Biotechnology and Bioprocess Engineering 2007, 12: 80-85
Screening of a Specific Monoclonal
Antibody against and Detection of Listeria
monocytogenes Whole Cells Using a Surface
Plasmon Resonance Biosensor
1 BioNanotechnology Research Center, KRIBB, Daejeon 305-806, Korea
2 Department of Chemical Engineering, Chungnam National University, Daejeon 305-764, Korea
3 Department of Food Science and Technology, Gyeongsang National University, Jinju 660-701, Korea
4 Department of Food Science and Technology, Chung-Ang University, Seoul 155-756, Korea
5 Department of Environmental Engineering and Biotechnology, Hankuk University of Foreign Studies, Yongin 449-791, Korea
6 Department of Biological Sciences, College of Natural Science, Sungkyunkwan University, Suwon 440-746, Korea
7 Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, Korea
Abstract In this study, a specific monoclonal antibody against Listeria monocytogenes was screened using an SPR biosensor.
Monoclonal antibodies were bound to protein L, after which the L. monocytogenes cells were subjected to an affinity as-
say. Protein L was immobilized on a carboxymethyl dextran (CM-Dex) surface via an amine coupling method, and util-
ized repeatedly by regeneration. The monoclonal antibody, ‘A18’, was selected and employed for the high-sensitivity de-
tection of L. monocytogenes. Under optimized conditions, 103 cells/mL or 50 cells were detected by the SPR biosensor.
Keywords: surface plasmon resonance (SPR), protein L, Listeria monocytogenes, antibody
Listeria monocytogenes causes listeriosis, and has been
detected in a variety of environments, including foods .
Listeriosis occurs around the world, with an annual inci-
dence of approximately 7 cases per million of the normal
population . Its incidence tends to be higher in pregnant
women, the elderly, and people with weakened or sup-
pressed immune systems [3,4]. The traditional culture-based
technique for the detection of L. monocytogenes requires
5~10 days for complete identification, and is a fairly labor-
intensive process. Several alternative detection methods have
been developed for the rapid and sensitive detection of L.
monocytogenes, including ELISA, PCR, DNA microarray,
Tel: +82-42-860-4445 Fax: +82-42-879-8594
and biosensor methods [5-7].
SPR biosensor technology is one of the most promising
detection methods currently in use for rapid pathogenic iden-
tification . It is sensitive to changes in the thickness or
refractive indices of biomaterials at the interface between a
thin gold film and an ambient medium, and is thus capable
of characterizing biomolecular interactions in real time with-
out the need for labeling [9,10]. As SPR sensors are capable
of detecting changes in a refractive index of several hundred
nanometers over the sensor surface, bacterial cell particles
specifically bound to an antibody can induce a large SPR
angle shift, as compared with biomolecules covering an
identical surface area . A number of SPR biosensors
have been developed for the direct detection and identifica-
tion of pathogens, including E. coli O157:H7 , Le-
gionella pneumophila , Salmonella typhimurium ,
and L. monocytogenes [6,15]. The lower detection limit var-
ies depending on the species, and its range has been deter-
Biotechnol. Bioprocess Eng. 81=
mined as 102~107 cells/mL.
In this study, we selected a specific monoclonal antibody
(mouse IgG1) against L. monocytogenes, and also attempted
to detect it directly. Leonard et al. reported that the detection
limit via indirect assay was 105 cells/mL, which measures
the amount of free anti-L. monocytogenes antibody existing
in the supernatant after the centrifugation of the mixture of
the anti-L. monocytogenes and a sample of L. monocyto-
genes . The direct detection of microbial cells is often
difficult, owing principally to their large size. In order to
assay whole L. monocytogenes cells with an SPR biosensor,
it is first necessary to find a high-affinity antibody for the
cell and to optimize the detection surface. In this study,
monoclonal anti-L. monocytogenes mouse antibodies were
screened with an SPR sensor. In the majority of relevant
reports, the selection of a specific antibody has been con-
ducted via ELISA, in which the cells are attached to plates
followed by the binding of candidate antibodies and a tagged
secondary antibody. The antibody selected via the ELISA
method, however, may not prove adequate for direct binding
to cells, due to its large size, and according to the report of
Hearty et al., this antibody proved insufficient to detect less
than 107 cells/mL of L. monocytogenes . In this study,
specific antibodies were screened via direct binding to cells
using an SPR biosensor. It was determined that the screened
antibody evidenced a high degree of sensitivity for the direct
detection of L. monocytogenes.
MATERIALS AND METHODS
HBS buffer (pH 7.4, 10 mM 4-[2-hydroxyethyl]pipera-
zine-1-ethanesulfonic acid, 150 mM sodium chloride, and
0.005% (v/v) surfactant P-20) and an amine coupling kit
hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 1.0
M ethanolamine-HCl (pH 8.5) were purchased from Biacore
AB (Uppsala, Sweden). Bovine serum albumin (BSA) and
dextran (M.W. 500,000) were purchased from Sigma (St.
Louis, MO, USA), and 11-mercaptoundecanoic acid (MUA),
11-mercapto-undecanol (MUOH), epicholrohydrin, diethyl-
ene glycol dimethyl ether, bomoacetic acid (99 + %), H2SO4,
and H2O2 were acquired from Aldrich (Milwaukee, WI,
USA). The gold chip for the SPR biosensor (2 nm of chro-
mium as an adhesion layer and 50 nm of gold deposited on a
25 mm circular glass) was obtained from K-MAC (Daejeon,
Korea). Protein L was purchased from Pierce (Rockford, IL,
Preparation of Monoclonal Antibodies
L. monocytogenes (ATCC 19114) was grown in tryptic
soy broth (TSB) medium (Difco, Ditroit, USA) for 24 h at
37oC to stationary phase. The formalin-killed L. monocyto-
genes (FKLM) samples were then prepared. In brief, the
bacteria were harvested via centrifugation at 5,000 rpm at
4oC and washed twice with PBS (10 mM NaH2PO4, 2 mM
KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4). The washed
cells were then resuspended in PBS containing 0.3% formal-
dehyde and maintained for 12 h at room temperature. The
cells were then washed three times via the previously de-
scribed method and suspended in PBS. The FKLM was
stored at −70oC until used. Five female BALB/c mice, each
8 weeks of age, were immunized with 109 FKLM cells in 0.1
mL of sterilized PBS, which was emulsified with an equal
volume of Freund’s complete adjuvant. Boost injections
were administered 2, 4, and 6 weeks afterward. One week
after the third injection, the sera were collected from the
caudal vein of each of the experimental mice. Titers of antis-
era were assessed via indirect non-competitive ELISA.
Three days prior to cell fusion, the mice from whom antisera
were collected which evidenced high titers were adminis-
tered an additional intraperitoneal boost injection without
adjuvant. Cell fusion was accomplished via the method de-
veloped by Kohler and Milstein . The mice whose antis-
era evidenced high titers were then sacrificed, and the im-
mune spleen cells were fused with myeloma cells (V653)
using 1 mL of 50% polyethylene glycol 1500. The fused
cells were then screened with Hypoxanthine Aminopterin
Thymidine (HAT) medium, composed of Dulbecco’s Modi-
fied Eagle Medium (DMEM) containing 20% FBS, 100
units/mL gentamicin, 100 μM hypoxanthine, 0.4 μM amin-
opterin, and 16 μM thymidine, for 10 days after fusion. The
cell solution was then centrifuged, and the supernatant was
utilized without further purification. The subclass of the
mouse antibody was identified as IgG1, and the light chain
was verified to be of the κ-type, using a commercial kit
(Boehringer Mannheim, Germany).
Preparation of Carboxymethyl Dextran (CM-Dex)
The bare gold chip was cleaned for 30 min with a solution
of H2SO4/H2O2 (3/1) at 60oC. It was then immersed in a 10
mM ethanolic solution of MUOH at room temperature for
20~24 h for the formation of a self-assembled MUOH
monolayer on the gold surface. It was then washed with
ethanol and water, and sonicated for 5 min in ethanol. In
order to activate the hydroxyl group of MUOH, the gold
chip was treated for 4 h with 0.6 M epichlorohydrin in a 1:1
mixture of 0.4 M NaOH and 2-methoxyethyl ether. After
washing with ethanol and water, the gold chip was immersed
for 20 h in 0.3 mg/mL of dextran solution in 0.1 M NaOH.
The dextran-coated surfaces were then treated for 16 h with
1 M bromoacetic acid in 2 M NaOH solution. The gold chip
was washed with DW and dried with N2 gas. It was then
maintained in a refrigerator (4oC) until use.
SPR measurements were conducted using a cuvette-based
AutoLab ESPRIT instrument (Eco Chemie, Utrecht, Nether-
lands). The disk gold chip was attached to a half-cylinder
prism with refractive-index matching oil (nD = 1.517), and
was inserted into the AutoLab SPR instrument. The volume
of the loaded sample was 50 μL. Following each step, the
gold chip was washed and equilibrated with a 10 mM
HEPES buffer containing 0.15 M NaCl and 0.05% Tween 20
(pH 7.4). All experiments were conducted at a mixing rate of
16.7 μL/sec and a mixing volume of 10 μL, at 25oC.
Screening of L. monocytogenes-specific Antibodies
Using the SPR Biosensor
The CM-Dex gold chip was inserted to the AutoLab sys-
tem and was activated for 7 min with a mixture of 0.4 M
EDC and 0.1 M NHS. Protein L (0.1 mg/mL) was allowed to
react for 30 min in 10 mM acetic acid, and 1 M ethanola-
mine (pH 8.5) was allowed to react for 5 min in order to
deactivate any unreacted surfaces. Monoclonal antibodies in
HAT medium were permitted to react for 15 min, after
which L. monocytogenes (109 cells/mL in TSB medium) was
allowed to react for 30 min. The surface was then regener-
ated for 5 min with 10 mM glycine buffer (pH 2.0). The se-
quential reaction entailing antibody binding, L. monocyto-
genes whole cell binding, and regeneration was conducted
on the same protein L surface at least 20 times.
Assay of L. monocytogenes Using a Selected
The bare gold chip was cleaned for 30 min with a solution
of H2SO4/H2O2 (3/1) at 60oC. It was then immersed in a 10
mM ethanolic solution of MUA at room temperature for
20~24 h for the formation of a self-assembled MUA
monolayer on the gold surface. It was then washed with
ethanol and sonicated in ethanol for 5 min. The self-
assembled MUA gold chip was subjected to the AutoLab
system, and was activated for 7 min by a mixture of 0.1 M
EDC and 0.025 M NHS. Protein L (0.1 mg/mL) was reacted
in 10 mM acetic acid for 30 min, and 1 M ethanolamine (pH
8.5) was allowed to react to deactivate any unreacted sur-
faces. A screened monoclonal antibody was allowed to react
for 15 min, and then whole L. monocytogenes cells were
allowed to react for 30 min. The surface was regenerated for
5 min with 10 mM glycine buffer (pH 2.0).
RESULTS AND DISCUSSION
Screening of a Specific Antibody against L.
Antibodies evidencing high titers were prescreened via the
method described in the Materials and Methods section, and
29 antibodies were selected in total. Protein L was employed
to immobilize antibodies followed by binding to the L.
monocytogenes cells. Protein L specifically binds the kappa
(κ) light chain of immunoglobulins without interfering with
the antigen-binding site . Mouse IgG1 exhibits higher
affinity with protein L than with protein G or protein A
[17,18]. Protein L was immobilized onto a CM-Dex surface
Fig. 1. A sensorgram showing the antibody affinity testing pro-
cedure using L. monocytogenes cells on a CM-Dex sur-
face. (a) EDC/NHS activation, (b) immobilization of pro-
tein L, (c) blocking with ethanolamine, (d) antibody im-
mobilization, (e) 109 L. monocytogenes cells, (f) regen-
via amine coupling . The CM-Dex surface is the most
frequently used in SPR biosensors, because it effectively
prevents nonspecific binding . Antibody immobilization,
cell binding tests, and regeneration were successively con-
ducted. The protein L immobilized onto the CM-Dex surface
could be employed more than 20 times without any loss of
binding affinity. A typical sensorgram, shown in Fig. 1, dis-
plays information regarding EDC/NHS activation, protein L
immobilization, blocking with ethanolamine, antibody im-
mobilization, the binding of L. monocytogenes cells, and
regeneration. The quantity of protein L bound to the CM-
Dex was 164 mD, equivalent to 1.3 ng per 1 mm2 of surface
area (1 mD of the AutoLab SPR system corresponds to 8 RU
of the Biacore Instruments system, and 1 RU is equivalent to
1 pg per 1 mm2 of surface area.) . The quantity of an
antibody bound to the protein L surface was 600 mD, a value
which indicates that 87% of protein L was used to bind the
antibody, considering that the molecular weights of protein L
and IgG were 35.8 and 150 kD, respectively. After the bind-
ing test was conducted on the L. monocytogenes cells, the
bound antibody and L. monocytogenes cells were removed
with glycine buffer at a pH of 2.0, and the protein L surface
was utilized again.
The amounts of prescreened antibodies immobilized onto
the protein L surface and L. monocytogenes whole cells
bound to the antibody surface are summarized in Fig. 2.
Several antibodies, including A6, A13, A14, A17, A18, and
A22, evidenced relatively high binding affinity with L.
monocytogenes cells. A negative shift in the SPR signal after
the pouring of the cell solution (cells suspended in the cul-
ture media, TSB) was observed with certain antibodies, such
as A7, A11, and A16, which appeared to have been removed
from the sensor surface. The A18 antibody, which exhibited
the most pronounced cell binding activity, was selected for
use in further investigations of detection sensitivity in the
direct whole cell binding assay.
Detection of Whole L. monocytogenes Cells Using
Assay conditions were optimized for the highly sensitive
Biotechnol. Bioprocess Eng. 83=
Fig. 2. SPR angle shift for binding of mouse monoclonal antibodies on protein L (A) and binding of 109 cells/mL L. monocytogenes to
the antibody (B).
Table 1. Comparison of three procedures for the cell binding with the A18 antibody
SPR angle shift (mD)
on protein L
Antibody on protein L
solution on antibody
Cells on antibody
CM-Dexa 164 13 561 01 29
MUA-1b 081 46 353 27 84
MUA-2c 081 46 353 02 29
aSame to the procedure used in screening antibodies.
bMUA surface was used instead of CM-Dex surface in the CM-Dex procedure.
cIn the MUA-1 procedure, PBST buffer was used instead of TSB medium as a cell suspending solution.
detection of L. monocytogenes cells, using the selected anti-
body (A18) as a capture probe. The previous assay condition
utilized in the screening of the specific antibody proved in-
feasible as a sensitive detection method, as the deviations in
separate measurements became prodigious at low cell con-
centrations (data not shown). It has been assumed that these
measurement deviations were caused by the 3-dimensional
properties of the CM-Dex surface, which may prevent L.
monocytogenes cells from approaching antibodies immobi-
lized onto the sensor surface, due to the random orientation
of the antibodies . As an alternative to the CM-Dex sur-
face, the self-assembled MUA monolayer, which is used
extensively in protein immobilization procedures, was as-
sessed for its feasibility as a protein L−immobilizing surface.
The immobilization of protein L on the MUA surface was
generally the same as that on the CM-Dex surface, and a
diluted EDC/NHS activation solution was employed. In the
cell binding test, PBST buffer (10 mM NaH2PO4, 2 mM
KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, pH
7.4), as well as TSB medium, was employed as a cell-
suspension solution. The experimental data comparing the
MUA and CM-Dex surfaces are shown in Table 1. The
MUA surface evidenced a higher level of nonspecific bind-
ing, whereas components contained within the DMEM solu-
tion were bound nonspecifically to protein L immobilized
onto the MUA surface (on the MUA surface: 46 mD, as
compared to 13 mD on the CM-Dex surface). The compo-
nents containing TSB solution, which was utilized as the
cell-cultivation medium, also bound abundantly to the anti-
bodies attached to the protein L−immobilized MUA surfaces.
The data generated in the MUA-1 procedure as compared
with the CM-Dex procedure revealed that, although the level
of nonspecific binding was higher than with the CM-Dex
surface, the binding of L. monocytogenes cells was also more
Fig. 3. SPR assay of L. monocytogenes cells, using A18 anti-
body as a capture probe.
pronounced. PBST buffer was utilized as a cell-suspension
solution in an effort to overcome the nonspecific binding
problem associated with the MUA-1 procedure. As is shown
in the MUA-2 procedure (Table 1), PBST solution resulted
in an absence of nonspecific binding signals, and the degree
of cell binding was almost identical to that observed in the
CM-Dex procedure. Thus, the MUA-2 procedure was se-
lected for the quantification of the L. monocytogenes cells, as
the nonspecificity issues observed in the MUA-1 procedure
and the non-reproducibility inherent to the CM-Dex proce-
dure were effectively overcome in the MUA-2 procedure.
Each concentration of L. monocytogenes (103~109 cells/mL)
was assayed in duplicate. As is shown in Fig. 3, 103 L.
monocytogenes cells/mL, or 50 cells (considering the sample
volume was 50 µL) were detectable. This degree of detection
sensitivity is significantly higher than has been reported in
other studies [6,15].
We have demonstrated an improved method for SPR bio-
sensor detection, evidencing superior sensitivity to whole L.
monocytogenes cells, using surface modification and anti-
body screening protocols. Monoclonal anti-L. monocyto-
genes mouse antibodies were screened with the SPR sensor.
Although in most such research, the ELISA method is em-
ployed for screening, a specific antibody was selected in this
case via the direct binding of cells to antibody candidates
using the SPR biosensor. It was determined that the antibody
selected in this study evidenced a high degree of sensitivity
for the direct detection of L. monocytogenes. Under opti-
mized conditions, 103 L. monocytogenes cells/mL, or 50
cells, were detectable.
Acknowledgements This study was supported by a grant
from the Korea Health 21 R&D Project, Ministry of Health
& Welfare, Republic of Korea (03-PJ1-PG1-CH11-0003)
and the KRIBB Research Initiative Program.
Received November 1, 2006; accepted January 23, 2007
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