaturing and reducingconditions. F(ab′)2 fragments were obtained frompurified antibodies by treatment with pepsin in acidicmedium. Fab′ fragments were obtained from F(ab′)2 byreduction in the presence of 0.02 M 2-mercaptoethyla-mine and subsequently coupled to AChE using theheterobifunctional reagent N-succinimidyl-4-(N-malei-midomethyl)-cyclohexane-1-carboxylate (SMCC) asdescribed previously (Grassi et al., 1989).

2.8. Labelling of mAbs with biotin

To determine the ‘binding complementarity’ of thedifferent mAbs, mAbs were labelled with biotin using anactivated N-hydroxysuccinimide ester of biotin thatreacts with the primary amino group of the antibodies.Briefly, 20 µl of a 10 mg/ml biotin–NHS ester inanhydrous DMFwere added to 500 µg of mAb dissolvedin 0.1 M borate buffer (pH 8.5). After 1-h incubation atroom temperature, 4 ml of EIA buffer were added. Thispreparation was stored frozen at −20 °C until use.

2.9. Screening of culture supernatants

Anti-Fc BoNT/A antibodies in hybridoma culturesupernatants was detected using an immunoenzymatictest. Briefly, 50 µl of each supernatant from 96-wellculture plates were transferred into microtitre platescoated with goat anti-mouse IgG/IgM antibodies, and50 µl of Fc BoNT/A-biotin (100 ng/ml) were added. Afterovernight incubation at 4 °C, the plates were washedand 100 µl of AChE-labelled streptavidin conjugate(2 EU/ml) were added to each well. After a further 4-hincubation at room temperature followed by severalwashings, 200 µl of Ellman's mediumwere added to eachwell. After 1-h incubation, the absorbance was measuredat 414 nm.

2.10. Determination of binding complementarity foreach pair of mAbs

The simultaneous binding of the different mAbs to asingle Fc BoNT/A or BoNT/A molecule was analyzedin immunometric tests, with one mAb immobilized onthe solid phase and the other used as a biotin-labelledtracer. Experiments were performed as follows: 100 µl

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of a 1 ng/ml Fc BoNT/A or 10 ng/ml BoNT/A solutionwere added with 100 µl of a 100 ng/ml biotin-labelledmAb solution to microtitre plate wells coated with oneof the mAbs. After 18-h incubation at 4 °C, the plateswere washed before adding 200 µl/well of AChE-labelled streptavidin conjugate (2 EU/ml). After afurther 1-h incubation at room temperature followedby several washings, 200 µl of Ellman's medium wereadded to each well.

2.11. Immunometric assays using mAb–AChE conjugates

Immunometric assays were performed in 96-wellmicrotitre plates coated with one of the mAbs aspreviously described (Creminon et al., 1993). Thepreliminary experiments were performed using a‘simultaneous procedure’, involving addition of 100 µlof BoNT/A solution (either purified or from C.botulinum culture supernatant) to each well togetherwith 100 µl of mAb–AChE conjugate at a concentrationof 5 EU/ml for an 18-h incubation at 4 °C. Each standardpreparation was prepared in duplicate and the blank(buffer alone) in 8-fold in order to determine theminimum detectable concentration. For further opti-mized protocols, a ‘sequential procedure’ was selected,corresponding to an initial 18-h reaction of the BoNT/Asolution in the wells at 4 °C followed by severalwashings and a further 2-h reaction of the mAb–AChEconjugate (100 µl/well at 5 EU/ml) at room temperature.After incubation of the tracer, the plates were extensivelywashed and solid-phase bound AChE activity wasrevealed by addition of 200 µl of Ellman's reagent fora 1-h reaction.

2.12. Determination of BoNT/A assay characteristics

Standard curves were expressed in terms of absor-bance values at 414 nm as a function of the BoNT/Aconcentration. The minimum detectable concentration(MDC) was taken as the concentration of standardinducing a significant increase (3 standard deviations) ofthe signal in the absence of BoNT/A standard (non-specific binding). The intra-assay (within-assay) coeffi-cients of variation were determined by assayingduplicates of four different BoNT/A concentrations ordilutions from C. botulinum culture supernatant, fivetimes on the same day. Inter-assay (between-assay)coefficients were determined by repeating this experi-ment on six different days.

The specificity of the assay was checked by establish-ing a standard curve for purified BoNT/B, /C, /E orBoNT/B, /C, /D or /E present in the corresponding

C. botulinum culture supernatants. Results were ex-pressed in percentage cross-reactivity (%CR), arbitrarilydefined as the ratio of the concentration of standard(BoNT/A) over the concentration of the other serotype(/B, /C, /D or /E) producing a 0.5 absorbance unit signal.

2.13. Chromatographic characterization of BoNT/Aimmunoreactivity

The immunoreactive material detected in C. botuli-num culture supernatant was fractionated via molecularsieve chromatography using an AKTÄ system and S300column (HiPrep 26/60 Sephacryl S-300 HR) (GEHealthcare Bio-Sciences AB Uppsala, Sweden). Elutionwas achieved with 0.1 M phosphate buffer (pH 7.4)containing 0.15 M NaCl and 0.01% NaN3 (buffer A) or0.1 M Tris buffer (pH 8) including 0.4 M NaCl and0.01% NaN3 (buffer B), with a flow rate of 1.3 ml/min.Two millilitres of a diluted C. botulinum culture super-natant (1/100 in buffer A or B) were injected and 2.5 mlfractions were collected. The immunoreactivity wasmeasured for each fraction using the immunometricassay previously described after a 1/100 dilution in EIAbuffer or in buffer B supplemented with 0.1% BSA. TheS300 column was calibrated with a mixture of bluedextran, thyroglobulin, ferritin, aldolase, and ovalbumin.Purified BoNT/A was eluted under the same conditionsas the standard.

2.14. Samples preparation

In this experiment, botulinum neurotoxin complexfrom supernatant was assayed by spiking differentmedia: 0.1 M phosphate buffer (pH 7.4) containing0.15 M NaCl, 0.1% bovine serum albumin (BSA) and0.01% sodium azide (control); tap water; bottled water;plasma; milk; orange juice and apple juice. The pH ofboth the orange and apple juice was adjusted to 7.4before adding the complex. Two millilitres of eachmedium were spiked with neurotoxin complex to reachfinal concentrations of 1000 and 1750 LD50 (approxi-mately 5 and 9 ng/ml). The solutions were mixed byshaking and incubated for 30 min at room temperaturebefore centrifugation at 7000 ×g for 30 min at 4 °C toremove solid particles or the lipid layer (milk). Theaqueous supernatants were carefully removed andsamples containing 1000 and 1750 LD50 were assayedafter diluting 2- and 5-fold in 0.1 M Tris buffer (pH 8)+0.4 M NaCl+0.1% BSA+0.01% NaN3, respectively. Asnegative control, the corresponding medium withoutneurotoxin complex was prepared and diluted under thesame conditions. These samples were quantified using a

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titrated C. botulinum culture supernatant as standard,diluted in 0.1 M Tris buffer (pH 8)+0.4 M NaCl+0.1%BSA+0.01% NaN3.

3. Results and discussion

3.1. Preparation of monoclonal anti-Fc BoNT/Aantibodies

Anti-Fc BoNT/A antibodies were raised in miceimmunized with Fc BoNT/A, and the mouse presentingthe highest titre (N1/50,000) via EIA was chosen forfusion. The corresponding spleen cells were fused withNS1 myeloma cells. One week after fusion, about 1083wells presented actively multiplying hybridomas. Thepresence of mouse anti-Fc BoNT/A antibodies waschecked by testing their capacity to bind biotin-labelledFc BoNT/A, allowing selection of 343 strongly positiveculture supernatants. The corresponding hybridomaswere subcloned by limiting dilution to give 14 stablesecreting lines that were expanding as ascitic tumours.Final purification of the different mAbs was achievedusing the caprylic acid precipitation method.

3.2. Development of a two-site immunometric assaywith purified BoNT/A

To establish the mAb complementarity, an exhaustiveanalysis of all possible combinations of mAb pairs (onefor capture and the other biotin-labelled as tracer) wasperformed. Of these 196 possibilities, only a single con-

Table 1Signals obtained for all possible combinations of mAb pairs

(A) Tests realised with Fc BoNT/A (1 ng/ml). (B) Tests realised with BoNT/signals between 2 and 1 Absorbance Units; light dots, signals N0.1 Absorba

centration of Fc BoNT/A (1 ng/ml) or BoNT/A (Sigma)(10 ng/ml) was tested in duplicate and compared to non-specific binding (NSB). The results of these experimentsare summarized in Table 1. Five groups of mAbsrecognizing five distinct epitopes on the Fc BoNT/Aprotein could be characterized. Groups A, B and Ccontain five (TA2, TA10, TA11, TA12 and TA14), four(TA4, TA7, TA13 and TA17) and three mAbs (TA1,TA5, TA15), respectively; groups D and E include only asingle mAb (TA9 and TA16, respectively). All the mAbsof one group presented binding compatible with themAbs of the other groups, but did not bind with them-selves or with mAbs of the same group. Preliminaryexperiments showed that 36 combinations provided uswith a good signal/NSB ratio for Fc BoNT/A, butamong these only five retained these good properties forBoNT/A. This discrepancy is probably due to conforma-tional differences between the entire BoNT/A and itsC-terminal fragment (binding domain).

Since the five best combinations for BoNT/Ameasure-ment involved the same three biotin-labelled mAbs astracer, these mAbs were selected and labelled with AChEto establish standard curves of BoNT/A using the othermAbs as capture antibody. Surprisingly, only minordifferences in terms of sensitivity were noted for a hugenumber of combinations, demonstrating that most of thesandwich assays detect less than 100 pg/ml of BoNT/Astandard in buffer (data not shown). However, since thisassay should be applied toBoNT/Ameasurement either inaqueous medium or possibly food extracts to detect thetoxin, we decided to select a sequential strategy involving

A (10 ng/ml). Black squares, signals N2 Absorbance Units; dark dots,nce Unit; white squares, no specific signal.

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a first incubation step on the solid phase followed bywashings to protect the enzyme activity from the possibledeleterious effects of the sample matrix. Using thisprocedure, the tracer TA4-AChE had the best properties(lowest NSB and highest signal) associated with differentmAbs for capture, i.e. TA-2, -3,-11, -12, -15 and -16, witha sensitivity similar to the initial simultaneous procedure(detection b100 pg/ml).

3.3. Measurement of BoNT/A in culture supernatant ofClostridium botulinum

The effective measurement of BoNT/A in complexedforms was investigated by assaying the toxin content in aC. botulinum culture supernatant previously titrated at 5106/ml lethal dose 50 (LD50) by mouse bioassay.Different dilutions of this culture supernatant in EIAbuffer were prepared and assayed using purified BoNT/A as standard. We thus obtained a 4 µg/ml BoNT/Aconcentration, which may correspond to 1.25×109 LD50

for 1 mg of BoNT/A. This is quite different from thevalue of 1.86×108 LD50/mg BoNT/A previouslyreported by DasGupta et al. (1966). Such a discrepancywas previously reported by Szilagyi et al. (2000) whenmeasuring BoNT/A alone or complexed using affinity-purified polyclonal antibodies obtained using a strategysimilar to that used here.Moreover, the same observationwas made by Poli et al. (2002) for serotype E usingantibody directed against the corresponding neurotoxinfragment. Chen et al. (1997) used several scFv thatbound specifically to different domains (translocation,catalytic, binding) of BoNT/A, and showed that theinteraction between HA and BoNT/A was probablymediated by the binding domain. Finally, although abuffer of pH above 7.2 (as the EIA buffer used here) was

Fig. 1. Effect of buffer composition on the immunoassay of purified BoNTpotassium phosphate buffer. All the following buffers contain 0.1% BSA andbuffer); (B) buffer pH 7; (C) (B)+0.15 M NaCl; (D) (B)+0.4 M NaCl; (E) buff(I) (H)+0.15 M NaCl; (J) (H)+0.4 M NaCl.

reported to dissociate the toxin/protein complex (Ferreiraet al., 2004), we assumed that the observed differencemay result from interference in the binding of the mAbsdue to the proteins complexed with BoNT/A, leading toan underestimation of the toxin concentration. To checkthis hypothesis, we decided to define a procedure todisrupt the BoNT/A–HA–NTNH complexes and toallow measurement of the ‘free’ neurotoxin.

Since ion-exchange chromatography proved usefulin purifying BoNT/A from its complex forms (e.g.DEAE-cellulose using a NaCl gradient in 0.15 M Tris–HCl buffer, pH 8) (DasGupta and Boroff, 1968), wepostulated that the separation of the toxin from HA andNTNH could only occur if the buffer used dissociatesthe complexes. To verify this assumption, BoNT/Aeither purified (1 ng/ml) or complexed (culture super-natant at 1/10000 dilution) was assayed after dilution indifferent buffers: 0.1 M Tris–HCl buffer (containing0.1% BSA) at different pHs (7, 8, 9) with different NaClconcentrations (0, 0.15, 0.4 M). To verify that a pHabove 7.2 releases the neurotoxin from the complex, thesame experiment was performed using potassiumphosphate and EIA buffers as control. To analyze theimpact of the different buffers only on the neurotoxin/protein complex dissociation (and not on the mAbaffinity), the results were expressed as a ratio (culturesupernatant signal/BoNT/A signal). As shown in Fig. 1,only minor changes were noted for the Tris–HCl buffer,whereas a marked decrease in the ratio was observedwhen the dilution of culture supernatant was performedin the different phosphate buffers at pH 7, 7.4 (EIAbuffer) or pH 8 with high NaCl concentration (0.4 M).These results demonstrated that neurotoxin in super-natant is better measured after dilution in the differentTris–HCl buffers or phosphate buffer at pH≥8, with

/A or complexed BoNT/A. White bars, Tris–HCl buffer; black bars,0.01% NaN3. (A) 0.1 M phosphate buffer (pH 7.4)+0.15 M NaCl (EIAer (pH 8); (F) (E)+0.15 0 NaCl; (G) (E)+0.4 M NaCl (H) buffer (pH 9);

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low NaCl concentrations. To determine if this increasecomes from the release of BoNT/A from the complex,molecular sieve chromatography of a diluted culturesupernatant was performed using either 0.1 M phos-phate buffer (pH 7.4)+0.15 M NaCl or 0.1 M Tris–HClbuffer (pH 8)+0.4 M NaCl as dilution and elution buffer.The collected fractions were assayed after dilution inboth elution buffers supplemented with 0.1% BSA and0.01% NaN3 using the immunometric assay. As shownin Fig. 2A, elution using phosphate buffer led to threeimmunoreactive peaks when the fractions were dilutedin phosphate buffer; an extra high molecular weightpeak was detected after dilution in Tris–HCl buffer. Theimmunoreactivity of the three other peaks was greaterafter dilution in Tris buffer. Conversely, the patternobtained using Tris–HCl buffer for elution (Fig. 2B)corresponded to a single peak (identified as the last peakin Fig. 2A) whose intensity was not modified by the

Fig. 2. Molecular sieve chromatography of Clostridium botulinum culture supphosphate buffer (pH 7.4)+0.15 M NaCl or (B) 0.1 M Tris–HCl buffer+0.4 Mbuffer (dotted line) or in 1 M Tris–HCl buffer (pH 8) containing 0.4 M NaCl, 0to the LL form (I), the L form (II), the M form (III), and the S form (IV).

buffer used for dilution. By reference to the molecularweight calibration performed for the column and theelution determined for purified BoNT/A, the differentpeaks were identified as the various BoNT/A-com-plexed forms, i.e. peak I corresponding to the LL form,peak II to the L form, peak III to the M form, and peakIV to the S form (toxin alone). As expected from theprevious results (see Fig. 1), the dilution of the fractionsin Tris–HCl buffer increased the immunoreactivity ofthe complexed forms, especially for the LL form, whichwas not detected in phosphate buffer. These experimentsdemonstrate that 0.1 M Tris–HCl buffer (pH 8)containing 0.4 M NaCl, 0.1% BSA and 0.01% NaN3

efficiently dissociates the BoNT/A–HA–NTNH com-plexes, since a single immunoreactive peak eluting astrue BoNT/A is recovered.

These results established that the optimal pH fordissociation of the toxin–protein complex depends on

ernatant using a Sepharose S300 column (26×60) eluted with (A) 0.1 MNaCl. The collected fractions were assayed after dilution either in EIA.1% BSA and 0.01%NaN3 (solid line). The different peaks correspond

Fig. 4. The immunoassay determination of BoNT/A complexes in foodor plasma samples. The samples were spiked with neurotoxincomplex, incubated for 30 min at room temperature, then diluted 2-or 5-fold in 0.1 M Tris–HCl buffer (pH 8) containing 0.4 M NaCl,0.1% BSA and 0.01% NaN3. White bars, 2-fold dilution; black bars,5-fold dilution.

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buffer composition. Thus, Tris–HCl buffer is efficient atall the pHs tested (7, 8, 9), whatever the NaCl concen-tration, and potassium phosphate buffer is efficient at apH≥8, with low NaCl concentration.

The BoNT/A content in culture supernatant wasassayed again using the 0.1 M Tris–HCl buffer (pH 8)containing 0.4 M NaCl, 0.1% BSA and 0.01% NaN3 fordilution. A 30 µg toxin/ml concentration was obtained,corresponding to 1.66×108 LD50/mg of toxin, a value inaccordance with the results of DasGupta et al., (1966).

This buffer was then selected for further dilution ofthe samples and for the immunometric assay.

3.4. Sensitivity, precision and specificity of theimmunometric assay

Fig. 3 presents the standard curves obtained using theselected pair of mAbs (TA-16 for capture immobilizedon the solid phase and TA-4-AChE as tracer) and asstandard purified BoNT/A (Sigma) or culture super-natant (diluted either in EIA buffer or in 0.1 M Tris–HClbuffer, pH 8, containing 0.4 M NaCl, 0.1% BSA and0.01% NaN3). As expected purified BoNT/A or culturesupernatant diluted in both dilution buffers providedsimilar standard curves; a lower immunoreactivity wasobserved for culture supernatant diluted in EIA buffer.

Fig. 3. Standard curve obtained with purified BoNT/A or culture supernatant dNaCl, 0.1% BSA and 0.01% NaN3, using the sequential procedure for the imtracer. Purified BoNT/A (5) and culture supernatant (n) diluted in EIA bufbuffer. The inset shows the low portion of the standard curves in detail.

This two-site immunometric assay reaches a minimumdetectable concentration (MDC) close to 40 pg/ml(270 atmol/ml) for purified BoNT/A and close to 8LD50/ml for culture supernatant. These results seem tobe close to the sensitivities reported in the very recentlypublished immunoassays (Szilagyi et al., 2000; Ahn-

iluted in EIA buffer or 0.1 M Tris–HCl buffer (pH 8) containing 0.4 Mmunometric assay with TA-16 as capture antibody and TA-4-AChE asfer, purified BoNT/A (q) and culture supernatant (z) diluted in EIA

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Yoon et al., 2004; Sharma et al., 2006; Attree et al.,2007).The present assay thus appears sensitive enoughto detect BoNT/A below the human lethal dose (oraldose), considered to be close to 70 µg of toxin for 70 kgof body weight (Arnon et al., 2001). We have alsocompared two purified BoNT/A, either from a commer-cial source (Sigma) or prepared by Dr Popoff. No effectof the dilution buffer was observed for both purifiedtoxins, demonstrating the absence of complexed formsbut the toxin purified by Dr Popoff provided a four timeshigher signal (data not shown). This discrepancy mayresult from the extent of toxin purity and/or differenttechniques used to quantify the standards and/or toxinproduced by different bacterial strains (different sub-types). These data further illustrate the need for aninternational standard for quantifying BoNT/A. Within-assay coefficients of variation (CV %) were 8.6, 3.5, 2and 2 for 200, 400, 1000 and 4000 pg/ml of purifiedBoNT/A, respectively, and 6.3, 2.8, 2 and 1.7 for 33,100, 250 and 1000 LD50 of culture supernatant, respec-tively. Between-assay coefficients of variation (CV %)were 17, 9, 5 and 6.4 for the same concentrations ofpurified BoNT/A, and 12.7, 5.5, 4.2 and 4.2 for the samedilutions of culture supernatant, respectively.

The specificity of the assay was evaluated byassaying purified BoNT/B, /C and /E or BoNT/B, /C, /D and /E from the corresponding C. botulinum culturesupernatants. We failed to detect these related proteins,demonstrating that the present sandwich assay isspecific to the BoNT/A toxin form.

3.5. Sample measurement

The quantification of neurotoxin complex in differentmedia (Fig. 4) shows that tap water, bottled water andorange juice did not induce interference whatever thedilution used. Nevertheless, milk induced a lowinterference for the 2-fold dilution, and plasma andapple juice showed interference for both dilutions. Weobtained a recovery yield of 68% for plasma and 74%for apple juice for the 5-fold dilution. This preliminarystudy only analyzed a few matrices compared with arecent publication (Sharma et al., 2006) but the presentimmunoassay allowed detection of BoNT/A in allmatrices, as reported by Sharma et al., and could alsogive an accurate quantification in 4 out of 6 matrices.

In conclusion, this work shows that the use of anappropriate dilution buffer permits an accurate quanti-fication of BoNT/A with a sandwich immunoassay,whatever the neurotoxin form. Preliminary experimentsshowed that the immunometric assay developed duringthis study could detect the neurotoxin in different

matrices, while correct quantification may require atleast a 10 fold dilution for some matrices. These resultsthus appeared promising although an extensive studyusing a larger range of alimentary matrices and othersubtypes of BoNT/A (possibly recognized differently bythe antibodies; Smith et al., 2005) is required to ensurefull validation of the method.

Acknowledgements

The authors are greatly indebted to Y. Frobert for herexpert technical assistance. This work was supportedby grants from the Commissariat à l'Energie Atomique(France).

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