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Applied and Environmental Microbiology, May 2000, p. 2278-2281, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Production and Characterization of Monoclonal
Antibodies against Vegetative Cells of Bacillus
cereus
Nadine
Charni,
Claude
Perissol,
Jean
Le Petit, and
Nathalie
Rugani*
Laboratoire de Microbiologie, Service 452, UPRES A 6116 CNRS, Faculté des Sciences et Techniques de St
Jérôme, Université Aix-Marseille, 13397 Marseille
Cedex 20, France
Received 20 December 1999/Accepted 3 March 2000
 |
ABSTRACT |
Two monoclonal antibodies (MAbs) against Bacillus
cereus were produced. The MAbs (8D3 and 9B7) were selected by
enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus
vegetative cells while failing to recognize B. cereus
spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa
antigen, while MAb 9B7 recognized two antigens with molecular masses of
approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination
to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.
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TEXT |
Vegetative cells and spores of
Bacillus cereus are present in the environment and can
frequently be found in many raw, dried, and processed foods (4,
13, 19, 23, 25, 31, 41). This bacterium has been implicated in
two different types of food poisoning, namely emetic and diarrheal
(26, 28), and is responsible for numerous cases of food
spoilage because of the production of lipases and proteases (7,
35). In addition, certain strains of B. cereus can
grow at temperatures as low as 4 to 6°C (41, 42), and
these psychrotrophic B. cereus strains are a health risk to
the consumer since vegetative cells can produce enterotoxins mainly in
the exponential phase (8, 15-17). It is impossible for the
food industry to exclude B. cereus from their products because, as many studies have shown, B. cereus cells can
survive heat processing and can grow in foods kept at refrigerated
storage conditions. Thus, it is important to develop methods to detect the presence of B. cereus in order to eliminate the threat
of food poisoning. Several selective plating methods described for detecting B. cereus require, with confirmatory testing, up
to 4 days to perform (21, 24, 32, 33, 39, 40). Other efforts
in B. cereus research have focused on detection of the organism by detection of enterotoxin-producing cells (1, 22, 29), and commercial kits designed to detect enterotoxic B. cereus via immunoassays have been developed (6, 7, 9).
In the food industry, immunoassays are also used to detect
spore-forming and non-spore-forming bacteria. Commercial
immunoassay-based kits that use either polyclonal antibodies
or monoclonal antibodies (MAbs) are available to detect
Salmonella, Listeria, and other organisms.
Immunoassays have been developed for the detection of
Bacillus and Clostridium spores by using
polyclonal antibodies and MAbs in enzyme-linked immunosorbent assays
(ELISAs) (11, 36, 37). Immunoassays have also been developed
for the vegetative cells of both spore-formers and nonsporeformers
(20). To date, however, there are no commercially available
ELISAs for the rapid detection of the vegetative cells of B. cereus in food products.
In this paper, we describe the production and characterization of two
MAbs against B. cereus. These antibodies were selected by
ELISA for their reactivity with B. cereus vegetative cells. The specificity of the selected antibodies was tested against bacterial
cells of a variety of species within and across genera and spores of
B. cereus. These antibodies, which are specific for
vegetative cells, can be used to develop a rapid and sensitive method
for the detection of strains of B. cereus in foods that potentially cause food poisoning.
Bacterial strains and culture conditions.
The bacterial
strains used in this study are shown in Table
1. All bacteria were grown at
30°C in Trypticase Soy medium (bioMerieux, Marcy l'Etoile,
France). A spore suspension of B. cereus cells was
prepared from an overnight culture of B. cereus vegetative cells that was inoculated on the sporulation medium described by
Faille et al. (10). To remove vegetative cell
remnants, spores were treated with a solution of thimerosal as
described by Norris and Wolf (34). After
centrifugation at 10,000 × g for 10 min, spores were
then incubated in TEL buffer containing 100 mM Tris-HCl (pH 8), 5 mM
EDTA, and 0.5% lysozyme at 50,000 U/mg for 1 h at 37°C.
For immunization procedure and immunochemical techniques, all bacterial
cells in an exponential growing stage and
B. cereus spores
were harvested by centrifugation at 10,000 ×
g for 10 min
at 4°C and washed twice in phosphate-buffered saline
(PBS).
Production of MAbs.
The MAbs were produced by the procedure
described by Galfre and Milstein (12). Vegetative cells of
B. cereus LMG 6923 (108/ml) were injected into
BALB/c mice. Hybridomas were screened for antibody production by ELISA
with vegetative cells of B. cereus LMG 6923 as antigens.
Selected hybridomas were cloned at least twice by limiting
dilution method. The MAb-secreting clones were propagated as ascitic
fluid by the procedure of Harlow and Lane (18). The
isotyping of MAbs was performed with a mouse monoclonal isotyping
kit according to the manufacturer's instructions. Antibodies were
concentrated by ammonium sulfate precipitation of ascites, and
immunoglobulin G (IgG) antibody was purified by using a protein A
column (18).
Immunochemical techniques. (i) ELISA.
The ELISA was done
according to the method described by Harlow and Lane (18),
with a few modifications. Briefly, microtiter plates (Immulon-1;
Dynatech, Chantilly, Va.) were coated overnight at 4°C with
108 bacterial cells or B. cereus spores per ml.
After blocking with PBS containing 3% nonfat dry milk and 0.05% Tween
20, the plates were incubated with the MAbs. Peroxidase-conjugated
goat anti-mouse IgG+IgM (Jackson Immunoresearch, Immunotech, Marseille,
France) and o-phenylenediamine (Sigma Chemical Co., St.
Quentin Fallavier, France) were used as secondary antibodies and
substrate, respectively. Absorbance was read at 490 nm by using a
microtiter plate reader (Metertech 
960 Instrument; BioBlock).
The ELISA procedure was also performed on plates coated with
trypsin-protease-treated
B. cereus vegetative cells to
determine
MAb specificity for protein antigens: wells at
10
8 cells/ml were treated with 1 mg of trypsin at
37°C for 4 h and
were treated again with 100 µg of protease at
37°C
overnight.
(ii) SDS-PAGE and immunoblotting.
B. cereus LMG 6923 (108 vegetative cells) was extracted by the method of Matar
et al. (30) and was subjected to electrophoresis through
sodium dodecyl sulfate (SDS)-12% polyacrylamide gels as described by
Laemmli (27).
Proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE)
were electroblotted by the method of Harlow and Lane (
18).
After transfer for 3 h at 1.2 A, the membrane was blocked for
1 h in PBS containing 5% nonfat dry milk and 0.5% Tween 20. MAbs
and peroxidase-conjugated goat anti-mouse IgG+IgM (Jackson
Immunoresearch)
were used as primary and secondary antibodies,
respectively. The
blots were visualized with chemiluminescence
(DuPont Co., Newtown,
Conn.).
For glycoprotein determination, proteins separated by SDS-PAGE were
exposed to periodic acid-Schiff staining (
38). The proteins
which stained positive as glycoproteins were determined and compared
to
the antigens determined by
immunoblotting.
Periodate oxidation was used to determine MAb specificity for
carbohydrate determinants (
2).
B. cereus cells
exposed to
various concentrations of sodium metaperiodate (0 to 0.05 M)
were
subjected to immunoblotting as indicated
above.
(iii) ELISA capture system.
ELISA capture system was performed
with MAb 9B7 used as a specific capture antibody and with biotinylated
MAb 8D3 used as a detector antibody (18). Briefly,
microtiter plates were coated with 2 µg of MAb 9B7 for 2 h at
37°C. All dilutions were performed in ELISA buffers. Vegetative cell
cultures of B. cereus were applied to wells for 1 h,
and detection of bound antigen was performed by application of
biotinylated MAb 8D3. Streptavidin-peroxidase (Sigma S 5512) was
applied for 0.5 h. Absorbance was read at 490 nm after addition of
the substrate o-phenylenediamine (Sigma Chemical Co.).
A total of nine hybridomas were screened by ELISA for their
reactivities with
B. cereus LMG 6923 vegetative cells. Of
these,
only two hybridomas secreted antibodies reactive with
B. cereus.
These MAbs, designated 8D3 and 9B7, were
found to be IgG1 and
IgM, with kappa light
chains.
The specificity of the MAbs was examined by ELISA with a panel of
select bacteria (Table
1). The results showed that the
MAbs recognized
not only vegetative cells of
B. cereus LMG 6923,
which was
used for immunization, but also the vegetative cells
of
B. cereus originating from food or environmental samples. MAb
8D3
reacted strongly with all
B. cereus strains and with
Bacillus thuringiensis subsp.
berliner and
B. thuringiensis subsp.
kurstaki.
MAb 8D3 reacted
weakly with
B. thuringiensis DSM 2046,
B. thuringiensis subsp.
israelensis, and
Bacillus
mycoides. MAb 9B7 reacted strongly
with
B. cereus
species and reacted weakly with
B. thuringiensis subsp.
berliner and
B. thuringiensis subsp.
kurstaki. However,
B. cereus is closely related
to
B. thuringiensis and
B. mycoides (
3,
5). These data could explain the cross-reactivity of
the
MAbs with vegetative cells of
B. thuringiensis or
B. mycoides.
None of these antibodies reacted with
B. cereus LMG 6923 spores.
In addition, both antibodies showed no
reactivity to several members
of the family of
Enterobacteriaceae (
Salmonella enteritidis,
Escherichia coli,
Proteus vulgaris,
Citrobacter freundii) and other bacteria
(
Micrococcus
luteus,
Pseudomonas stutzeri,
Listeria
monocytogenes).
The antigenic reactivity of the MAbs was destroyed when ELISA plates
were coated with trypsin-protease-treated vegetative
cells of
B. cereus. These results show that the antigens recognized
by MAbs
8D3 and 9B7 are proteinaceous in
nature.
MAbs were analyzed for the antigenic specificity by SDS-PAGE
followed by immunoblotting (Fig.
1).
B. cereus LMG 6923 vegetative
cell extracts were used for
electrophoretic studies. MAb 8D3 recognized
an antigen with a
molecular mass of 22 kDa. The antigens which
react with MAb 9B7
have molecular masses of approximately 58 and
62 kDa. Periodic
acid-Schiff staining indicated that the proteins
which react with MAb
9B7 may be glycoproteins. Treatment of
B. cereus cells with
sodium metaperiodate had no effect on the detection
of
B. cereus by MAb 9B7, as assessed by immunoblotting. This indicates
that the antigens which react with MAb 9B7 are not carbohydrates.
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FIG. 1.
Immunoblotting with MAbs against B. cereus
LMG 6923 vegetative cells. Shown are molecular mass standards (in
kilodaltons) (A) and B. cereus vegetative cells with MAbs
8D3 (B) and 9B7 (C).
|
|
ELISA experiments indicate that the proteins recognized by MAbs 8D3 and
9B7 are specific to vegetative cells, since the antibodies
did not
react with spores of
B. cereus. Furthermore, as shown
by
SDS-PAGE and immunoblotting analysis, MAbs 8D3 and 9B7 react
with
different proteins on the vegetative cells. This result indicates
that
these MAbs are able to capture vegetative cells of
B. cereus in an immunoassay. Our future research will focus on the use of
these
antibodies to develop immunoassays which will detect
B. cereus cells in food
products.
We need a rapid and reliable method to detect vegetative cells of
B. cereus, since checking for this pathogenic organism is
critical to ensure food safety. It is impossible for the food
industry
to completely avoid the presence of
B. cereus in their
products, and the consumption of foods containing 10
5
vegetative cells of
B. cereus per ml will result in food
poisoning.
Thus, the detection method should be sensitive enough to be
able
to detect low numbers of
B. cereus organisms
(
14).
We have therefore developed the following method to detect vegetative
cells of
B. cereus, which we tested in pure culture.
We used
MAbs 8D3 and 9B7 to develop an ELISA capture system. MAb
9B7 was
used as a specific capture antibody and MAb 8D3 was used
as a detector
antibody. The results show that the ELISA capture
system can detect and
quantify vegetative cells of
B. cereus (Table
2). We determined that the lower
detection limit was on the order
of 10
2 cells per ml and
the upper detection limit was on the order of
10
8 cells per
ml, with absorbance values of 0.224 to 1.805. So, this
work appears to
demonstrate the feasibility of detecting this
pathogenic organism in
food products with our ELISA capture system.
| |
ACKNOWLEDGMENTS |
We thank J. Perrier for valuable discussions and helpful
suggestions regarding this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Microbiologie, Service 452, UPRES A 6116 CNRS, Faculté des
Sciences et Techniques de St Jérôme, Université
Aix-Marseille, Avenue Escadrille Normandie Niemen, 13397 Marseille Cedex 20, France. Phone: (33) 4 91 28 81 90. Fax: (33) 4 91 28 80 30. E-mail:
Nathalie.Rugani{at}Microbio.u-3mrs.fr.
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Applied and Environmental Microbiology, May 2000, p. 2278-2281, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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