www.elsevier.com/locate/ijfoodmicro

International Journal of Food Mic

A rapid chemiluminescent slot blot immunoassay for the detection

and quantification of Clostridium botulinum neurotoxin type E,

in cultures

Brigitte Cadieuxa, Burke Blanchfielda, James P. Smithb, John W. Austina,*

aMicrobiology Research Division, Bureau of Microbial Hazards, Food Directorate,

Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada, K1A 0L2bMcGill University, Macdonald Campus, Department of Food Science and Agricultural Chemistry,

21 111 Lakeshore, Ste-Anne-de-Bellevue, Quebec, Canada, H9X 3V9

Received 18 March 2004; received in revised form 29 July 2004; accepted 7 October 2004

Abstract

A simple, rapid, cost-effective in vitro slot blot immunoassay was developed for the detection and quantification of

botulinum neurotoxin type E (BoNT/E) in cultures. Culture supernatants of 36 strains of clostridia, including 12 strains

of Clostridium botulinum type E, 12 strains of other C. botulinum neurotoxin serotypes, and 12 strains of other

clostridial species were tested. Samples containing BoNT/E were detected using affinity-purified polyclonal rabbit

antisera prepared against BoNT/E with subsequent detection of secondary antibodies using chemiluminescence. All

strains of C. botulinum type E tested positive, while all non C. botulinum type E strains tested negative. The sensitivity

of the slot blot immunoassay for detection of BoNT/E was approximately four mouse lethal doses (MLD). The intensity

of chemiluminescence was directly correlated with the concentration of BoNT/E up to 128 MLD, allowing quantification

of BoNT/E between 4 and 128 MLD. The slot blot immunoassay was compared to the mouse bioassay for detection of

BoNT/E using cultures derived from fish samples inoculated with C. botulinum type E, and cultures derived from

naturally contaminated environmental samples. A total of 120 primary enrichment cultures derived from fish samples, of

which 103 were inoculated with C. botulinum type E, and 17 were uninoculated controls, were assayed. Of the 103

primary enrichment cultures derived from inoculated fish samples, all were positive by mouse bioassay, while 94 were

also positive by slot blot immunoassay, resulting in a 7.5% false-negative rate. All 17 primary enrichment cultures

derived from the uninoculated fish samples were negative by both mouse bioassay and slot blot immunoassay. A total

of twenty-six primary enrichment cultures derived from environmental samples were tested by mouse bioassay and slot

blot immunoassay. Of 13 primary enrichment cultures positive by mouse bioassay, 12 were also positive by slot blot

immunoassay, resulting in a 3.8% false-negative rate. All 13 primary enrichment cultures that tested negative by mouse

bioassay also tested negative by slot blot immunoassay. The slot blot immunoassay could be used routinely as a

0168-1605/$ - s

doi:10.1016/j.ijf

* Correspondi

E-mail addr

robiology 101 (2005) 9–16

ee front matter D 2004 Elsevier B.V. All rights reserved.

oodmicro.2004.10.038

ng author. Tel.: +1 613 957 0902; fax: +1 613 941 0280.

ess: John_Austin@hc-sc.gc.ca (J.W. Austin).

B. Cadieux et al. / International Journal of Food Microbiology 101 (2005) 9–1610

positive screen for BoNT/E in primary enrichment cultures, and could be used as a replacement for the mouse bioassay

for pure cultures.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Clostridium botulinum; Botulinum neurotoxin type E (BoNT/E); Immunoassay; Detection; Quantification

1. Introduction

Clostridium botulinum produces botulinum neuro-

toxin (BoNT), which is the most toxic substance

known, as it is lethal to humans in nanogram quantities.

There are seven different BoNTs, types A through G,

based on their serological specificity (Hatheway,

1993). Types A, B, E, and F are responsible for all

outbreaks of human foodborne botulism.

C. botulinum spores are ubiquitous in soils and

sediments worldwide and their numbers and types vary

depending on the region (Austin and Dodds, 2000). C.

botulinum type E is most often implicated in foodborne

botulism outbreaks in northern coastal regions, such as

Alaska, Japan, northern Canada, Norway, and Den-

mark, with the predominant incriminated foods being

fish and sea mammals (Austin, 2003). The majority of

botulism cases occurring in Canada are caused by C.

botulinum type E.

At present, the mouse bioassay is the only official

method used to detect BoNTs due to its reliability and

sensitivity (Kautter and Solomon, 1977). Although the

mouse bioassay method can detect as little as 5 pg of

BoNT, it has a number of limitations: (i) it uses live

animals; (ii) it is expensive, since animal facilities and

trained personnel are required; (iii) it is time-consu-

ming, taking up to 3 days for completion. As a result, a

more rapid and cost-effective in vitro test is needed.

Many in vitro assays have been investigated as

alternatives to the mouse bioassay for detection of

BoNT. Most of these are based on immunochemical

detection where an antibody recognizes and binds to

BoNT (Boroff and Shu-Chen, 1973; Shone et al., 1985;

Dezfulian and Bartlett, 1991; Doellgast et al., 1993;

Potter et al., 1993; Szilagyi et al., 2000; Ferreira et al.,

2003). A secondary antibody, linked to an enzyme or a

radioactive tag, recognizes and binds to the primary

antibody. Addition of the substrate results in a

detectable signal at the site of antigen–antibody

complex. Other assays developed are based on the

endopeptidase activity of BoNT (Ekong et al., 1997;

Wictome et al., 1999). BoNT cleaves the specific

peptide leaving a C-terminal cleavage fragment to be

detected by a dual antibody system as described above.

These in vitro assays can be used to detect BoNT, both

qualitatively and quantitatively.

However, most of these assays have proven to be

time-consuming, complex, and/or expensive, often

requiring reagents that are not commercially available.

A more rapid, simpler, and less-expensive method to

detect BoNT/E seems to be required. Here we

describe an assay with such characteristics.

2. Materials and methods

2.1. Bacterial strains and culture conditions

Twelve strains of C. botulinum type E, three strains

of C. botulinum type A, three strains of group I C.

botulinum type B, three strains of group II C.

botulinum type B, three strains of group II C.

botulinum type F, and 12 other Clostridium species

were used in this study (Table 1). All cultures were

grown anaerobically at optimum temperatures for 4

days in special peptone–peptone–glucose–yeast extract

broth, SPGY (5% special peptone (Oxoid, Basing-

stoke, Hampshire, England), 0.5% peptone (Becton

Dickinson, Sparks, MD, USA), 0.4% glucose (Difco,

Detroit, MI, USA), 2% yeast extract (Difco), 0.1%

sodium thioglycollate (Sigma, St. Louis, MO, USA),

pH 7.2). Culture supernatants were harvested by

centrifuging the cultures at 23,500�g for 30 min at 4

8C, and serially diluted in gelatin phosphate buffer

(0.2% gelatin (Difco), 0.4% Na2HPO4 (BDH Inc.,

Toronto, ON, Canada), pH 6.2) as needed.

2.2. Production of polyclonal rabbit antiserum

against BoNT/E

The production of polyclonal rabbit antiserum

against BoNT/E was done according to the method of

Table 1

Bacterial strains and growth temperature

Species Strain Serotype Group Growth

temperature

(8C)

C. botulinum Russa E II 25

C. botulinum Bennetta E II 25

C. botulinum Gordona E II 25

C. botulinum 8550a E II 25

C. botulinum LG9604E2a E II 25

C. botulinum FE9508EMAa E II 25

C. botulinum MU9708EJGa E II 25

C. botulinum IG4MB02E3a E II 25

C. botulinum SW280Ea E II 25

C. botulinum SOKR-23E1a E II 25

C. botulinum SP455/456E2a E II 25

C. botulinum F108a E II 25

C. botulinum A6a A I 35

C. botulinum 17Aa A I 35

C. botulinum 62Aa A I 35

C. botulinum 368Ba B I 35

C. botulinum 13983IIBa B I 35

C. botulinum IB1-Ba B I 35

C. botulinum 17Ba B II 25

C. botulinum 2Ba B II 25

C. botulinum DB-2a B II 25

C. botulinum 70Fa F II 25

C. botulinum 190Fa F II 25

C. botulinum 610Fa F II 25

C. aurantibutyricum ATCC 17777b n/a n/a 35

C. baratii 4624c n/a n/a 35

C. beijerinckii A401d n/a n/a 35

C. bifermentans ATCC 638b n/a n/a 35

C. butyricum ATCC 19398b n/a n/a 35

C. difficile ATCC 9689b n/a n/a 35

C. hastiforme ATCC33268b n/a n/a 35

C. novyi B ATCC27606b n/a n/a 35

C. perfringens ATCC13124b n/a n/a 35

C. sordellii ATCC9714b n/a n/a 35

C. sporogenes ATCC 3584b n/a n/a 35

C. tetani A064d n/a n/a 35

n/a: not applicable, these strains do not produce BoNT.a Strains come from the Botulism Reference Service, Bureau of

Microbial Hazards, Health Products and Food Branch, Health

Canada, Ottawa, Ontario, Canada.b Strains come from the American Type Culture Collection,

Manassas, Virginia, U.S.A.c Strains come from Dr. L.V. Holdeman, Virginia Polytechnic

Institute and State University, Blacksburg, Virginia, U.S.A.d Strains come from the Laboratory Centre for Disease Control,

Population and Public Health Branch, Health Canada, Ottawa,

Ontario, Canada.

B. Cadieux et al. / International Journal of Food Microbiology 101 (2005) 9–16 11

Harlow and Lane (1988). 10 Ag of purified botulinal

toxoid type E (Wako Pure Chemicals Industries, Chuo-

Ku, Osaka, Japan) wasmixed with 900 Al of aluminium

hydroxide adjuvant (Alhydrogelk, Cedarlane Labo-

ratories Limited, Hornby, ON, Canada). Half of the

preparation was injected subcutaneously while the

other half was injected intramuscularly into the hind

leg of a female New Zealand white rabbit (Charles

River Canada, St-Constant, Quebec, Canada) weighing

2.5 kg. Five additional booster injections, of the same

composition as the first injection, were given every 45

days. The rabbit was bled 10 days after the final booster

injection, blood was collected by cardiac puncture, and

the serum was collected and stored in individual vials

(1 ml) at �20 8C.

2.3. Preparation of affinity-purified antibodies

A sulfhydryl-affinity column (Pierce, Rockford,

IL, USA) was prepared according to the manufactur-

er’s instructions by coupling 0.5 mg of C. botulinum

type E neurotoxin toxoid complex (Wako Pure

Chemicals Industries) to the column gel. Subse-

quently, 0.5 ml of antiserum was applied to the

prepared column and allowed to bind. The column

was washed with phosphate-buffered saline, PBS,

(100 mM NaCl, 3 mM KCl, 800 mM Na2HPO4, 1

mM KH2PO4, pH 7.2) and the antibodies were eluted

with 3 ml of glycine buffer (100 mM glycine, pH 2.5)

resulting in a sixfold dilution of the antibodies from

the original serum. The eluted affinity-purified anti-

bodies were immediately neutralized with 1 M Tris

buffer, pH 9.5.

2.4. Slot blot immunoassay

During slot blotting, 0.5 ml of culture supernatant

was applied to a slot blot manifold (Hoefer Scientific

Instruments, San Francisco, CA, USA) and drawn by

vacuum through a pre-wetted polyvinylidene fluoride

(PVDF) membrane (Schleicher and Schuell, Keene,

NH, USA). Unbound membrane sites were blocked

for 1 hour with 2% skim milk powder in Tris

buffered saline containing Tween-20 (0.05 M Tris–

HCl, 0.2 M NaCl, pH 7.4, 0.1% Tween-20, TBST).

The membrane was then incubated 2 h at room

temperature or, alternatively overnight at 4 8C, with a

1:1000 dilution (using whole antiserum) or 1:200

dilution (using affinity-purified antibodies) of rabbit

anti-BoNT/E antiserum diluted in 1% skim milk

powder in TBST. The membrane was washed three

B. Cadieux et al. / International Journal of Food Microbiology 101 (2005) 9–1612

times with TBST, and was incubated 1 hour at room

temperature with a 1:5000 dilution of horseradish

peroxidase-conjugated donkey anti-rabbit immuno-

globulin G (Amersham Pharmacia Biotech, Baie

d’Urfe, Quebec, Canada) diluted in TBST. Again,

the membrane was washed three times with TBST

and the blot was processed using the ECL+kchemiluminescence kit (Amersham Pharmacia Bio-

tech) according to the manufacturer’s instructions,

using the detection reagents supplied in the chem-

iluminescence kit. Chemiluminescence was detected

with a Kodak Imagestation 440 (Eastman Kodak,

Rochester, NY, USA) and the intensity of each band

on the slot blot was measured using the Kodak ID

Image Analysis Softwarek (Eastman Kodak).

2.5. Primary enrichment cultures

Inoculated fish samples: A variety of fish

samples including Alaskan pollock, smoked salmon,

Atlantic salmon, and Pacific salmon were weighed

(25 g) and inoculated with C. botulinum type E

(strains Russ, Gordon, Bennett, and 8550) at 103

spores/g. Each sample was homogenized separately

with 75 ml of gelatin phosphate buffer in a

Stomacher Lab blender 400 (Seward UAC House,

London, UK) for 4 minutes and centrifuged at

15,000�g for 20 min at 4 8C. Then, 50 ml of

SPGY broth was added to the pellet and the

samples were incubated anaerobically at 25 8C for

7 days. After incubation, the cultures were centri-

fuged at 23,500�g for 30 min at 4 8C, and

supernatants were filter-sterilized using a 0.45-Amsyringe filter (Whatman, Clifton, NJ, USA). Sam-

ples were assayed for BoNT/E using the mouse

bioassay and the slot blot immunoassay.

Environmental samples: Soil samples naturally

contaminated with C. botulinum type E spores were

collected around the coastline of Hudson Bay and

Ungava Bay, Northern Quebec, Canada. These

samples were weighed (30 g) and 30 ml of SPGY

broth was added. The samples were heated at 60 8Cfor 20 min and incubated anaerobically at 25 8C for 7

days. The cultures were centrifuged at 3700�g for 5

min at 4 8C and supernatants were filter-sterilized

using a 0.45-Am syringe filter (Whatman). Samples

were assayed for BoNT/E using the mouse bioassay

and the slot blot immunoassay.

2.6. Mouse bioassay

The mouse bioassay was performed as described

previously (Kautter and Solomon, 1977; Austin and

Blanchfield, 1997; Solomon and Lilly, 1998). Samples

were trypsinized and 0.55 ml was injected intraper-

itoneally into each of two 20-g mice (Charles River

Canada) per sample. Mice were observed for develop-

ment of botulism symptoms for up to 72 h. Mice

displaying a pinchedwaist and laboured breathingwere

euthanized. Samples found to be toxic by mouse

bioassay were subsequently neutralized with specific

C. botulinum type E monovalent antiserum (Aventis,

Toronto, ON, Canada) as described by Austin and

Blanchfield (1997) and Solomon and Lilly (1998), to

confirm that toxicity in mice resulted from BoNT/E.

3. Results

3.1. Specificity of the slot blot immunoassay

The specificity of whole polyclonal rabbit anti-

serum was tested against C. botulinum type E and

different Clostridium species. Pure cultures of C.

botulinum type E were grown from strains isolated

from clinical, food or environmental samples. BoNT/E

was detected in supernatants of every C. botulinum

type E culture (Fig. 1). Cross-reactivity was observed

with various proteins found in supernatants of C.

botulinum group I, type A (A6 and 62A), and type B

(368B, 13983IIB), C. botulinum group II, type F (70F,

190F, 610F), Clostridium bifermentans, Clostridium

butyricum, Clostridium hastiforme, Clostridium novyi

B, Clostridium perfringens, and Clostridium tetani

(Fig. 1). There was no cross-reaction with sterile

growth medium (SPGY broth) or dilution buffer

(gelatin phosphate buffer). Purification of the poly-

clonal rabbit antiserum was found to be necessary to

increase its specificity to BoNT/E.

A sulfhydryl-affinity column was prepared with C.

botulinum type E toxoid to affinity-purify specific

antibodies to BoNT/E from the polyclonal rabbit

antiserum. The specificity of the affinity-purified

antibodies was tested against C. botulinum type E

and different clostridial species. The affinity-purified

antibodies reacted only with culture supernatants from

C. botulinum type E (Fig. 2).

Fig. 2. Detection of BoNT/E by slot blot immunoassay using

affinity-purified antibodies. Slot blot of culture supernatants o

various strains of clostridia diluted 1/10 in gelatin phosphate buffe

and blotted onto a PVDF membrane developed by chemilumines

cence. Row A, columns 1–12: C. botulinum type E; Row B

columns 1–3: C. botulinum type A; columns 4–6: C. botulinum type

B (group I); columns 7–9: C. botulinum type B (group II); columns

10–12: C. botulinum type F; Row C, column 1: C. aurantibutyr

icum; 2: C. baratii; 3: C. beijerinckii; 4: C. bifermentans; 5

C. butyricum; 6: C. difficile; 7: C. hastiforme; 8: C. novyi B; 9: C

perfringens; 10: C. sordellii; 11: C. sporogenes; 12: C. tetani; Row

D, columns 1–10: 2-fold serial dilution of BoNT/E interna

standards 256 MLD to 0.5 MLD; column 11: SPGY, 1/10; column

12: gelatin phosphate buffer.

Fig. 1. Detection of BoNT/E by slot blot immunoassay using

polyclonal rabbit antiserum. Slot blot of culture supernatants of

various strains of clostridia diluted 1/10 in gelatin phosphate buffer

and blotted onto a PVDF membrane developed by chemilumines-

cence. Row A, columns 1–12: C. botulinum type E; Row B,

columns 1–3: C. botulinum type A; columns 4–6: C. botulinum type

B (group I); columns 7–9: C. botulinum type B (group II); columns

10–12: C. botulinum type F; Row C, column 1: C. aurantibutyr-

icum; 2: C. baratii; 3: C. beijerinckii; 4: C. bifermentans; 5:

C.butyricum; 6: C. difficile; 7: C. hastiforme; 8: C. novyi B; 9: C.

perfringens; 10: C. sordellii; 11: C. sporogenes; 12: C. tetani; Row

D, columns 1–10: 2-fold serial dilution of BoNT/E internal

standards 256 MLD to 0.5 MLD; column 11: SPGY, 1/10; column

12: gelatin phosphate buffer.

B. Cadieux et al. / International Journal of Food Microbiology 101 (2005) 9–16 13

3.2. Sensitivity of the slot blot immunoassay

The sensitivity of the slot blot immunoassay using

affinity-purified antibodies was compared to the

sensitivity of the mouse bioassay for detection of

BoNT/E. Serially, twofold diluted culture supernatant

of C. botulinum type E Russ was injected intra-

peritoneally into mice to determine the titre of BoNT/

E in mouse lethal dose (MLD). One MLD corre-

sponds to the highest dilution of BoNT/E found to be

toxic in two out of two mice. Each dilution was also

analyzed using the slot blot immunoassay to compare

the sensitivity of the method to the mouse bioassay

(Fig. 3a). Use of affinity-purified antibodies with the

slot blot routinely allowed detection of BoNT/E to a

sensitivity of 4 MLD.

3.3. Quantification of BoNT/E by slot blot

immunoassay

The serial dilutions used to determine the sensitivity

of the assay were also used as standards for quantifi-

cation of BoNT/E by slot blot immunoassay. Since the

toxicity (in MLD) of each standard dilution had been

titrated, it was possible to compare the toxicity of each

dilution to the intensity of chemiluminescence on the

slot blot (Fig. 3b). A direct correlation was observed

between the intensity of chemiluminescence on the

slot blot and the toxicity of the standards between 4

and 128 MLD. At levels greater than 128 MLD, the

intensity of chemiluminescence became saturated and

reached a plateau.

3.4. Detection of BoNT/E in primary enrichment

cultures

The slot blot immunoassay using affinity-purified

antibodies was evaluated and compared to the mouse

bioassay for detection of BoNT/E in primary enrich-

ment cultures. A total of 120 enrichment cultures

derived from fish samples, of which 103 were

inoculated with C. botulinum type E, and 17 were

uninoculated controls, were assayed. Of the 103

enrichment cultures derived from inoculated fish

samples, all were positive by mouse bioassay, while

f

r

-

,

-

:

.

l

Fig. 3. (a) Sensitivity of the slot blot immunoassay using affinity-purified antibodies. Slot blot of 2-fold serial dilution (256 MLD to 0.5 MLD)

of C. botulinum type E Russ culture supernatant blotted onto a PVDF membrane developed by chemiluminescence. (b) Standard curve used for

quantification of BoNT/E, expressed in MLD, based on the intensity of chemiluminescence.

B. Cadieux et al. / International Journal of Food Microbiology 101 (2005) 9–1614

94 were also positive by slot blot immunoassay. The

other nine enrichment cultures were negative by slot

blot immunoassay, resulting in a 7.5% rate of false-

negative results. All 17 cultures derived from unin-

oculated fish samples were negative by both mouse

bioassay and slot blot immunoassay. Therefore, no

false-positive results were detected. Similar results

were obtained with 26 primary enrichment cultures

derived from environmental samples, of which 13 were

naturally contaminated with C. botulinum type E

spores. All 13 enrichment cultures derived from

naturally contaminated environmental samples were

positive by mouse bioassay, while 12 were positive by

slot blot immunoassay, resulting in a 3.8% false-

negative rate. All of the 13 enrichment cultures derived

from non-contaminated environmental samples were

negative by both mouse bioassay and slot blot

immunoassay. All samples positive by mouse bioassay

were neutralized with monovalent antiserum type E,

indicating that all positive samples were a result of

BoNT/E.

4. Discussion

The slot blot immunoassay could be used to detect

BoNT/E in either pure or primary enrichment cultures

of C. botulinum type E. Although cross-reaction was

detected when using polyclonal rabbit antiserum, it

was removed using affinity-purification to isolate only

the antibodies that specifically reacted with BoNT/E.

The cross-reaction observed in this study was similar

to that observed by Sakaguchi et al. (1974) who

reported low levels of cross-reaction between C.

botulinum type E and type F using the reversed

passive hemagglutination assay. Notermans et al.

(1982), Dezfulian and Bartlett (1991), and Dezfulian

(1993) also demonstrated cross-reaction of BoNT/E

antitoxin with C. botulinum type A and type B when

using different ELISA methods.

The sensitivity of the slot blot immunoassay using

affinity-purified antibodies was 4 MLD, similar to

that obtained by other immunoassays developed in

recent years. Dezfulian and Bartlett (1984), Shone et

al. (1985), Hallis et al. (1996), and Ferreira et al.

(2003), all reported sensitivities between 5 and 10

MLD using an enzyme-linked immunosorbent assay

(ELISA). Doellgast et al. (1993) achieved a sensi-

tivity of less than 1 MLD using an ELISA with

signal amplification via enzyme-linked coagulation

assay (ELCA). Others, such as Ekong et al. (1997)

and Wictome et al. (1999) were also able to obtain a

sensitivity of less than 1 MLD using an endopepti-

dase assay. Although, the sensitivity of the slot blot

B. Cadieux et al. / International Journal of Food Microbiology 101 (2005) 9–16 15

immunoassay using affinity-purified antibodies is

less than that of the mouse bioassay. The slot blot

immunoassay detected BoNT/E in all pure cultures

tested, indicating that it could be used as a replace-

ment for the mouse bioassay when testing pure

cultures.

Using the correlation between the intensity of

chemiluminescence on the slot blot and the toxicity of

the sample, BoNT/E could be quantified in any

sample containing between 4 and 128 MLD. Samples

containing more than 128 MLD could be diluted for

further quantification. To prevent possible chemilu-

minescence variations between slot blots, standards

were applied to each slot blot immunoassay.

Finally, the slot blot immunoassay was used to

detect BoNT/E in primary enrichment cultures derived

from various types of fish inoculated with C.

botulinum type E, and also from naturally contami-

nated environmental samples. While no false-positive

results were detected, some false-negative results were

obtained with both inoculated fish and naturally

contaminated environmental samples. These false-

negative results may have been due to some cultures

containing less BoNT/E than the sensitivity threshold

of the slot blot assay.

The slot blot immunoassay appears to have

advantages over other assays for detection of

BoNT/E. It is a simple assay requiring inexpensive

reagents that are all commercially available. This

assay is simpler than many other immunoassays. For

example, the ELCA–ELISA assay (Doellgast et al.,

1993) works by multiple amplification steps requir-

ing many different enzymes and substrates while the

endopeptidase assay (Wictome et al., 1999) requires

that all samples run through an immunoaffinity

column before detection by ELISA. The slot blot

immunoassay is also rapid, taking less than 6 h for

completion, while other assays in which comparable

sensitivities and specificities can be obtained gen-

erally take between 20 and 24 h (Dezfulian and

Bartlett, 1985; Shone et al., 1985; Doellgast et al.,

1993; Ekong et al., 1997; Ferreira et al., 2003).

Additionally, during a single slot blot assay, up to 36

samples can be tested; and the slot blot immunoassay

can be used to rapidly quantify BoNT/E in any

sample. The speed of this assay and the potential for

screening a large number of samples are critical

aspects to the food industry.

While the slot blot immunoassay has high specif-

icity and sensitivity approaching that of the mouse

bioassay, it cannot replace the mouse bioassay as the

official method for detection of BoNT/E. However, it

can be used as a screening tool for the detection of

strongly positive samples prior to testing them by the

mouse bioassay. Further studies need to be done to

improve the sensitivity of the slot blot immunoassay

in order to detect samples with low levels of BoNT/E,

such as contaminated foods.

Acknowledgements

This study was supported by Health Canada,

McGill University, and le Ministere de l’agriculture

des pecheries et de l’alimentation du Quebec

(MAPAQ). The authors would like to acknowledge

A. Minkley for preparing polyclonal rabbit antiserum

against BoNT/E as well as S. Rutherford and D.

Leclair for technical assistance.

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