BACKGROUND
the foodborne pathogen listeria monocytogenes causes listeriosis—a severe illness that ranges from mild gastroenteritis to invasive infection in immunocompromised people, neonates, and the elderly
<cit> .
in pregnant women, it causes premature births, miscarriages, and neonatal sepsis or fetal deaths.
l. monocytogenes is ubiquitous and found in food-processing environments
<cit>  and food products, including ethnic soft cheese
<cit> , sliced lunch meats
<cit>  and frankfurters, and seafood
<cit> .
it has been implicated in numerous food outbreaks and recalls, including a large outbreak involving cantaloupe in the us, which caused  <dig> deaths and  <dig> miscarriage
<cit> .
listeriosis has an estimated 19% fatality rate and ranks third among all fatalities resulting from foodborne infections in the usa
<cit> .
therefore, many countries have established a “zero tolerance” policy towards l. monocytogenes in rte foods
<cit> .
food recalls have increased each year, placing an economic burden on food manufacturers and growers.
rapid and accurate detection methods may alleviate some of these problems.
the genus listeria consists of  <dig> species: l. monocytogenes, l. ivanovii, l. seeligeri, l. welshimeri, l. innocua, l. grayi, and two new species, l. marthii <cit>  and l. rocourtiae <cit> .
l. monocytogenes and l. ivanovii are pathogenic to humans and animals
<cit> .
many virulence and structural genes or gene products in listeria could be used as targets for antibody- or nucleic acid-based assay development
<cit> .
l. monocytogenes expresses several virulence proteins
<cit> , including internalin a , which promotes bacterial adhesion and invasion of the host cell
<cit> .
inla possesses n-terminal leucine-rich repeats that facilitate anchoring to the bacterial cell wall, while the most distal extracellular domain binds to e-cadherin, which is crucial for host cell–cell adhesion and maintenance of tissue architecture.
both pathogenic and non-pathogenic listeria species can be found in the same environment or food
<cit> .
however, when an enrichment step is used, the non-pathogenic species may overgrow and outcompete l. monocytogenes <cit> , leading to false-negative results.
l. innocua is the most frequently found bacteria in listeria-contaminated foods
<cit> , thus presenting a challenge for the specific capture and detection of pathogenic listeria <cit> .
hence, it is essential to develop methods that are capable of detecting pathogenic species in the presence of non-pathogenic species.
immunological approaches to detect pathogens in food are attractive; however, assay performance depends on the quality and specificity of the antibodies used
<cit> .
for detection of listeria, two types of assay specificities are desired: listeria genus- or l. monocytogenes-specific tests.
anti-listeria antibodies available from research laboratories or commercial vendors are associated with problems of low affinity
<cit> , reaction to heterologous antigens
<cit> , lack of reaction towards all serotypes of l. monocytogenes <cit> , lack of reaction due to physiological stress induced by growth media or assay parameters
<cit> , and lack of compatibility with certain bioassay platforms
<cit> .
thus, there is a need for continued efforts to produce high-quality antibodies.
the recovery of low numbers of pathogens from complex food matrices also impedes their rapid and sensitive detection
<cit> .
antibodies are routinely used as affinity ligands to separate and concentrate the target analyte from sample matrices using paramagnetic beads
<cit>  and also as recognition or reporter molecules on immunoassay platforms
<cit> .
the pmb-captured cells may be presumptively identified by plating them on selective or differential media
<cit> , or their identity may be confirmed by pcr
<cit> , flow cytometry
<cit> , or cytotoxicity assay
<cit> .
the use of a biosensor to detect cells captured by immunomagnetic separation  is an attractive approach due to increased speed, accuracy, and detection of a low number of targets
<cit> .
fiber-optic sensors utilize laser excitation to generate an evanescent wave in order to quantify biomolecules immobilized on an optical waveguide
<cit> .
a capture antibody is immobilized on the waveguide and a fluorescent -labeled second antibody is used as a reporter for the target analyte.
once the laser beam  travels through the optical waveguide, it undergoes total internal reflection, and the fluorophore on the reporter antibody bound to the analyte is excited, thereby generating an evanescent wave.
the signal is propagated back up to the fiber and is detected in real time by a fluorometer.
this format has been successfully applied to many foodborne microorganisms and toxins, however, the limit of detection largely depends on the antibody and the reagents used
<cit> .
in the present study, monoclonal antibodies  against l. monocytogenes and listeria spp.
were generated, characterized, and employed to concentrate l. monocytogenes using pmbs.
finally, mabs were used on the fiber optic sensor to detect l. monocytogenes from inoculated food products .
in parallel, qpcr and light-scattering sensor methods were performed to confirm the results.
RESULTS
mab production and characterization by elisa and western blotting
we selected  <dig> stable hybridomas, of which  <dig>  reacted with both rinla and l. monocytogenes cells, and  <dig>  reacted with l. monocytogenes, l. innocua, and l. seeligeri.
after another round of screening of mabs-2d <dig>  -3b <dig>  -4e <dig>  and -3f <dig> against rinla or l. monocytogenes cells  by elisa, we chose mab-2d <dig>  and mab-3f <dig>  for future use.
an elisa  revealed that, among the anti-inla antibodies, mabs-2d <dig> and -3b <dig> strongly reacted  with l. monocytogenes 4b cells, while mab-4e <dig> gave slightly lower reaction values .
the listeria genus-specific mab-3f <dig> gave strong elisa values  when tested against other listeria spp., without producing significant cross-reactions with other bacterial species .
in the western blot, mab-2d <dig> reacted with an 80-kda protein band  from l. monocytogenes and l. ivanovii, but it did not react with other listeria spp., including l. marthii or l. rocourtiae .
mab-2d <dig> was reactive with all  <dig> serotypes; however, a relatively weak reaction with  <dig> strains of serotype 1/2c  was observed.
mab-2d <dig> also reacted with a 66-kda band from serotype 3c , which is presumably a truncated inla-protein variant
<cit> .
mab-2d12-reactive inla was distributed in the secreted, cell wall, and intracellular protein fractions of bacteria .
immunofluorescence microscopy confirmed the specific binding of anti-inla antibody  to the surface of l. monocytogenes cells, but it did not react with l. innocua .
mab-3f <dig> showed a strong reaction with a single protein band of ~30 kda  from all listeria spp.
with the exception of l. welshimeri .
in addition, this mab showed strong reactions with protein preparations from all  <dig> serotypes of l. monocytogenes .
bacterial capture using antibody-coated paramagnetic beads
pmbs with mab-2d <dig> had higher capture efficiency than those with mab-3f <dig>  using the same antibody, the smaller-sized  myone beads displayed significantly higher capture efficiency than the dynabeads m- <dig>  for l. monocytogenes 4b  and l. ivanovii  .
the capture efficiency curve with different concentrations of l. monocytogenes cells  was bell-shaped; the highest capture  was obtained at 105 cfu/ml, while the lowest capture was obtained at concentrations of 103 cfu/ml and at 107–108 cfu/ml .
at initial l. monocytogenes concentrations of  <dig>   <dig>  and 106 cfu/ml, myone-2d <dig> captured  <dig> %,  <dig> %, and  <dig> % of cells, respectively, while m-280-2d <dig> captured 15%,  <dig> %, and  <dig> %, respectively.
these values were significantly different  from mab-3f <dig> conjugated to myone or m- <dig> .
a similar trend was seen for l. ivanovii, but the values obtained were lower than those for l. monocytogenes.
therefore, the capture efficiency depends on antibody performance, bead size, and initial bacterial concentration.
abacteria were grown in tsb-ye for 18 h at 37 °c.
the data are average of  <dig> experiments analyzed in duplicate.
values labeled with different letters  in a row or in a column are significantly different at p <  <dig> .
all subsequent ims experiments were performed using myone beads.
the fluorescence microscopic image in figure
4b shows the capture of l. monocytogenes by myone-2d <dig>  the capture efficiency of myone-2d <dig> and myone-3f <dig> was evaluated with bacteria grown in the recommended enrichment broths, leb or fb.
myone-2d <dig> showed significantly higher  capture of l. monocytogenes and l. ivanovii than other listeria spp., and the capture efficiency was similar for leb or fb .
the capture efficiency for myone-2d <dig> was comparable for the l. monocytogenes serotypes tested, including 4b , 1/2a , and 1/2b , as well as for a strain of l. ivanovii , and negligible capture of other listeria spp.
was observed .
myone-3f <dig> displayed similar capture efficiency for all listeria spp.
tested, irrespective of the enrichment broths used .
when the capture efficiency of myone-2d <dig>  myone-3f <dig>  and dynabeads anti-listeria was compared against a listeria panel, myone-2d <dig> captured the most pathogenic listeria.
for all other listeria spp., both myone-3f <dig> and dynabeads anti-listeria had similar values .
thus, myone-2d <dig> is highly specific for the capture of pathogenic listeria, and myone-3f <dig> and dynabeads anti-listeria displayed similar capture efficiency for all listeria spp.
tested.
the capture efficiency of pmbs for l. monocytogenes in a co-culture with l. innocua was also determined .
the bacteria were grown in fb, mixed  in pbs to achieve concentrations of ~1 × 105 cfu/ml each and the capture efficiency was determined by plating followed by bardot-based colony identification.
myone-2d <dig> captured ~104 cfu/ml  of bacteria, of which most colonies  were confirmed to be l. monocytogenes by bardot .
myone-3f <dig> captured ~ <dig>  ×  <dig> cells , and ~50% were confirmed to be l. monocytogenes.
dynabeads anti-listeria captured ~ <dig>  × 103 cfu/ml , of which 40% were l. monocytogenes.
we also investigated the capture efficiency of bacteria from inoculated food matrices.
hotdogs were inoculated with ~10 cfu/g each of l. monocytogenes 4b and l. innocua as a mono- or co-culture and enriched for 18 h at 37°c.
myone-2d <dig> showed higher capture of l. monocytogenes  than l. innocua  in the monocultures, but in the co-culture experiment the total bacterial capture dropped to  <dig> %.
myone-3f <dig> captured  <dig> % of the l. monocytogenes cells in the monoculture experiment, while the commercial dynabeads anti-listeria captured only  <dig> % .
dynabeads anti-listeria also captured a numerically  higher percentage of l. innocua  compared with l. monocytogenes  .
overall, these data show that myone-2d <dig> captured 10-fold more l. monocytogenes than l. innocua, while myone-3f <dig> captured  <dig> -fold more l. monocytogenes than l. innocua.
dynabeads anti-listeria had the highest capture efficiency for l. innocua from hotdogs.
the capture of listeria was also investigated with soft cheese made from goat’s milk in a co-culture experiment .
cheese samples were inoculated with l. monocytogenes 4b  and l. innocua  and enriched in fb for 18 h until the total count reached ~ <dig>  × 108 cfu/ml.
the bacterial capture using myone-2d <dig> was  <dig>  ±  <dig> %, while myone-3f <dig>  and dynabeads anti-listeria  showed significantly  lower capture efficiency .
capture of l. monocytogenes colonies on bhi agar plates was verified by a light-scattering sensor, with l. monocytogenes and l. innocua producing distinct scatter patterns .
specificity and limit of detection of the fiber-optic sensor
the specificity and limit of detection  of the fiber optic sensor were analyzed by using mab-2d <dig> as capture antibody and cy5-labeled mab-2d <dig> as a reporter.
the sensor generated strong signals against l. monocytogenes and l. ivanovii, with a maximum signal of  <dig>  pa. in contrast, non-pathogenic listeria produced a maximum signal of  <dig> – <dig>  pa , and non-listeria bacteria, including salmonella typhimurium; e. coli o157:h7; and background food contaminant isolates, staphylococcus aureus, s. epidermidis, enterobacter cloacae, and lactococcus lactis <cit> , produced signals of ~ <dig>  pa .
similar results were obtained when mab-3f <dig> was used as the capture and mab-2d <dig> as the reporter molecule .
in the mixed cultures containing l. monocytogenes, l. innocua, and e. coli o157:h <dig> , the signals for mab-2d <dig> and mab-3f <dig> were  <dig>  ±  <dig>  pa and  <dig>  ±  <dig> pa, respectively, which were significantly  higher than the values obtained for l. innocua  or e. coli  alone .
the background control  values ranged from 504–  <dig> pa. therefore, both fiber-optic sensor configurations, 2d12–2d <dig> and 3f8–2d <dig>  are highly specific for pathogenic listeria, and specificity was contributed primarily by anti-inla mab-2d <dig>  other combinations did not produce satisfactory results .
the lod was also evaluated by using pure cultures of l. monocytogenes and l. ivanovii serially diluted in pbs .
using mab-2d <dig> as the capture molecule, the signals increased proportionately as the bacterial concentration increased until a cell concentration of 1 × 106 cfu/ml was reached, which gave the maximum signal , almost reaching the threshold of the analyte  <dig> fluorometer.
the lowest cell concentration that was considered positive  was 3 × 102 cfu/ml for l. monocytogenes  and 1 × 103 cfu/ml for l. ivanovii .
these values were at least 2-fold higher than those produced by the samples with  <dig> cells or pbs .
when mab-3f <dig> was used as capture antibody, the lod for l. monocytogenes  and l. ivanovii  was ~1 × 105 cfu/ml .
ims coupled with a fiber-optic sensor for detection of l. monocytogenes
bacteria captured by myone-2d <dig> or myone-3f <dig> were detected by the mab-2d12-coated fiber-optic sensor  and yielded signals of  <dig>  ±  <dig>  pa and  <dig>  ±  <dig>  pa, respectively .
the mab-3f <dig> fiber optic sensor  produced signals of  <dig>  ±  <dig>  pa and  <dig>  ±  <dig>  pa, respectively .
the fiber optic signal value for myone-2d <dig> and -3f <dig> captured l. monocytogenes was about  <dig> to 3-fold higher than the signals obtained from the lod concentrations  .
these data indicate that l. monocytogenes detection using mab-2d <dig> for ims and a fiber optic sensor gave better results compared with those obtained using mab-3f <dig>
in soft cheese-containing co-culture of l. monocytogenes and l. innocua, both myone-2d <dig> and myone-3f <dig> captured bacteria and produced signals of  <dig>  ±  <dig>  pa and  <dig>  ±  <dig>  pa, respectively .
bacteria captured with dynabeads anti-listeria gave the lowest fiber-optic signals .
in listeria-inoculated hotdog samples, only myone-2d <dig> was used for ims and assayed by fiber optic sensor.
the signal from the sample containing both l. monocytogenes and l. innocua was  <dig>  ±  <dig>  pa, while that from l. monocytogenes- and l. innocua-inoculated food was  <dig>  ±  <dig>  pa and  <dig>  ±  <dig>  pa, respectively .
for both food samples, the fiber optic signal values for myone-2d <dig> and -3f <dig> captured l. monocytogenes but not the l. innocua were higher than the signals obtained from the lod cell concentrations  .
therefore, the ims and fiber optic sensor can be used together for detection of l. monocytogenes from enriched food samples, even in presence of l. innocua or other bacteria.
real-time qpcr for validation
real-time qpcr targeting hlya was used to quantify pmb-captured listeria from hotdogs and goat’s cheese artificially contaminated with l. monocytogenes and l. innocua .
when ims was applied to the cheese samples followed by qpcr, myone-2d <dig> showed cell counts that were  <dig> times higher than those of myone-3f <dig> and dynabeads anti-listeria.
in hotdog samples, myone-2d <dig> produced cell counts that were 2– <dig> times higher than those of the other  <dig> types of beads.
aqpcr analysis is based on hlya.
primers to 16s gene sequences were used as internal control.
bdata are average of  <dig> experiments run in triplicate.
values labeled with letters  in a column are significantly different at p <  <dig> .
discussion
the recovery of low numbers of target pathogens from complex food matrices is a challenge for sensitive detection methods
<cit> .
ims using pmbs is used to separate and concentrate target pathogens from food samples before detection by plating, immunoassay, pcr, or biosensor methods
<cit> .
antibodies
<cit>  or alternative molecules
<cit>  are used as capture molecules for ims, and improvements in reagents and assay platform development are essential to enhance assay performance.
the specific detection of whole cells of l. monocytogenes using immunological methods relies on highly specific antibodies with a strong affinity for bacterial surface antigens
<cit> .
the antigen target should be uniformly distributed on the target organism, covalently anchored to the cell wall, and accessible to the antibody
<cit> .
inla is a well-characterized protein that is highly specific to l. monocytogenes and l. ivanovii, and it has all the desirable properties of an antigen
<cit> .
thus, we produced mabs against inla  and p <dig> .
the resulting mabs were employed in ims to capture and concentrate bacteria from food followed by fiber-optic sensor-based detection.
to the best of our knowledge, this is the first demonstration of the combined use of these two approaches.
inla-specific antibody production was facilitated by the use of whole cells of l. monocytogenes and purified rinla as immunogens.
hybrid b-lymphocyte clones secreted antibodies with a strong reaction towards live whole cells, but a weaker reaction was observed with heat-killed cells .
since rinla was soluble, denaturing agents were not required before immunization.
thus, the native structure of inla during the immune response was preserved, and the resulting antibody recognized the native protein on the surface of bacteria.
the inla-specific mab-2d <dig> reacted with all known l. monocytogenes serotypes, whereas previously reported mabs failed to recognize all  <dig> serotypes
<cit> .
only serotype 1/2c showed a weak reaction with mab-2d <dig>  however, this strain has been involved in a few sporadic cases of listeriosis
<cit>  and is rarely found.
moreover, none of the  <dig> strains of serotype 1/2c expressed a functional, full-length inla
<cit> , which may explain why mab-2d <dig> displayed a reduced reaction to 1/2c.
when tested with serotype 3c, mab-2d <dig> reacted strongly with a ~66 kda band instead of the normal 80-kda inla band.
the smaller band may represent truncated inla, which results from inla mutation
<cit> .
generally, such strains are less invasive and are less likely to cause systemic infection as confirmed in animal models
<cit> .
we also generated a listeria species-specific mab by immunization with whole cells of l. monocytogenes.
mab-3f <dig>  reacted with a ~30-kda protein  present in all eight listeria species.
therefore, mab-3f <dig> may aid tracking of listeria contamination in foods or the food-production environment.
the separation of target organisms following primary enrichment using ims is faster than using selective secondary enrichment
<cit> .
thus, we performed ims using two different sizes of commercial beads.
antibody-coated 1-μm myone t <dig> exhibited significantly higher capture efficiency than the  <dig> -μm m- <dig> beads .
similarly, foddai et al.
<cit>  used six different magnetic beads, including the two types used in this study, to capture mycobacterium avium.
myone displayed better capture efficiency than that of m- <dig>  but the overall capture efficiency was low .
in the present study, the capture efficiency for myone-2d <dig> and m280-2d <dig> was  <dig> % and  <dig> % , respectively while  <dig> % for myone-3f <dig> and  <dig> % for m280-3f <dig>  paoli et al.
<cit>  used m-280-coated scfv antibody to acta and reported a maximum capture of 19% for l. monocytogenes.
walcher et al.
<cit>  reported a capture range of 46%–122% using a bacteriophage endolysin specific for listeria spp.
coated on m-280; however, the long capture incubation time  may have allowed bacterial growth, thereby producing a higher capture rate.
furthermore, the binding of bacteriophage to host cells is an irreversible process, which may lead to higher capture efficiency than with antibody-coated pmbs.
koo et al.
<cit>  used hsp60-coated m- <dig>  which showed a capture efficiency for l. monocytogenes of  <dig> %– <dig> %.
the capture efficiency also depended on the initial bacterial concentration.
the highest capture  with myone-2d <dig> or myone-3f <dig> was seen at a bacterial concentration of 105 cfu/ml .
this is important for meaningful comparisons to be made between the performances of ims in different studies, which may use a wide range of initial bacterial concentrations.
collectively, ims data indicate that beads with a smaller diameter  have better capture efficiency than larger beads  due to higher surface area to mass ratio and smaller beads can bind more antibody per mg of beads  .
furthermore, the antibody affinity, the distribution/expression of antigens on the surface of bacteria, and the initial bacterial concentration also significantly affect capture efficiency
<cit> .
here, the abundant expression of inla on the surface of l. monocytogenes cells coupled with the use of smaller sized pmb was most likely responsible for increased capture efficiency.
however, the assay performance may be affected if pmb followed by fiber optic sensor was applied to food samples directly without an enrichment step.
in such situation, food matrices may affect bacterial antigen expression or antibody affinity
<cit> .
we tested the capture efficiency of l. monocytogenes in a co-culture experiment in buffer or food.
food contaminated with l. monocytogenes may contain other listeria spp.
and background competitive microflora
<cit> .
l. monocytogenes grows slowly and is a poor competitor; hence, lower cell numbers are expected in food samples
<cit> .
in a mixed population, l. monocytogenes may be outgrown by other species of listeria during enrichment
<cit> .
here, ims using myone-2d <dig> efficiently captured l. monocytogenes, in the presence of l. innocua while both myone-3f <dig> and dynabeads anti-listeria captured more l. innocua cells than l. monocytogenes .
furthermore, the capture efficiency for myone-2d <dig> using a co-culture in buf-fer or food varied from  <dig> %– <dig> % .
less than optimal level of capture was attributed largely to the presence of higher initial concentrations of bacteria  in the sample and the presence of interfering agents  in food matrices, particularly in soft cheese.
furthermore, the increased capture of l. monocytogenes in hotdog compared to pbs was possibly due to increased expression of mab-2d12-reactive antigen  during enrichment while cells used in pbs were originally cultured in bhi, which may have caused reduced inla expression resulting in reduced l. monocytogenes capture .
l. ivanovii is an opportunistic human pathogen that is associated with gastroenteritis and bacteremia in humans
<cit> ; therefore, the development of methods to detect this pathogen is also essential.
mab-2d <dig> reacted with l. ivanovii, which was successfully detected by using ims and a fiber-optic sensor.
hearty et al.
<cit>  reported the inla-specific mab-2b3; however, this antibody was unable to detect l. ivanovii in their assay setup.
mab-2b <dig> may be specific for an epitope of inla on l. monocytogenes that is absent on l. ivanovii.
pmb-captured cells were also identified by bardot and qpcr.
bardot is a light-scattering sensor that detects and identifies bacterial colonies on agar plates with a high degree of precision in minutes, since each species has a distinctive scatter-fingerprint signature
<cit> .
bardot allowed quantitative estimation of capture rate for l. monocytogenes and l. innocua on bhi or mox plates  instantly based on colony scatter patterns and it is easy to perform without the requirement for any additional reagents or probes.
real-time qpcr confirmed that l. monocytogenes capture and detection from food by myone-2d <dig> was 13%–16%, which is significantly higher than that by myone-3f <dig> and dynabeads anti-listeria .
these estimations are slightly higher than the plate count and the light-scattering data obtained in this study.
yang et al.
<cit>  used nanoparticles for ims and showed better capture and detection of l. monocytogenes in milk with real-time pcr  compared with plate counts .
this may be because qpcr detects dna from nonviable or viable but non-culturable cells, which may not otherwise be detected by traditional plating methods
<cit> .
the fiber-optic sensor operates based on the principles of antibody-antigen interaction and is marketed by research international.
it is currently used for foodborne or biothreat agent detection
<cit> .
the antibody  used in this study on the optical waveguide made the assay highly specific for l. monocytogenes and l. ivanovii, with the detection limit of 3 × 102 cfu/ml, a significant improvement over previous reports.
geng et al.
<cit>  used mab-c11e <dig> to show cross-reaction with some l. innocua strains with lod of  <dig>  × 103 cfu/ml.
using a polyclonal anti-listeria capture antibody and an inla-specific aptamer as a reporter, ohk et al.
<cit>  reported specific detection of l. monocytogenes with a lod of 103 cfu/ml.
CONCLUSIONS
we developed highly specific anti-inla mab  against pathogenic listeria: l. monocytogenes and l. ivanovii and anti-p <dig> mab  against all listeria spp.
including the two new species .
anti-inla antibody allowed specific detection of low levels  of l. monocytogenes and l. ivanovii when used on ims and a fiber-optic sensor in the presence of other bacteria from buffer, soft cheese or hotdogs inoculated with low levels of cells  following enrichment.

 the foodborne pathogen listeria monocytogenes causes listeriosis—a severe illness that ranges from mild gastroenteritis to invasive infection in immunocompromised people, neonates, and the elderly. in pregnant women, it causes premature births, miscarriages, and neonatal sepsis or fetal deaths. l. monocytogenes is ubiquitous and found in food-processing environments and food products, including ethnic soft cheese, sliced lunch meats and frankfurters, and seafood. it has been implicated in numerous food outbreaks and recalls, including a large outbreak involving cantaloupe in the us, which caused deaths and miscarriage. listeriosis has an estimated 19% fatality rate and ranks third among all fatalities resulting from foodborne infections in the usa. therefore, many countries have established a “zero tolerance” policy towards l. monocytogenes in rte foods. food recalls have increased each year, placing an economic burden on food manufacturers and growers. rapid and accurate detection methods may alleviate some of these problems. the genus listeria consists of species: l. monocytogenes, l. ivanovii, l. seeligeri, l. welshimeri, l. innocua, l. grayi, and two new species, l. marthii and l. rocourtiae. l. monocytogenes and l. ivanovii are pathogenic to humans and animals. many virulence and structural genes or gene products in listeria could be used as targets for antibody- or nucleic acid-based assay development. l. monocytogenes expresses several virulence proteins, including internalin a, which promotes bacterial adhesion and invasion of the host cell. inla possesses n-terminal leucine-rich repeats that facilitate anchoring to the bacterial cell wall, while the most distal extracellular domain binds to e-cadherin, which is crucial for host cell–cell adhesion and maintenance of tissue architecture. both pathogenic and non-pathogenic listeria species can be found in the same environment or food. however, when an enrichment step is used, the non-pathogenic species may overgrow and outcompete l. monocytogenes, leading to false-negative results. l. innocua is the most frequently found bacteria in listeria-contaminated foods, thus presenting a challenge for the specific capture and detection of pathogenic listeria. hence, it is essential to develop methods that are capable of detecting pathogenic species in the presence of non-pathogenic species. immunological approaches to detect pathogens in food are attractive; however, assay performance depends on the quality and specificity of the antibodies used. for detection of listeria, two types of assay specificities are desired: listeria genus- or l. monocytogenes-specific tests. anti-listeria antibodies available from research laboratories or commercial vendors are associated with problems of low affinity, reaction to heterologous antigens, lack of reaction towards all serotypes of l. monocytogenes, lack of reaction due to physiological stress induced by growth media or assay parameters, and lack of compatibility with certain bioassay platforms. thus, there is a need for continued efforts to produce high-quality antibodies. the recovery of low numbers of pathogens from complex food matrices also impedes their rapid and sensitive detection. antibodies are routinely used as affinity ligands to separate and concentrate the target analyte from sample matrices using paramagnetic beads and also as recognition or reporter molecules on immunoassay platforms. the pmb-captured cells may be presumptively identified by plating them on selective or differential media, or their identity may be confirmed by pcr, flow cytometry, or cytotoxicity assay. the use of a biosensor to detect cells captured by immunomagnetic separation is an attractive approach due to increased speed, accuracy, and detection of a low number of targets. fiber-optic sensors utilize laser excitation to generate an evanescent wave in order to quantify biomolecules immobilized on an optical waveguide. a capture antibody is immobilized on the waveguide and a fluorescent -labeled second antibody is used as a reporter for the target analyte. once the laser beam travels through the optical waveguide, it undergoes total internal reflection, and the fluorophore on the reporter antibody bound to the analyte is excited, thereby generating an evanescent wave. the signal is propagated back up to the fiber and is detected in real time by a fluorometer. this format has been successfully applied to many foodborne microorganisms and toxins, however, the limit of detection largely depends on the antibody and the reagents used. in the present study, monoclonal antibodies against l. monocytogenes and listeria spp. were generated, characterized, and employed to concentrate l. monocytogenes using pmbs. finally, mabs were used on the fiber optic sensor to detect l. monocytogenes from inoculated food products. in parallel, qpcr and light-scattering sensor methods were performed to confirm the results.  mab production and characterization by elisa and western blotting we selected stable hybridomas, of which reacted with both rinla and l. monocytogenes cells, and reacted with l. monocytogenes, l. innocua, and l. seeligeri. after another round of screening of mabs-2d -3b -4e and -3f against rinla or l. monocytogenes cells by elisa, we chose mab-2d and mab-3f for future use. an elisa revealed that, among the anti-inla antibodies, mabs-2d and -3b strongly reacted with l. monocytogenes 4b cells, while mab-4e gave slightly lower reaction values. the listeria genus-specific mab-3f gave strong elisa values when tested against other listeria spp., without producing significant cross-reactions with other bacterial species. in the western blot, mab-2d reacted with an 80-kda protein band from l. monocytogenes and l. ivanovii, but it did not react with other listeria spp., including l. marthii or l. rocourtiae. mab-2d was reactive with all serotypes; however, a relatively weak reaction with strains of serotype 1/2c was observed. mab-2d also reacted with a 66-kda band from serotype 3c, which is presumably a truncated inla-protein variant. mab-2d12-reactive inla was distributed in the secreted, cell wall, and intracellular protein fractions of bacteria. immunofluorescence microscopy confirmed the specific binding of anti-inla antibody to the surface of l. monocytogenes cells, but it did not react with l. innocua. mab-3f showed a strong reaction with a single protein band of ~30 kda from all listeria spp. with the exception of l. welshimeri. in addition, this mab showed strong reactions with protein preparations from all serotypes of l. monocytogenes. bacterial capture using antibody-coated paramagnetic beads pmbs with mab-2d had higher capture efficiency than those with mab-3f using the same antibody, the smaller-sized myone beads displayed significantly higher capture efficiency than the dynabeads m- for l. monocytogenes 4b and l. ivanovii. the capture efficiency curve with different concentrations of l. monocytogenes cells was bell-shaped; the highest capture was obtained at 105 cfu/ml, while the lowest capture was obtained at concentrations of 103 cfu/ml and at 107–108 cfu/ml. at initial l. monocytogenes concentrations of and 106 cfu/ml, myone-2d captured %, %, and % of cells, respectively, while m-280-2d captured 15%, %, and %, respectively. these values were significantly different from mab-3f conjugated to myone or m-. a similar trend was seen for l. ivanovii, but the values obtained were lower than those for l. monocytogenes. therefore, the capture efficiency depends on antibody performance, bead size, and initial bacterial concentration. abacteria were grown in tsb-ye for 18 h at 37 °c. the data are average of experiments analyzed in duplicate. values labeled with different letters in a row or in a column are significantly different at p < . all subsequent ims experiments were performed using myone beads. the fluorescence microscopic image in figure  4b shows the capture of l. monocytogenes by myone-2d the capture efficiency of myone-2d and myone-3f was evaluated with bacteria grown in the recommended enrichment broths, leb or fb. myone-2d showed significantly higher capture of l. monocytogenes and l. ivanovii than other listeria spp., and the capture efficiency was similar for leb or fb. the capture efficiency for myone-2d was comparable for the l. monocytogenes serotypes tested, including 4b, 1/2a, and 1/2b, as well as for a strain of l. ivanovii, and negligible capture of other listeria spp. was observed. myone-3f displayed similar capture efficiency for all listeria spp. tested, irrespective of the enrichment broths used. when the capture efficiency of myone-2d myone-3f and dynabeads anti-listeria was compared against a listeria panel, myone-2d captured the most pathogenic listeria. for all other listeria spp., both myone-3f and dynabeads anti-listeria had similar values. thus, myone-2d is highly specific for the capture of pathogenic listeria, and myone-3f and dynabeads anti-listeria displayed similar capture efficiency for all listeria spp. tested. the capture efficiency of pmbs for l. monocytogenes in a co-culture with l. innocua was also determined. the bacteria were grown in fb, mixed in pbs to achieve concentrations of ~1 × 105 cfu/ml each and the capture efficiency was determined by plating followed by bardot-based colony identification. myone-2d captured ~104 cfu/ml of bacteria, of which most colonies were confirmed to be l. monocytogenes by bardot. myone-3f captured ~  ×  cells, and ~50% were confirmed to be l. monocytogenes. dynabeads anti-listeria captured ~  × 103 cfu/ml, of which 40% were l. monocytogenes. we also investigated the capture efficiency of bacteria from inoculated food matrices. hotdogs were inoculated with ~10 cfu/g each of l. monocytogenes 4b and l. innocua as a mono- or co-culture and enriched for 18 h at 37°c. myone-2d showed higher capture of l. monocytogenes than l. innocua in the monocultures, but in the co-culture experiment the total bacterial capture dropped to %. myone-3f captured % of the l. monocytogenes cells in the monoculture experiment, while the commercial dynabeads anti-listeria captured only %. dynabeads anti-listeria also captured a numerically higher percentage of l. innocua compared with l. monocytogenes. overall, these data show that myone-2d captured 10-fold more l. monocytogenes than l. innocua, while myone-3f captured -fold more l. monocytogenes than l. innocua. dynabeads anti-listeria had the highest capture efficiency for l. innocua from hotdogs. the capture of listeria was also investigated with soft cheese made from goat’s milk in a co-culture experiment. cheese samples were inoculated with l. monocytogenes 4b and l. innocua and enriched in fb for 18 h until the total count reached ~  × 108 cfu/ml. the bacterial capture using myone-2d was  ±  %, while myone-3f and dynabeads anti-listeria showed significantly lower capture efficiency. capture of l. monocytogenes colonies on bhi agar plates was verified by a light-scattering sensor, with l. monocytogenes and l. innocua producing distinct scatter patterns. specificity and limit of detection of the fiber-optic sensor the specificity and limit of detection of the fiber optic sensor were analyzed by using mab-2d as capture antibody and cy5-labeled mab-2d as a reporter. the sensor generated strong signals against l. monocytogenes and l. ivanovii, with a maximum signal of pa. in contrast, non-pathogenic listeria produced a maximum signal of – pa, and non-listeria bacteria, including salmonella typhimurium; e. coli o157:h7; and background food contaminant isolates, staphylococcus aureus, s. epidermidis, enterobacter cloacae, and lactococcus lactis, produced signals of ~ pa. similar results were obtained when mab-3f was used as the capture and mab-2d as the reporter molecule. in the mixed cultures containing l. monocytogenes, l. innocua, and e. coli o157:h, the signals for mab-2d and mab-3f were  ±  pa and  ±  pa, respectively, which were significantly higher than the values obtained for l. innocua or e. coli alone. the background control values ranged from 504– pa. therefore, both fiber-optic sensor configurations, 2d12–2d and 3f8–2d are highly specific for pathogenic listeria, and specificity was contributed primarily by anti-inla mab-2d other combinations did not produce satisfactory results. the lod was also evaluated by using pure cultures of l. monocytogenes and l. ivanovii serially diluted in pbs. using mab-2d as the capture molecule, the signals increased proportionately as the bacterial concentration increased until a cell concentration of 1 × 106 cfu/ml was reached, which gave the maximum signal, almost reaching the threshold of the analyte fluorometer. the lowest cell concentration that was considered positive was 3 × 102 cfu/ml for l. monocytogenes and 1 × 103 cfu/ml for l. ivanovii. these values were at least 2-fold higher than those produced by the samples with cells or pbs. when mab-3f was used as capture antibody, the lod for l. monocytogenes and l. ivanovii was ~1 × 105 cfu/ml. ims coupled with a fiber-optic sensor for detection of l. monocytogenes bacteria captured by myone-2d or myone-3f were detected by the mab-2d12-coated fiber-optic sensor and yielded signals of  ±  pa and  ±  pa, respectively. the mab-3f fiber optic sensor produced signals of  ±  pa and  ±  pa, respectively. the fiber optic signal value for myone-2d and -3f captured l. monocytogenes was about to 3-fold higher than the signals obtained from the lod concentrations. these data indicate that l. monocytogenes detection using mab-2d for ims and a fiber optic sensor gave better results compared with those obtained using mab-3f in soft cheese-containing co-culture of l. monocytogenes and l. innocua, both myone-2d and myone-3f captured bacteria and produced signals of  ±  pa and  ±  pa, respectively. bacteria captured with dynabeads anti-listeria gave the lowest fiber-optic signals. in listeria-inoculated hotdog samples, only myone-2d was used for ims and assayed by fiber optic sensor. the signal from the sample containing both l. monocytogenes and l. innocua was  ±  pa, while that from l. monocytogenes- and l. innocua-inoculated food was  ±  pa and  ±  pa, respectively. for both food samples, the fiber optic signal values for myone-2d and -3f captured l. monocytogenes but not the l. innocua were higher than the signals obtained from the lod cell concentrations. therefore, the ims and fiber optic sensor can be used together for detection of l. monocytogenes from enriched food samples, even in presence of l. innocua or other bacteria. real-time qpcr for validation real-time qpcr targeting hlya was used to quantify pmb-captured listeria from hotdogs and goat’s cheese artificially contaminated with l. monocytogenes and l. innocua. when ims was applied to the cheese samples followed by qpcr, myone-2d showed cell counts that were times higher than those of myone-3f and dynabeads anti-listeria. in hotdog samples, myone-2d produced cell counts that were 2– times higher than those of the other types of beads. aqpcr analysis is based on hlya. primers to 16s gene sequences were used as internal control. bdata are average of experiments run in triplicate. values labeled with letters in a column are significantly different at p < . discussion the recovery of low numbers of target pathogens from complex food matrices is a challenge for sensitive detection methods. ims using pmbs is used to separate and concentrate target pathogens from food samples before detection by plating, immunoassay, pcr, or biosensor methods. antibodies or alternative molecules are used as capture molecules for ims, and improvements in reagents and assay platform development are essential to enhance assay performance. the specific detection of whole cells of l. monocytogenes using immunological methods relies on highly specific antibodies with a strong affinity for bacterial surface antigens. the antigen target should be uniformly distributed on the target organism, covalently anchored to the cell wall, and accessible to the antibody. inla is a well-characterized protein that is highly specific to l. monocytogenes and l. ivanovii, and it has all the desirable properties of an antigen. thus, we produced mabs against inla and p. the resulting mabs were employed in ims to capture and concentrate bacteria from food followed by fiber-optic sensor-based detection. to the best of our knowledge, this is the first demonstration of the combined use of these two approaches. inla-specific antibody production was facilitated by the use of whole cells of l. monocytogenes and purified rinla as immunogens. hybrid b-lymphocyte clones secreted antibodies with a strong reaction towards live whole cells, but a weaker reaction was observed with heat-killed cells. since rinla was soluble, denaturing agents were not required before immunization. thus, the native structure of inla during the immune response was preserved, and the resulting antibody recognized the native protein on the surface of bacteria. the inla-specific mab-2d reacted with all known l. monocytogenes serotypes, whereas previously reported mabs failed to recognize all serotypes. only serotype 1/2c showed a weak reaction with mab-2d however, this strain has been involved in a few sporadic cases of listeriosis and is rarely found. moreover, none of the strains of serotype 1/2c expressed a functional, full-length inla, which may explain why mab-2d displayed a reduced reaction to 1/2c. when tested with serotype 3c, mab-2d reacted strongly with a ~66 kda band instead of the normal 80-kda inla band. the smaller band may represent truncated inla, which results from inla mutation. generally, such strains are less invasive and are less likely to cause systemic infection as confirmed in animal models. we also generated a listeria species-specific mab by immunization with whole cells of l. monocytogenes. mab-3f reacted with a ~30-kda protein present in all eight listeria species. therefore, mab-3f may aid tracking of listeria contamination in foods or the food-production environment. the separation of target organisms following primary enrichment using ims is faster than using selective secondary enrichment. thus, we performed ims using two different sizes of commercial beads. antibody-coated 1-μm myone t exhibited significantly higher capture efficiency than the -μm m- beads. similarly, foddai et al. used six different magnetic beads, including the two types used in this study, to capture mycobacterium avium. myone displayed better capture efficiency than that of m- but the overall capture efficiency was low. in the present study, the capture efficiency for myone-2d and m280-2d was % and %, respectively while % for myone-3f and % for m280-3f paoli et al. used m-280-coated scfv antibody to acta and reported a maximum capture of 19% for l. monocytogenes. walcher et al. reported a capture range of 46%–122% using a bacteriophage endolysin specific for listeria spp. coated on m-280; however, the long capture incubation time may have allowed bacterial growth, thereby producing a higher capture rate. furthermore, the binding of bacteriophage to host cells is an irreversible process, which may lead to higher capture efficiency than with antibody-coated pmbs. koo et al. used hsp60-coated m- which showed a capture efficiency for l. monocytogenes of %– %. the capture efficiency also depended on the initial bacterial concentration. the highest capture with myone-2d or myone-3f was seen at a bacterial concentration of 105 cfu/ml. this is important for meaningful comparisons to be made between the performances of ims in different studies, which may use a wide range of initial bacterial concentrations. collectively, ims data indicate that beads with a smaller diameter have better capture efficiency than larger beads due to higher surface area to mass ratio and smaller beads can bind more antibody per mg of beads. furthermore, the antibody affinity, the distribution/expression of antigens on the surface of bacteria, and the initial bacterial concentration also significantly affect capture efficiency. here, the abundant expression of inla on the surface of l. monocytogenes cells coupled with the use of smaller sized pmb was most likely responsible for increased capture efficiency. however, the assay performance may be affected if pmb followed by fiber optic sensor was applied to food samples directly without an enrichment step. in such situation, food matrices may affect bacterial antigen expression or antibody affinity. we tested the capture efficiency of l. monocytogenes in a co-culture experiment in buffer or food. food contaminated with l. monocytogenes may contain other listeria spp. and background competitive microflora. l. monocytogenes grows slowly and is a poor competitor; hence, lower cell numbers are expected in food samples. in a mixed population, l. monocytogenes may be outgrown by other species of listeria during enrichment. here, ims using myone-2d efficiently captured l. monocytogenes, in the presence of l. innocua while both myone-3f and dynabeads anti-listeria captured more l. innocua cells than l. monocytogenes. furthermore, the capture efficiency for myone-2d using a co-culture in buf-fer or food varied from %– %. less than optimal level of capture was attributed largely to the presence of higher initial concentrations of bacteria in the sample and the presence of interfering agents in food matrices, particularly in soft cheese. furthermore, the increased capture of l. monocytogenes in hotdog compared to pbs was possibly due to increased expression of mab-2d12-reactive antigen during enrichment while cells used in pbs were originally cultured in bhi, which may have caused reduced inla expression resulting in reduced l. monocytogenes capture. l. ivanovii is an opportunistic human pathogen that is associated with gastroenteritis and bacteremia in humans; therefore, the development of methods to detect this pathogen is also essential. mab-2d reacted with l. ivanovii, which was successfully detected by using ims and a fiber-optic sensor. hearty et al. reported the inla-specific mab-2b3; however, this antibody was unable to detect l. ivanovii in their assay setup. mab-2b may be specific for an epitope of inla on l. monocytogenes that is absent on l. ivanovii. pmb-captured cells were also identified by bardot and qpcr. bardot is a light-scattering sensor that detects and identifies bacterial colonies on agar plates with a high degree of precision in minutes, since each species has a distinctive scatter-fingerprint signature. bardot allowed quantitative estimation of capture rate for l. monocytogenes and l. innocua on bhi or mox plates instantly based on colony scatter patterns and it is easy to perform without the requirement for any additional reagents or probes. real-time qpcr confirmed that l. monocytogenes capture and detection from food by myone-2d was 13%–16%, which is significantly higher than that by myone-3f and dynabeads anti-listeria. these estimations are slightly higher than the plate count and the light-scattering data obtained in this study. yang et al. used nanoparticles for ims and showed better capture and detection of l. monocytogenes in milk with real-time pcr compared with plate counts. this may be because qpcr detects dna from nonviable or viable but non-culturable cells, which may not otherwise be detected by traditional plating methods. the fiber-optic sensor operates based on the principles of antibody-antigen interaction and is marketed by research international. it is currently used for foodborne or biothreat agent detection. the antibody used in this study on the optical waveguide made the assay highly specific for l. monocytogenes and l. ivanovii, with the detection limit of 3 × 102 cfu/ml, a significant improvement over previous reports. geng et al. used mab-c11e to show cross-reaction with some l. innocua strains with lod of  × 103 cfu/ml. using a polyclonal anti-listeria capture antibody and an inla-specific aptamer as a reporter, ohk et al. reported specific detection of l. monocytogenes with a lod of 103 cfu/ml.  we developed highly specific anti-inla mab against pathogenic listeria: l. monocytogenes and l. ivanovii and anti-p mab against all listeria spp. including the two new species. anti-inla antibody allowed specific detection of low levels of l. monocytogenes and l. ivanovii when used on ims and a fiber-optic sensor in the presence of other bacteria from buffer, soft cheese or hotdogs inoculated with low levels of cells following enrichment. 
subsequently, we captured l. monocytogenes by myone-2d and myone-3f from hotdogs inoculated with mono- or co-cultures of l. monocytogenes and l. innocua, enriched for 18 h and detected by fiber-optic sensor and confirmed by plating, light-scattering, and qpcr assays. furthermore, capture efficiency for myone-2d was highly specific for l. monocytogenes and l. ivanovii. the detection limit for l. monocytogenes and l. ivanovii by the fiber-optic immunosensor was 3 × 102 cfu/ml using mab-2d as capture and reporter antibody. ims coupled with a fiber-optic sensor using anti-inla mab is highly specific for l. monocytogenes and l. ivanovii and enabled detection of these pathogens at low levels from buffer or food. in this study, monoclonal antibodies against internalin a and p were generated and used on paramagnetic beads of varying diameters for concentration, as well as on fiber-optic sensor for detection. anti-inla mab-2d was specific for listeria monocytogenes and l. ivanovii, and p30-specific mab-3f was specific for the genus listeria. selective media plating, light-scattering, and qpcr assays confirmed the ims and fiber-optic results. immunomagnetic separation and immunoassays are widely used for pathogen detection. listeria monocytogenesinternalin amonoclonal antibodyimmunomagnetic separationfiber optic sensorlight scattering sensorqpcrdetectionbiosensor
 immunomagnetic separation and immunoassays are widely used for pathogen detection. however, novel technology platforms with highly selective antibodies are essential to improve detection sensitivity, specificity and performance. in this study, monoclonal antibodies against internalin a and p were generated and used on paramagnetic beads of varying diameters for concentration, as well as on fiber-optic sensor for detection.  anti-inla mab-2d was specific for listeria monocytogenes and l. ivanovii, and p30-specific mab-3f was specific for the genus listeria. at all bacterial concentrations tested in the ims assay; the 1-μm diameter myone beads had significantly higher capture efficiency than the -μm diameter m- beads with both antibodies. the highest capture efficiency for myone-2d was significantly higher than that of myone-3f and dynabeads anti-listeria antibody. furthermore, capture efficiency for myone-2d was highly specific for l. monocytogenes and l. ivanovii. subsequently, we captured l. monocytogenes by myone-2d and myone-3f from hotdogs inoculated with mono- or co-cultures of l. monocytogenes and l. innocua, enriched for 18 h and detected by fiber-optic sensor and confirmed by plating, light-scattering, and qpcr assays. the detection limit for l. monocytogenes and l. ivanovii by the fiber-optic immunosensor was 3 × 102 cfu/ml using mab-2d as capture and reporter antibody. selective media plating, light-scattering, and qpcr assays confirmed the ims and fiber-optic results.  ims coupled with a fiber-optic sensor using anti-inla mab is highly specific for l. monocytogenes and l. ivanovii and enabled detection of these pathogens at low levels from buffer or food. listeria monocytogenesinternalin amonoclonal antibodyimmunomagnetic separationfiber optic sensorlight scattering sensorqpcrdetectionbiosensor 