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Critical Reviews in Microbiology
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Listeria monocytogenes: review of pathogenesis and
virulence determinants-targeted immunological
assays
Leonardo Lopes-Luz, Marcelo Mendonça, Matheus Bernardes Fogaça, André
Kipnis, Arun K. Bhunia & Samira Bührer-Sékula
To cite this article: Leonardo Lopes-Luz, Marcelo Mendonça, Matheus Bernardes Fogaça,
André Kipnis, Arun K. Bhunia & Samira Bührer-Sékula (2021): Listeria�monocytogenes: review
of pathogenesis and virulence determinants-targeted immunological assays, Critical Reviews in
Microbiology, DOI: 10.1080/1040841X.2021.1911930
To link to this article: https://doi.org/10.1080/1040841X.2021.1911930
Published online: 24 Apr 2021.
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REVIEW ARTICLE
Listeria monocytogenes: review of pathogenesis and virulence
determinants-targeted immunological assays
Leonardo Lopes-Luz
a
, Marcelo Mendonc¸a
b
, Matheus Bernardes Fogac¸a
a
, Andr
e Kipnis
a
, Arun K. Bhunia
c,d,e
and Samira B€
uhrer-S
ekula
a
a
Instituto de Patologia Tropical e Sa
ude P
ublica, Universidade Federal de Goi
as, Goi^
ania, Brasil;
b
Curso de Medicina Veterin
aria,
Universidade Federal do Agreste de Pernambuco, Garanhuns, Brasil;
c
Department of Food Science, Purdue University, West Lafayette,
IN, USA;
d
Department of Comparative Pathobiology, Purdue University, West Lafayette, IN, USA;
e
Purdue Institute of Inflammation,
Immunology and Infectious Disease, Purdue University, West Lafayette, IN, USA
ABSTRACT
Listeria monocytogenes is one of the most invasive foodborne pathogens and is responsible for
numerous outbreaks worldwide. Most of the methods to detect this bacterium in food require
selective enrichment using traditional bacterial culture techniques that can be time-consuming
and labour-intensive. Moreover, molecular methods are expensive and need specific technical
knowledge. In contrast, immunological approaches are faster, simpler, and user-friendly alterna-
tives and have been developed for the detection of L. monocytogenes in food, environmental,
and clinical samples. These techniques are dependent on the constitutive expression of L. mono-
cytogenes antigens and the specificity of the antibodies used. Here, updated knowledge on
pathogenesis and the key immunogenic virulence determinants of L. monocytogenes that are
used for the generation of monoclonal and polyclonal antibodies for the serological assay devel-
opment are summarised. In addition, immunological approaches based on enzyme-linked
immunosorbent assay, immunofluorescence, lateral flow immunochromatographic assays, and
immunosensors with relevant improvements are highlighted. Though the sensitivity and specifi-
city of the assays were improved significantly, methods still face many challenges that require
further validation before use.
ARTICLE HISTORY
Received 27 August 2020
Revised 19 March 2021
Accepted 29 March 2021
Published online 20 April
2021
KEYWORDS
Listeria monocytogenes;
pathogenesis; listeriosis;
virulence; antibodies;
antigens; serological
methods; lateral flow
immunoassay; detection
1. Introduction
Listeria monocytogenes is a Gram-positive, invasive,
foodborne bacterial pathogen that causes listeriosis, a
systemic disease resulting from the ingestion of conta-
minated food. The systemic spread of the pathogen
from the gastrointestinal tract depends on its ability to
cross intestinal, blood-brain, and placental barriers
(Radoshevich and Cossart 2018; Drolia and Bhunia
2019). Localised infection in the gastrointestinal tract
may lead to the onset of a rare form of gastroenteritis
characterised by diarrhoea, abdominal cramp, and flu-
like symptoms in healthy adults, whereas the systemic
invasive disease is manifested by fever, headache, men-
ingitis, encephalitis, liver abscess, abortion, premature
birth, stillbirth, and neonatal infection with sepsis and
pneumonia in immunocompromised hosts (Drolia and
Bhunia 2019; Schlech 2019). The mortality rate in
immunocompromised hosts including the elderly, preg-
nant women, and their foetuses, neonates, cancer
patients, and chemotherapy recipients is 20–30%
(Swaminathan and Gerner-Smidt 2007). In particular,
pregnant women and their foetuses are highly suscep-
tible to listeriosis and 16–27% of all L. monocytogenes
infections are reported in pregnant women (Lamont
et al. 2011; Craig et al. 2019). A recent study suggested
that L. monocytogenes infection in the first trimester
presents the greater risk of fetal loss than in the third
trimester (Wolfe 2017). Contaminated ready-to-eat-
foods such as hotdogs, soft cheeses mainly made with
unpasteurised milk, smoked fish, ice cream, p^
at
e,
polony, cantaloupe, apple, and vegetables were impli-
cated to be the primary vehicle for transmission
(Buchanan et al. 2017; Smith et al. 2019). The infectious
dose varies widely (<100 cells to 10
11
cells) and
depends on the amount of contaminated food con-
sumed, the bacterial strain, and the immunological sta-
tus of the host (Pouillot et al. 2016; Buchanan
et al. 2017).
The genus Listeria has 20 species and is subdivided
into two major groups: (i) Listeria sensu stricto
CONTACT Arun K. Bhunia bhunia@purdue.edu Department of Food Science, Purdue University, West Lafayette, IN, USA
ß2021 Informa UK Limited, trading as Taylor & Francis Group
CRITICAL REVIEWS IN MICROBIOLOGY
https://doi.org/10.1080/1040841X.2021.1911930
comprising of the species L. monocytogenes, L. innocua,
L. seeligeri, L. ivanovii, L. welshimeri and L. marthii and
(ii) Listeria sensu lato including L. grayi, L. fleischmannii,
L. weihenstephanensis, L. rocourtiae, L. floridensis, L. cor-
nellensis, L. riparia. L. aquatica, L. grandensis, L. newyor-
kensis, L. goaensis, L. booriae, L. costaricensis and L.
thailandensis (N
u~
nez-Montero et al. 2018; Leclercq et al.
2019). Only L. monocytogenes and L. ivanovii are consid-
ered pathogenic infecting humans and animals,
respectively. L. monocytogenes species can be further
subdivided into four lineages (I-IV), with lineage sero-
types I and II (1/2a, 1/2b, and 4b) involved in most lis-
teriosis cases reported worldwide (Weller et al. 2015).
The dissemination of L. monocytogenes through
foods is facilitated by the fact that the pathogen is
resistant and adaptable to adverse environmental con-
ditions such as high salt concentration (Gandhi and
Chikindas 2007), broad temperature range (1–45 C),
low pH (Tasara and Stephan 2006), and oxygen-limiting
conditions (Lungu et al. 2009; Roberts et al. 2020),
which are also encountered in human gastrointestinal
tract thus exacerbating L. monocytogenes infection in
humans (Horn and Bhunia 2018). Through the forma-
tion of biofilms (Lee et al. 2019; Bai et al. 2021) and
osmoadaptation, L. monocytogenes also resist desicca-
tion and sanitising agents (Pang et al. 2019). Thus, L.
monocytogenes can easily multiply on several surfaces,
increasing the chance of listeriosis outbreaks through
contaminated foods (Ferreira et al. 2014).
Traditional techniques for the listeriosis diagnostic
and L. monocytogenes detection in food include pre-
enrichment, selective enrichment, and isolation steps
that are time-consuming and labour-intensive (Le
Monnier et al. 2011; Bhunia 2014; Malica et al. 2015).
Most of the detection methods need pre-enrichment
cultivation to increase the pathogen concentration to
detectable levels of 10
4
to 10
5
CFU/mL (Gasanov et al.
2005;Wu2019). As alternatives to these problems,
many methods based on bacterial nucleic acid detec-
tion have been developed, like polymerase chain reac-
tion (PCR) and DNA hybridisation. Though these
molecular methods are highly sensitive, they are expen-
sive and need specialised technical knowledge and
equipment (Wang and Salazar 2016). PCR methods are
also highly susceptible to inhibitors and may not differ-
entiate viable from dead cells unless RNA has been
used as a target or cells are treated with propidium
monoazide before assay (Chen et al. 2017; Zheng et al.
2018). Therefore, immunological techniques (Figure 1),
exemplified mainly by enzyme-linked immunosorbent
assay (ELISA), immunosensors, and lateral flow immuno-
chromatographic assays (LFIA), are alternative
approaches to L. monocytogenes detection because of
their low cost, high sensitivity, and specificity, and sim-
plicity in data interpretation (Banada and Bhunia 2008;
Liu, Du, et al. 2017; Lv et al. 2019; Xu et al. 2021).
Immunological methods are developed using poly-
clonal (PAb) and monoclonal (MAb) antibodies pro-
duced against various target antigens of L.
monocytogenes (Bhunia 1997; Banada and Bhunia 2008;
Lathrop et al. 2014). However, some methods have
reported low specificity due to high genetic diversity
among L. monocytogenes strains (Dussurget et al. 2004).
Moreover, Listeria antigen expression may severely be
affected by the enrichment broths or growth conditions
used for culturing the bacteria (Nannapaneni et al.
1998; Lathrop et al. 2008) or acid, salt, and tempera-
ture-induced stressors before immunological detection
(Geng et al. 2003; Hahm and Bhunia 2006). Vital target
proteins should be strongly associated with the cell sur-
face and should be expressed by all L. monocytogenes
serotypes (Paoli et al. 2007). Furthermore, some avail-
able methods recognise antigenic targets shared by
other Listeria species and/or other non-Listeria bacteria
(Meyer et al. 2011). Thus, studies of surface biomarkers
of L. monocytogenes have been carried out in the past
several years, aiming to develop new immunological
approaches to pathogen control, prevention and to
help eliminate selective pre-enrichment steps (Boivin
et al. 2016; Mendonc¸a et al. 2016; Zhang et al. 2016;
Phraephaisarn et al. 2017).
The L. monocytogenes antigens for the generation
and selection of monoclonal and polyclonal antibodies
were reviewed previously (Bhunia 1997; Banada and
Bhunia 2008; Bhunia 2018) and are summarised in
Table 1. We will highlight the application of highly sen-
sitive and specific antibodies combined with relevant
and recent improvements of immunological
approaches to L. monocytogenes detection thus ena-
bling the improvement in the assay sensitivity.
Furthermore, we will provide a brief updated informa-
tion on L. monocytogenes pathogenesis and the viru-
lence factors (Table 1) that play central roles in
pathogenesis. Immunological assays based on virulence
determinants provide a solid foundation for the devel-
opment of a pathogen-specific diagnostic or detection
tool (Figure 1).
2. Virulence factors of Listeria monocytogenes
and their antigenic potentials
2.1. Listeria monocytogenes pathogenesis
After ingestion of contaminated food by the host, L.
monocytogenes overcome an acidic environment in the
2 L. LOPES-LUZ ET AL.
Table 1. Listeria monocytogenes virulence factors.
Virulence factors Size (kDa) Host cell receptor Function References
Protein regulatory
factor (PrfA)
27 –Regulation of many virulence protein expression (Dussurget et al. 2002; Johansson and
Freitag 2019)
Sigma B (r
B
) 30.5 –Regulation of stress and virulence genes (Liu et al. 2019)
Adhesion Proteins
Listeria adhesion
protein (LAP)
104 Hsp60
(chaperone protein)
Adhesion to intestinal epithelial cells; Disruption of
intestinal epithelial barrier
(Drolia et al. 2018; Drolia and Bhunia 2019)
Listeria adhesion
protein B (LapB)
184 Unknown Adhesion and invasion into host cells (Boivin et al. 2016)
Autolysin
amidase (Ami)
99 Unknown Adhesion to hepatocytes (Asano et al. 2011)
Fibronectin binding
protein (FbpA)
55.3, 48.6, 46.7, 42.4,
and 26.8
Fibronectin Adhesion to cells and also serves as a chaperone to
stabilise and secretion of LLO, InlB
(Osanai et al. 2013; Gelb
ı
cov
a et al. 2016)
Internalin J (InlJ) 92 Mucus Adhesion to epithelial cells and binds to human
intestinal mucin-2 (MUC2)
(Lind
en et al. 2008)
Internalin F (InlF) 90 Vimetin protein The crossing of blood-brain barrier (Dramsi et al., 1997; Ghosh et al. 2018)
Autolysin IspC 86 Unknown Adhesion to non-phagocytic cells (Ronholm et al. 2013)
Lmo1656 12 sorting nexin
6–BAR complex
Transcytosis in goblet cells (David et al. 2018)
Invasion
Internalin (InlA) 88 E-cadherin (tight
junction protein)
Promotes bacterial internalisation into enterocytes and
bacterial transcytosis across the intestinal barrier
(Ribet and Cossart 2015; Phelps et al. 2018;
Drolia and Bhunia 2019)
Internalin B (InlB) 65 Met (tyrosine kinase
receptor c-Met),
gC1q-R/p32
Acts in the invasion of enterocytes and passage
through M-cells of Peyer’s patches
(Bierne and Cossart 2002; Lathrop et al. 2008;
Auriemma et al. 2010)
Virulence invasion
protein (Vip)
43 Gp96
(chaperone protein)
Invasion of epithelial cells (Cabanes et al. 2005)
LAP 104 Hsp60 Induces junctional protein dysregulation and increases
epithelial permeability (translocation)
(Drolia et al. 2018; Drolia and Bhunia 2019)
Lysis of Vacuole
Listeriolysin (LLO) hlyA 58–60 Cholesterol A haemolysin that disrupts the vacuolar membrane and
releases the bacteria into the cytoplasm
(Phelps et al. 2018)
Phospholipase (plcA –
PI-PLC; plcB –
PC-PLC)
29–33 –Lyses of vacuole membrane (Poussin and Goldfine 2005; Suryawanshi
et al. 2017)
Cell-to-cell Spread
Actin polymerisation
protein (ActA)
88–92 –Initiates host cell actin polymerisation for bacterial
movement inside the cytoplasm
(Lathrop et al. 2008; Radoshevich and
Cossart 2018)
PC-PLC 29–32 Cholesterol Lyses of vacuole membrane (Grundling et al. 2003)
Metalloprotease (Mpl) 29 –Helps synthesis of PLC (Bhunia 1997; Yeung et al. 2005)
Miscellaneous
Activities
P60 (cell
wall hydrolase)
60 –Invasion of non-professional phagocytic cells (Luo and Cai 2012; Lingala and Ghany 2015)
Bile salt
hydrolase (BSH)
36 –Survival in gut (Dussurget et al. 2002)
Fructose-1,6-
bisphophate
aldolase (FAB)
30 –Moonlighting protein: i) adhesion to the host’s cells and
ii) role in the pathogenesis
(Mendonc¸a et al. 2016)
Internalin C (InlC) 33 Human intestinal
mucin-2 (MUC2)
Perturbs apical cell junctions (Rajabian et al. 2009)
(continued)
CRITICAL REVIEWS IN MICROBIOLOGY 3
stomach by regulating bacterial pH homeostasis. The
glutamate decarboxylase system (Karatzas et al. 2012;
Paudyal et al. 2018), arginine deiminase pathway
(Cheng et al. 2017), and an agmatine deiminase system
(Ryan et al. 2009) are important mechanisms for bacter-
ial resistance to acids. Bile salt hydrolase (BSH) and bile
salt exclusion (BSE) proteins defend against the anti-
microbial activity of bile (Gahan and Hill 2014). Being a
facultative anaerobe, L. monocytogenes can also prolif-
erate under an oxygen-limiting environment in the
intestine (Burkholder et al. 2009; Lungu et al. 2009;
Roberts et al. 2020). Resident gut microbiota provides
little resistance against L. monocytogenes colonisation
and spread (Quereda et al. 2016; Becattini et al. 2017).
The crossing of the intestinal epithelial barrier is crit-
ical for the systemic spread of the infection which can
occur through both the small intestine and large intes-
tine (Drolia and Bhunia 2019; Bai et al. 2021). Listeria
adhesion protein (LAP), also known as acetaldehyde
alcohol dehydrogenase (AdhE) promotes bacterial pas-
sage through the epithelial barrier by opening the tight
junction barrier following interaction with the host cell
receptor, heat shock protein 60 (Hsp60), and conse-
quent activation of nuclear factor-jB (NF-jB) and
myosin light chain kinase (MLCK) early in the infection
process (Burkholder and Bhunia 2010; Drolia et al. 2018,
2020). While Internalin A (InlA) facilitates bacterial pas-
sage by interacting with the host cell E-cadherin via
intracellular route by transcytosis (Nikitas et al. 2011;
Bou Ghanem et al. 2012). F-actin-mediated bacterial
adhesion independent of interaction with E-cadherin
also promotes L. monocytogenes invasion into epithelial
cells (Ortega et al. 2017). M cells overlying Peyer’s
patches also help bacterial invasion, but in the absence
of M cells, L. monocytogenes still can reach lamina prop-
ria using the aforementioned virulence factors (Pron
et al. 1998). Besides, the Internalin B (InlB), a non-cova-
lently anchored surface protein interacts with the tyro-
sine kinase receptor c-Met/HFGR (Hepatocyte Growth
Factor Receptor) and also with glycosaminoglycans on
epithelial cells (Shen et al. 2000; Bierne and Cossart
2002; Quereda et al. 2019). InlB is also required for pas-
sage through M-cells of Peyer’s patches and also helps
bacterial internalisation into enterocytes (Cabanes et al.
2002; Sobyanin et al. 2017; Phelps et al. 2018). To cross
the blood-brain barrier, L. monocytogenes employs
Internalin (InlF) as the primary invasion protein (Ghosh
et al. 2018), while LLO and ActA are reported to play an
important role during olfactory epithelium invasion for
neurolisteriosis in neonates via the intranasal route of
infection (P€
agelow et al., 2018).
Table 1. Continued.
Virulence factors Size (kDa) Host cell receptor Function References
Internalin H (InlH) 58 –Contributes to systemic listeriosis (Markkula et al. 2011)
Autolysin
amidase (Ami)
99 Unknown Bacteriolysin: enhances the host immune response (Asano et al. 2011)
LAP 104 Hsp60 Upregulates TNF-a and IL-6 expression in intestinal Cells (Drolia and Bhunia 2019)
Listeriolysin (LLO) hlyA 58–60 Cholesterol Induces lymphocyte apoptosis and suppresses
proinflammatory cytokines
(Pattabiraman et al. 2017)
Listeria nuclear-
targeted protein
A (IntA)
23 BAHD1 Decreases the host’s immune response (Lebreton et al. 2011)
Listeriolysin S <5–Hemolytic and cytotoxic; bacteriocin (bactericidal) (Cotter et al. 2008; Quereda et al. 2016)
4 L. LOPES-LUZ ET AL.
Internalised L. monocytogenes are trapped in the
vacuole or phagosome which is disrupted by listerioly-
sin O (LLO), phosphatidylcholine-specific phospholipase
(PC-PLC), and phosphatidylinositol-specific phospholip-
ase C (PI-PLC), releasing the bacteria in the cytoplasm
(Phelps et al. 2018). In some cases, bacteria can persist
in the vacuole thus prolonging the incubation period
(Kortebi et al. 2017; Bierne et al. 2018). In the absence
of ActA, vacuolar bacteria may maintain a viable but
nonculturable state (Bierne et al. 2018). Once in the
cytoplasm, the bacterium replicates rapidly and spreads
by inducing actin nucleation and polymerisation by
inducing ActA expression (Theriot et al. 1992;
Radoshevich and Cossart 2018; Johansson and Freitag
2019). At this time of infection, L. monocytogenes may
produce extracellular vesicles that will release virulence
factors and toxins to overcome the autophagy system
of the host cells (Cheng et al. 2018; Coelho et al. 2019).
Besides, it has been shown that LLO can induce
lymphocyte apoptosis, increasing pathogenesis by sup-
pressing proinflammatory cytokines (Interleukin 6 and
TNFa) and increasing IL-10 expression induced by
innate apoptotic immunity (Pattabiraman et al. 2017).
Target organ infection may depend on the spread
dynamics of the L. monocytogenes population. Within a
population, there are bacteria called “rare pioneer”that
determine the boundaries of infection foci and may
promote the persistence of bacterial infection. The cell-
to-cell spread is not homogeneous and the “rare pio-
neer”bacteria can infect non-adjacent cells (Ortega
et al. 2019). Although gut microbiota act by preventing
the spread of the pathogen in the liver and spleen
(Becattini et al. 2017; Pickard et al. 2017), both organs
tend to have the same bacterial transport capacity
found in the small intestine and colon. The gallbladder,
however, is the organ that has shown higher bacterial
density (Zhang, Abel, et al. 2017) probably due to the
poor ability to recruit immune effector cells (Gonzalez-
Escobedo and Gunn 2013) and bacterial resistance to
biles (Gahan and Hill 2014).
2.2. Innate immune response
The innate immune system is the first line of defense
orchestrated by resident microbiota, mucus, epithelial
cells, and phagocytes which produce inflammatory
cytokines for recruitment and activation of immune
cells for controlling L. monocytogenes. Recent studies
have shown that IL-6 receptor depletion in inflamma-
tory monocytes decreased the expression of CD38
ectoenzyme that is important in phagocytosis and early
control of L. monocytogenes infection (Hoge et al. 2013).
Among phagocytes, neutrophils play an essential role
in the initial clearance of L. monocytogenes in the liver
(Shi et al. 2011). In addition, a recent study showed that
L. monocytogenes infected hepatocytes act on macro-
phage migration to the liver through TLR-2 signalling,
promoting bacterial clearance and resistance to infec-
tion (Wang et al. 2019). However, the bacterial clear-
ance by macrophages can be affected by the induction
Figure 1. Immunoassay formats include ELISA (Enzyme- Linked Immunosorbent Assay), Immunofluorescence Assay, and Lateral
Flow Immunochromatographic Assay.
CRITICAL REVIEWS IN MICROBIOLOGY 5
of actin polymerisation that protects the bacteria from
autophagic recognition (Cheng et al. 2018).
2.3. Virulence factors and antigenic potential
L. monocytogenes express an array of virulence factors
(Table 1) that are essential for its survival and persist-
ence in the gastrointestinal tract and crossing of the
intestinal, blood-brain, and placental barriers (Bhunia
2018). The specific steps in the infection process involve
bacterial adhesion and intestinal barrier crossing (Drolia
and Bhunia 2019), epithelial cell invasion (Camejo et al.
2011), cell-to-cell spread (Travier et al. 2013; Travier and
Lecuit 2014; Bhunia 2018) and systemic dissemination
(Coelho et al. 2019).
2.3.1. Listeria adhesion protein
Among the virulence factors, L. monocytogenes surface
protein LAP interacts with the host cell receptor, Hsp60
for adhesion and translocation across the intestinal epi-
thelial barrier (Jagadeesan et al. 2011; Drolia et al. 2018;
Bai et al. 2021). LAP is a 104 kDa bifunctional house-
keeping enzyme (acetaldehyde alcohol dehydrogenase,
AdhE) and is present in all Listeria species of sensu
stricto group (Jagadeesan et al. 2010; Kim et al. 2015).
LAP is a highly immunogenic protein and a monoclonal
antibody raised against it has been used to study the
expression of this protein in Listeria under various
growth conditions (Pandiripally et al. 1999; Santiago
et al. 1999; Jaradat and Bhunia 2002).
Alternative to antibodies, Hsp60 is useful as a cap-
ture molecule that was coupled to paramagnetic beads
(Koo et al. 2011) or on a microfluidic device (Koo et al.
2009) for L. monocytogenes detection. Hsp60 is a human
cell receptor that interacts with LAP and the affinity
constant for Hsp60-LAP is 1.68 10
8
M, equivalent to
antigen-antibody interaction (Kim et al. 2006;
Jagadeesan et al. 2011). Thus, LAP-Hsp60 are useful sur-
face proteins for the development of immunoassays for
the detection of L. monocytogenes.
2.3.2. Internalin proteins
Members of the internalin multigene family cover sur-
face proteins important in bacterial adhesion and inva-
sion (Cossart and Helenius 2014; Quereda et al. 2019),
being highly immunogenic molecules that generate
antibodies useful for applications in the diagnosis and
detection of L. monocytogenes in foods (Heo et al. 2007;
Paoli and Brewster 2007). The two most studied inter-
nalins, InlA, and InlB contain a leucine-rich repeat (LRR)
in the amino-terminal region followed by a conserved
inter-repeated (IR) region (Cabanes et al. 2002).
InlA is an 88 kDa surface protein covalently linked to
the cell wall and binds to the eukaryotic receptor, E-
cadherin, promoting bacterial internalisation into enter-
ocytes (Radoshevich and Cossart 2018) and bacterial
transcytosis across the intestinal barrier (Nikitas et al.
2011). InlA is abundantly expressed and uniformly dis-
tributed on the surface of L. monocytogenes cells and is
anchored covalently to the cell wall, while InlB has short
chains with a helical region and a loop that allows spe-
cific recognition of molecules within the host cell
(Gessain et al. 2015).
InlA protein is highly antigenic and antibodies
(Mendonca et al. 2012) or aptamers (Ohk et al. 2010)
were developed for sensitive detection using immuno-
sensors. Antibodies were also developed against InlB
protein (65 kDa) and used in surface plasmon resonance
sensors (Leonard et al. 2005; Hearty et al. 2006). A com-
parative genomics approach was also used to generate
a PAb against InlB (Lathrop et al. 2008,2014).
Two studies have focussed on the use of antibodies
against variable and immunodominant regions of inter-
nalins. Hearty et al (Hearty et al. 2006) produced anti-
InlA antibody (MAb-2B3) that reacted strongly with
strains belonging to serotype 1/2a, but weakly with 1/
2c, 4a, and 4b. Since most of the listeriosis outbreaks
involve serotype 4b, the MAb-2B3 is not a desirable
candidate for use in food screening. Mendonca et al
(Mendonca et al. 2012) also produced MAb-2D12
against InlA and its characterisation showed high speci-
ficity for both L. monocytogenes and L. ivanovii.
Antibodies, bi-functional antibodies, and antibody frag-
ments against InlB have been used in the development
of immunology-based techniques useful in the detec-
tion of L. monocytogenes in human, environmental, and
food samples with suspected contamination (Hearty
et al. 2006; Tully et al. 2008; Lathrop et al. 2014; Owais
et al. 2014; Gene et al. 2015). Despite the similar protein
structures with InlA, InlB protein can elicit more specific
antibodies against the L. monocytogenes since it is
unique to its genome (Glaser et al. 2001; Hurley
et al. 2019).
2.3.3. Haemolysin, phospholipases and ActA
As mentioned above, internalised L. monocytogenes
secrete LLO, PC-PLC, and PI-PLC to disrupt the vacuolar
membrane and are released into the cytoplasm. L.
monocytogenes also express ActA that initiates host cell
actin polymerisation facilitating bacterial movement
from cell-to-cell (Tilney and Portnoy 1989;K
€
uhn and
Enninga 2020). These virulence proteins have been
expressed in vitro as recombinant antigens because
they have immunogenic sequences that are useful in
6 L. LOPES-LUZ ET AL.
the generation of antibodies. Antibodies generated
against a sequence of 17 amino acids from the N-ter-
minal region of LLO (58 kDa) have shown high specifi-
city for L. monocytogenes in the immunodetection of
the bacterium (Day and Hammack 2019). Due to its
high immunogenicity, LLO has also been used as an
antigen in the development of immunological
approaches for the diagnosis of listeriosis in both ani-
mals and humans (Rekha et al. 2006; Shoukat et al.
2013; Suryawanshi et al. 2017).
Antibodies generated against PI-PLC (29–32 kDa)
(Poussin and Goldfine 2005) and ActA (88–92 kDa)
(Paoli and Brewster 2007; Lathrop et al. 2008,2014;
Travier and Lecuit 2014) found application in L. monocy-
togenes detection. PI-PLC is highly conserved among
the Listeria genus and other non-Listeria species, such
as Bacillus cereus, B. anthracis,Staphylococcus aureus,
and Clostridium spp., presenting 51–56% identity
among these species (Daugherty and Low 1993) and
inducing high cross-reactivity in diagnostic tests
(Suryawanshi et al. 2017). These anti-PI-PLC antibodies
possibly will have cross-reactivity in assays for the
detection of L. monocytogenes in food. Highly specific
PAb against ActA, on the other hand, have the potential
to be used in immunological assays for specific detec-
tion of L. monocytogenes (Lathrop et al. 2014). Coupling
anti-ActA single-chain antibody with magnetic beads
was also effective for the separation of L. monocyto-
genes from food matrices (Paoli et al. 2007).
2.3.4. Autolysins
Among the autolysins, a protein of 60 kDa (P60, also
known as cell wall hydrolase - CWH) is highly conserved
among the Listeria genus (80%–90%) (Bubert et al.
1992; Wieckowska-Szakiel et al. 2002; Yu et al. 2004)
and the generated antibodies are highly reactive to
each member. Thus, the L. monocytogenes detection
using MAb against the P60 protein (CWH) structure
showed high cross-reactivity (Wang et al. 2017).
However, the expression of a peptide composed of 11
amino acids unique to the P60 protein has allowed the
generation of highly specific antibodies that are suit-
able for the detection of L. monocytogenes (Etty et al.
2019,2020).
Antibodies were also generated against another
autolysin amidase, called peptidoglycan muramidase or
N-acetylmuramidase (encoded by murA)inL. monocyto-
genes (Bhunia et al. 1991; Bhunia and Johnson 1992;
Carroll et al. 2003). These MAbs were reactive against
antigenic epitopes that are highly selective for L. mono-
cytogenes with very little or no reaction with other
Listeria spp. (Bhunia et al. 1991; Bhunia and Johnson
1992). These antibodies have been used on various
immunosensor platforms (Table 2) for the detection of
this pathogen from food (Geng et al. 2004; Koo et al.
2009; Ohk et al. 2010; Ohk and Bhunia 2013; Rodr
ıguez-
Lorenzo et al. 2019). Besides, the MAb-C11E9 was also
used for the delivery of nanocargo loaded with the anti-
cancer drug on the surface of L. monocytogenes target-
ing solid tumours (Akin et al. 2007).
2.3.5. Miscellaneous proteins
Cell surface proteins also play an essential role as spe-
cific antibody-inducing molecules (Zhang et al. 2016).
Surface autolysin (IspC; 86 kDa) has immunodominant
epitopes for L. monocytogenes serotype 4b (Ronholm
et al. 2013) and Fructose 1,6-Bisphosphate Aldolase
(FAB; 30 kDa) has strong immunogenicity (Mendonc¸a
et al. 2016). Listeria adhesion protein B (LapB; 184 kDa)
that is absent in non-pathogenic species, has the
potential to generate species-specific antibodies (Boivin
et al. 2016). Thus, IspC, FAB, LapB, and LAP are useful
surface proteins for the development of immunoassays
for the detection of L. monocytogenes.
3. Listeria monocytogenes detection:
immunological approaches
Antibodies generated against the key antigens of L.
monocytogenes have been useful in the development of
new immunological techniques for listeriosis control
and prevention (Yu et al. 2004; Hearty et al. 2006; Etty
et al. 2020). Techniques based on applied immunology
and biotechnology have shown quick, simple, and inex-
pensive applications (Li, Jing, et al. 2018; Lv et al. 2019).
MAbs and PAbs are the essential key elements for
immunological assay development such as ELISA,
immunofluorescence, lateral flow immunoassay, and
immunosensors (Banada and Bhunia 2008;
Jeyaletchumi et al. 2010; Law et al. 2014;V
€
alimaa et al.
2015; Xu et al. 2021). Important improvements based
on biotechnology and antibody engineering have been
performed, such as techniques based on single-chain
antibody (scFv) fragments and heavy-chain antibodies
(V
H
H fragments). scFv and V
H
H antibodies have similar
antigen-binding affinities as whole antibodies, however,
those can be produced in large quantities using micro-
bial expression systems and have easy handling and
extensive application range (Paoli et al. 2004; Liu,
Xiong, et al. 2017; King et al. 2018).
CRITICAL REVIEWS IN MICROBIOLOGY 7
3.1. Elisa and immunofluorescence-based methods
to detect Listeria
ELISA and immunofluorescence assays have been
widely used in the diagnosis of many diseases by
detecting specific antigens or antibodies. The results
are obtained through the enzymatic reaction of chrom-
ogens and fluorescent chemical compounds in ELISA
and immunofluorescence, respectively (Boonham et al.
2014; Diercks et al. 2017). The application of these
immunological assays has expanded to detect harmful
substances found in foods such as mycotoxins
(Leszczy
nska et al. 2013), allergens (Ito et al. 2016), bac-
terial toxins or antigens, and whole cells of gastro-
enteric bacteria (Zhao et al. 2017). Thus, these assays
have become an important tool in the prophylaxis of
several diseases, including listeriosis. Rapid detection
and identification of L. monocytogenes antigens in food
samples or food preparation/production premises can
help introduce proper Listeria control strategy and
potentially reduce listeriosis outbreaks (Cho et al. 2015;
Wang et al. 2015).
In the last decades, several methodologies based on
ELISA or immunofluorescence assays have been devel-
oped (Table 2). Notably, these immunoassays have
reduced the time for pathogen detection procedures
from 4 to 5 days to just 1 day compared to conventional
methodologies (Cavaiuolo et al. 2013; Bhunia 2014).
Regarding specificity, enzymatic and fluorescent-based
immunoassays have reached important improvements
for the accurate detection of L. monocytogenes in sam-
ples with suspected contamination. The production of
highly specific MAbs against L. monocytogenes antigens
ensures the specificity, especially the antibodies pro-
duced against epitopes of InlA (Mendonca et al. 2012),
InlB (Karamonov
a et al. 2003) or LLO (Prommajan et al.
2016) and recombinant p60 protein, that excluded con-
served regions between the genus Listeria (Coutu et al.
2014). PAbs can be used as efficient capture molecules
due to the heterogeneity of recognition while MAbs
assure the specificity of the method (Zhang et al. 2016).
In ELISA-based methods, the assay format can also
influence the specificity. Karamonov
a et al.
(Karamonov
a et al. 2003) demonstrated that the sand-
wich-type format, in which bacterial cells are free, was
more specific than the competitive format that keeps
bacteria immobilised. Also, both formats have distinct
antibody binding kinetics that can influence specificity.
In the sandwich-type format, it was possible to
decrease the limit of detection (LOD) through the use
of antibodies such as an anti-InlB antibody
(Karamonov
a et al. 2003), adaptations with piezoelectric
cantilever sensor (Sharma and Mutharasan 2013), use of
p60 recombinant antigens (Coutu et al. 2014) or use of
silica nanoparticles carrying polyacrylic acid (Chen
et al. 2015).
Tu et al. (Tu et al. 2016) showed that the use of V
H
H
fragments in indirect ELISA could decrease costs and
time for the detection of L. monocytogenes in food com-
pared to the use of conventional MAb or PAb. Liu,
Xiong, et al. (2017) showed that the application of scFv
fragments as a capture agent was sufficient to increase
the sensitivity of the assays, in addition to being a key
reagent of easy manipulation and with wide
application.
Although ELISA and immunofluorescence-based
methods are simpler than molecular techniques, they
are not considered rapid (Hearty et al. 2006; Jiang et al.
2019) because of the various washing and incubation
steps, in addition to the need for equipment to read or
visualise the reaction. These steps make the technique
more expensive, complex, and time-consuming (Bruno
et al. 2015; Chen et al. 2015). Thus, ELISA and immuno-
fluorescence assays may have limitations in the emer-
gency screening of food or production facilities with
suspected contamination by L. monocytogenes.
3.2. Lateral flow immunochromatographic-based
methods: rapid tests
Rapid tests are widely used in diagnostic medicine to
detect specific antibodies or pathogens and are charac-
terised by the availability of results in a few minutes (5
to 30 min) (Koczula and Gallotta 2016). In addition to
speed, rapid tests are inexpensive, simple, and easy to
perform. Thus, rapid tests have been applied in several
other areas such as environmental (Singh et al. 2015),
veterinary medicine (Jain et al. 2018), quality control in
industries, and food safety through the detection of
toxins, allergens (Ito et al. 2016) or even pathogens
such as L. monocytogenes (Cho and Irudayaraj 2013; Shi
et al. 2015; Wang et al. 2017). A rapid test that can diag-
nose a disease or detect pathogens in places with lim-
ited resources or when no pre-treatment or complex
processing of suspect samples is required, is classified
as a point-of-care test (POCT) (Koczula and
Gallotta 2016).
The lateral flow assay, also known as lateral flow
immunochromatographic assay (LFIA), is the immuno-
logical technique most used as a rapid test or POCT.
The assay principle is based on the sample containing
the analyte of interest introduction in the sample pad.
The sample moves laterally by the action of capillarity
through different zones of a strip. The first zone is the
conjugated pad which contains molecules conjugated
8 L. LOPES-LUZ ET AL.
Table 2. Immune approaches based on ELISA, immunofluorescence and immunosensors developed to detect L. monocytogenes
and other foodborne pathogens.
Immunoassay platforms Detected species
LOD (CFU/mL or CFU/
g)
(only L. monocytogenes) Food matrix
Improvement,
combination or antibody/
label used References
Multicolour and
ultrasensitive ELISA
E. coli O157:H7
Salmonella serotype
Choleraesuis
L. monocytogenes
7.0 10
1
in food
sample
(no enrichment)
milk Multicolour platform /
multicolour
fluorescence
hybridisation chain
reaction concatemers
(Lv et al. 2019)
Sandwich assay L. monocytogenes 6.5 in food sample
(48 h enrichment)
spiked pork and
pasteurised milk
Well-oriented single-chain
Fv (scFv)
antibody fragment
(Liu, Xiong, et al. 2017)
Dot-ELISA L. monocytogenes 1.0 10
6
in food
sample
(5 h enrichment)
pork meat and
fresh vegetables
MAb LMF3-238 (Prommajan
et al. 2016)
Sandwich assay L. monocytogenes 1.0 10
4
in food
sample
(no enrichment)
pasteurised milk Heavy-chain antibodies
(VHHs) (species-
specific nanobodies)
(Tu et al. 2016)
Plasmonic ELISA L. monocytogenes 8.0 10
1
in food
sample
(no enrichment)
spiked Lettuce Silica nanoparticles
carrying
polyacrylic acid
(Chen et al. 2015)
Sandwich assay Cross-reactivity
between:
L. monocytogenes
L. innocua
E. coli
S. Typhimurium
S. aureus
1.0 10
3
in pure
culture
(no enrichment)
–Antibodies against
recombinant
p60 protein
(Coutu et al. 2014)
Indirect assay L. monocytogenes
E. coli O157
10
3
in food sample
(no enrichment)
cucumber Commercial antibodies
from Abcam: anti-E.
coli O157 (ab20976)
and anti- L.
monocytogenes
(LZA2) (ab11439)
(Cavaiuolo et al. 2013)
Sandwich assay L. monocytogenes 10
3
in food sample
(no enrichment)
milk A novel piezoelectric
cantilever sensor
(Sharma and
Mutharasan 2013)
Competitive fluorescence
immunoassay
L. monocytogenes 1.0 in pure culture
(18 h enrichment)
–MAbs against
recombinant
p60 protein
(Beauchamp
et al. 2012)
Sandwich assay L. monocytogenes 5.0 in food sample
(24 h enrichment)
milk Anti-internalin B antibody (Karamonov
a
et al. 2003)
SERS spectroscopy
combined with
microfluidic
L. monocytogenes 110
5
in pure culture
(no enrichment)
–MAb-C11E9/
aminopeptidase
(66 kDa)
(Rodr
ıguez-Lorenzo
et al. 2019)
Custom-built multichannel
surface plasmon
resonance
(SPR) biosensor
L. monocytogenes
E. coli O157H:7
S. Enteritidis
28 CFU/25 g in food
sample
(24 h enrichment)
chicken PAb anti-Listeria (Zhang, Tsuji,
et al. 2017)
Multiplex fibre
optic biosensor
L. monocytogenes,
E. coli O157:H7
S. enterica
10
3
in food sample
(24 h enrichment)
ready-to-eat
meat samples
PAb-66 anti-Listeria
MAb-C11E9 anti-
aminopeptidase
(66 kDa)
(Ohk and Bhunia 2013)
Fibre optic immunosensor
coupled with
immunomagnetic
separation
L. monocytogenes
L. ivanovii
310
2
in food sample
(16–18 h
of enrichment)
hotdog and
soft cheese
MAb-2D12 anti-InlA (Mendonca et al. 2012)
Antibody–aptamer
functionalised fibre-
optic biosensor
L. monocytogenes 10
3
in pure culture
and 10
2
CFU/25 g in
food samples
(18 h enrichment)
ready-to-eat
meat products
Aptamer-A8 anti-InlA (Ohk et al. 2010)
Surface plasmon
resonance
(SPR) biosensor
L. monocytogenes 210
6
in pure culture
(no enrichment)
–scFv antibody/ActA (Nanduri et al. 2007)
Automated fibre
optic biosensor
L. monocytogenes 210
3
in PBS and
510
5
in food sample
(20 h enrichment)
Frankfurter sample PAb anti-Listeria
PAb-66 anti-Listeria
(Nanduri et al. 2006)
Fibre-optic immunosensor L. monocytogenes 4.310
3
in pure
culture
(20 h enrichment)
Hot dog and Bologna MAb-C11E9 anti-
aminopeptidase
(66 kDa)
(Geng et al. 2004)
Resonant mirror biosensor L. monocytogenes 110
6
in pure culture
(no enrichment)
–MAb-C11E9 anti-
aminopeptidase
(66 kDa)
(Lathrop et al. 2003)
CRITICAL REVIEWS IN MICROBIOLOGY 9
to coloured or fluorescent labels (Hsieh et al. 2017). In
conventional methods, colloidal gold nanoparticles
conjugated to specific antigens or antibodies that act
as detection agents are commonly used (Mao et al.
2009). Then, there is the detection zone formed by por-
ous membranes, such as the nitrocellulose (NC) mem-
brane, containing specific biological reagents
immobilised in test and control lines (Koczula and
Gallotta 2016). Polyclonal antibodies are widely used in
the test lines as a capture agent for the conjugate ana-
lyte (Banerjee and Jaiswal 2018; Jacinto et al. 2018). The
recognition of the analyte in the sample promotes
staining in the test line and the result can be read with
the naked eyes. The test operation is certified by
observing colour in the control line (Sajid et al. 2015;
Hsieh et al. 2017).
To ensure food safety and for foodborne pathogen
and toxin detection, the development of LFIA as a rapid
and inexpensive test has exploded in recent decades
(Table 3). LFIA is an essential detection tool in the food
industry and has aided the industry to eliminate and
prevent contamination by L. monocytogenes in process-
ing environments and in food products (Phraephaisarn
et al. 2017). Specifically, the greatest advantage of LFIA
compared to ELISA and immunofluorescence-based
methods are that a person with minimal technical
knowledge can perform the test without the need for a
laboratory or specialised equipment (Wang et al. 2015).
To be used as a tool to prevent listeriosis outbreaks,
an LFIA should be able to detect the lowest possible
number of bacterial cells per gram or millilitre. The min-
imum infection dose varies widely (<100 cells to 10
11
cells) among neonates, elderly, immunocompromised,
and healthy individuals (Sim et al. 2002; Ooi and Lorber
2005; Pouillot et al. 2016). The LFIA available for the
detection in pure culture or food samples have low
accuracy and the LOD of commercial tests ranges from
110
5
to 1 10
6
CFU/mL (Kitao et al. 2010; Cho and
Irudayaraj 2013). Combining LFIA with molecular tech-
niques, such as recombinase polymerase amplification
lateral flow (RPA-LF), Du et al. (Du et al. 2018) reported
LOD to be 1.5 10
1
CFU/mL in pure culture within
15 min: 10 min for DNA amplification and 5 min for visu-
alisation of the amplicons. In different food samples,
after 6 h of enrichment, the researchers reported the
LOD to be 12–36 CFU/mL while Liu, Du, et al. (2017)
reported a LOD of 22 to 36 CFU/mL in food sample
within 30 min.
Application of LFIA in food samples can be limited
by the types of food matrix used, which often affects
the biological activity of reagents and bacterial anti-
gens, decreasing the sensitivity of conventional tests (Li
et al. 2017). Bacterial separation and concentration
methodologies have been used as sample pre-treat-
ment to reduce the food matrix interference. Li et al.
(Li, Li, et al. 2018) developed a nucleic acid lateral flow
biosensor with an immunomagnetic separation plat-
form based on a biotin-streptavidin system that
resulted in a LOD of 3.5 10
3
in pure culture and LOD
of 3.5 10
4
CFU/g in lettuce, both after 6 h of enrich-
ment. Biotinylated antibodies show better capture cap-
acity in an immunoseparation system due to the high
affinity of streptavidin for biotin. In this system, the
binding sites of antibodies coupled to magnetic nano-
particles are targeted. Combining lateral-flow chroma-
tography enzyme immunoassay platform with
immunomagnetic separation and concentration, Cho
and Irudayaraj (Cho and Irudayaraj 2013) found a LOD
of 10
2
CFU/mL in pure culture and artificially contami-
nated milk samples in 2 h. In addition to reducing the
interference of the food matrix, immunomagnetic sep-
aration or concentration decreases other steps of the
pre-treatment process, maintains the biological activity
of the samples, and can also be used to emit signals for
quantitative analysis when the magnetic nanoparticles
are conjugated to monoclonal antibodies (Mendonca
et al. 2012; Cho and Irudayaraj 2013; Shi et al. 2015).
Employing a pathogen enrichment device (PED)
coupled with lateral-flow immunoassay, Hahm et al.
(Hahm et al. 2015) reported LOD of 3.2 10
2
to
5.6 10
4
for L. monocytogenes from food samples. The
LOD values varied depending on the initial inoculation
and the enrichment time (8–24 h), and the LOD was cal-
culated by counting the bacteria on MOX agar plates.
Combining surface-enhanced Raman scattering
(SERS) with bacteriophage amplification, Stambach
et al. (Stambach et al. 2015) demonstrated a LOD of
510
4
CFU/mL to 1 10
7
CFU/mL, after enrichment of
28 h. Using a similar approach, Wu (Wu 2019)
reported LOD of 75 CFU/mL in pure culture and about
200 CFU/mL in artificially contaminated milk spiked
with different concentrations of L. monocytogenes. They
used SERS-based LFIA for the simultaneous detection of
L. monocytogenes and Salmonella enterica serovar
Typhimurium.
3.3. Immunosensors
Immunological approaches based on biosensors also
use antibodies as key molecules for the recognition of
analytes in different types of samples. Thus, biosensors
that use antibodies are called immunosensors. In this
technique, the antigen-antibody bond is converted into
a measurable physical signal through a signal
10 L. LOPES-LUZ ET AL.
Table 3. Lateral flow-based methods for L. monocytogenes detection and their technical features.
Lateral flow-based methods/labelling Target species
LOD (CFU/mL or CFU/
g) in pure culture j
runtime (only L.
monocytogenes)
LOD (CFU/mL or CFU/g) in food matrix
jruntime (only L. monocytogenes) Improvement or labelling or antibody References
SERS-based lateral flow
immunochromatographic assay
L. monocytogenes
Salmonella
Typhimurium
75 j229 CFU in milk jA novel SERS using two different Raman
reporters:
anti-L. monocytogenes antibody and
DTNB-modified AuNPs
anti-S. Typhimurium antibody and 4-
MBA-modified AuNPs
(Wu 2019)
Nucleic acid lateral flow biosensor L. monocytogenes 3.5 10
3
j6 h 3.5 10
4
in lettuce j6 h Combination: PCR-based single specific
nucleic acid sequence amplification
with nucleic acid lateral flow
biosensor; pre-treatment platform:
immunomagnetic separation based on
the streptavidin-biotin system
(Li, Li, et al. 2018)
Lateral flow strip L. monocytogenes 1.5 10
1
j15 min 12–36 in milk, chicken breast, bread,
cereal milk, milk tea, and latte
coffee j15 min after 6 h
of enrichment
Recombinase polymerase amplification (Du et al. 2018)
SERS-based lateral flow strip biosensor L. monocytogenes
Salmonella serotype
Enteritidis
19 j5 min 22 to 36 in milk, chicken breast and
beef j5–30 min
Recombinase polymerase amplification (Liu, Du, et al. 2017)
Fluorescence
immunochromatographic assay
L. monocytogenes 4.0 10
4
to 4.0 10
5
j110
4
in sausage, pork and milk 100
times enriched j3h
Immunomagnetic separation and
polyclonal antibodies generated from
whole killed L. monocytogenes cells
(Li, Jing, et al. 2018)
Gold nanoparticle-based paper sensor Listeria sp. 1 10
3
to 1 10
4
j1 to 9 in milk j10 min after
8 h enrichment
Monoclonal antibody against P60 protein (Wang et al. 2017)
Lateral flow immunochromatography
assay coupled to pathogen enrichment
device (PED)
Escherichia coli
O157:H7, Salmonella
enterica,L
monocytogenes
–3.2 10
2
to 5.6 10
4
in spinach,
ground beef, hotdogs, and e.g.gs j
8–24 h of enrichment
Pathogen enrichment device (PED)
containing a growth chamber, filters,
and an ion-exchange cartridge to
deliver bacteria directly onto the
detection platforms
(Hahm et al. 2015)
SERS-lateral flow immunochromatography L. monocytogenes 510
4
to 1 10
7
j2–8h
–Bacteriophage amplification (phage A511) (Stambach et al. 2015)
Superparamagnetic lateral flow
immunoassay
L. monocytogenes 110
4
j20 min Various food sources, including
chicken, fish, flammulina
velutipes, etc.
Superparamagnetic particles and a pair of
monoclonal antibodies
(Shi et al. 2015)
Lateral-flow enzyme
immunoconcentration
L. monocytogenes 95 j2 h 97 in reduced-fat milk j2 h Immunomagnetic separation and
concentration step
(Cho and
Irudayaraj 2013)
Nucleic acid lateral flow immunoassay L. monocytogenes
Listeria spp.
–110
5
in dairy products, ready-to-eat
salad, sprouted seeds, nutrition for
children, etc j15 min after 24 h
(enrichment) and 3.5 h (isolation of
template DNA and a PCR
amplification step)
A duplex PCR (two labelled primer sets) (Bla
zkov
a et al. 2009)
Runtime not mentioned.
¶Not mentioned.
CRITICAL REVIEWS IN MICROBIOLOGY 11
transducer coupled to the biosensor (Banada and
Bhunia 2008; Bhunia 2008; Singh and Bhunia 2018;Xu
et al. 2021). Compared to lateral flow-based methods,
immunosensors also have flexibility in use, many are
portable and allow the simultaneous detection of sev-
eral biomolecules. The effectiveness of an immunosen-
sor depends on the high specificity and sensitivity that
are directly related to the type, quality, and reactivity of
the antibodies used and other combined techniques
(Ohk et al. 2010; Smolsky et al. 2017). Various immuno-
sensor platforms are developed for specific detection of
L. monocytogenes which are summarised in Table 2.
Fibre optic biosensor utilises sandwich immunoassay
configuration and the specificity of the assay depends
on the antibody pair used and the assay sensitivity on
the generation of the evanescent wave signal. Fibre
optic sensor has been developed using antibodies to N-
acetyl muramidase (MAb-C11E9) (Geng et al. 2004) and
InlA (Mendonca et al. 2012) and the assay was highly
specific and the LOD varied from 10
2
–10
4
CFU/mL.
Polyclonal anti-Listeria antibodies have heterogeneous
recognition of pathogen antigens and are useful as cap-
ture agents when immobilised on the fibre optic bio-
sensor platform (Nanduri et al. 2006,2007; Zhang, Tsuji,
et al. 2017). MAb-C11E9 application has improved the
specificity of immunosensors based on SERS
(Rodr
ıguez-Lorenzo et al. 2019), multiplex fibre optic
biosensor (Ohk and Bhunia 2013), and resonant mirror
biosensor (Lathrop et al. 2003). Although MAb-C11E9
reacts weakly with L. innocua antigens under certain
conditions of cultivation and bacterial lysis (Lathrop
et al. 2003), it was demonstrated that the application of
the MAb-C11E9 in the SERS-based method can distin-
guish L. monocytogenes from L. innocua (Rodr
ıguez-
Lorenzo et al. 2019).
The SERS-based method is a quantitative, stable, sim-
ple, and specific technique that allows improving L.
monocytogenes detection and has been extensively
studied. This technique is used to detect isolated mole-
cules on surfaces with high sensitivity through Raman
dispersion (Smolsky et al. 2017; Liu, Du, et al. 2017;
Oravec et al. 2018;). Recently, gold nanostars conju-
gated to MAb-C11E9 were used for the detection of L.
monocytogenes using SERS (Rodr
ıguez-Lorenzo et al.
2019). A surface plasmon resonance sensor was also
developed for the detection of L. monocytogenes using
MAb (Lathrop et al. 2003; Nanduri et al. 2006) or phage-
displayed single chain antibodies (Nanduri et al. 2007).
Zhang et al (Zhang, Tsuji, et al. 2017) developed a
multichannel SPR using PAb for the detection of L.
monocytogenes,S. Enteritidis, and E. coli O157:H7. After
24 h of enrichment, the three pathogens were detected
at a concentration of >10
5
CFU/mL in pure culture, and
LOD was estimated to be 2.3 10
5
CFU/mL for L. mono-
cytogenes in a chicken sample.
The use of highly specific MAbs, PAbs, or other
reagents based on antibody engineering is essential for
developing highly specific immunosensors. An aptamer
specific for internalin A was used in combination with
polyclonal anti-Listeria P66 in a sandwich format for
detection of L. monocytogenes in a fibre-optic biosensor
(Ohk et al. 2010). It is important to note that immuno-
logical approaches that use some type of pre-treatment
or separation process with magnetic nanoparticles
improve the sensitivity of the assays. However, these
combined approaches require additional more complex
analyses when compared to results interpreted with the
naked eye (Fisher et al. 2009; Uusitalo et al. 2016).
4. Conclusions and future perspectives
Comprehensive knowledge of the pathogenesis and
the role of each virulence factor in pathogenicity pro-
vides a solid foundation for developing a robust diag-
nostic or detection tool for a pathogen. Targeting a
virulence factor as an antigen also helps to develop an
assay that can detect only the pathogenic species (ex.
L. monocytogenes) within a genus that may have non-
pathogenic members (ex. L. innocua,L. welshimeri,L.
seeligeri.L. marthii, and others) that share high genetic
sequence similarities. In this review we provide updated
information on the pathogenesis of L. monocytogenes,
the immunogenicity of virulence determinants and
their expression in food or food testing reagents (such
as in enrichment broths) that are key to the generation
and selection of highly specific monoclonal and poly-
clonal antibodies for immunological assay
development.
The immunological assays include ELISA, immuno-
fluorescence, lateral flow immunochromatography, and
immunosensors. Though immunological methods are
perceived as less sensitive than molecular-based meth-
ods (such as PCR), their affordability, ease of use, rapid
results, and portability (point-of-care use) make them
highly attractive. Furthermore, the limit of detection
varies among the immunoassay platforms: a traditional
ELISA requires a minimum of 1 10
6
CFU, and commer-
cial lateral flow-based assays require about 1 10
7
CFU.
The application of improved fluorogenic or colorimetric
substrates in ELISA and nanoparticles and fluorophores
in lateral flow-based assays can improve the assay sen-
sitivity significantly, i.e. a reduced detection limit.
Remarkably, the most improvement in immunoassays
in recent years has been observed in biosensor
12 L. LOPES-LUZ ET AL.
(immunosensor) platforms, where a detection limit of
110
2
110
3
CFU was achieved for L. monocyto-
genes. However, such sensitive assays are more vulner-
able to inhibitors, background microflora, and food
matrix thus sample preparation needs extreme care
which, therefore, may prolong the assay time (i.e. sam-
ple-to-result) and cost per test.
It is generally accepted that most food products,
especially those that are ready-to-eat are free of L.
monocytogenes or its level is very low and a majority of
aforementioned assay platforms are unable to provide
test results when applied to food samples directly.
Therefore, an enrichment step, either growth-based or
physical concentration method (IMS, centrifugation) is
needed to meet the detection threshold. The growth-
based enrichment method is common since it helps
resuscitate injured or stressed bacterial cells, increase
bacterial numbers, dilute inhibitors and reduce back-
ground microbial competitors providing reliable assay
results, but prolongs the assay time. However, the
length of enrichment can be shortened with an assay
that has a very low detection limit. On the other hand,
IMS is useful but samples containing high levels of food
particles or low pH may interfere with bacterial enrich-
ment efforts. Irrespective of detection platforms used, a
highly efficient sample preparation step is critical for
success especially for those that promote resuscitation
and growth and remove interfering agents from the
test samples to meet the detection threshold for
that assay.
Several immunoassay platforms were presented in
this review, as well as improvements that have been
studied and applied in combination with these
immunological techniques. This combination has
increased the specificity and sensitivity of the techni-
ques, making them important tools in listeriosis diagno-
sis, control, and prevention. However, the
methodologies still face challenges in the removal of
food matrices and inhibitors as part of efficient sample
preparation. Furthermore, due to the low sensitivity of
most tests, sample enrichment steps are imperative to
increase bacterial concentration to a detectable level.
Thus, there is a need for further investigation on meth-
odology optimisation and the selection of more specific
antibodies for the development of a more effective,
fast, and accessible method.
Disclosure statement
The authors declare no conflict of interest.
Funding
The work in the authors’laboratories was supported by
funds from the Brazilian Coordination for the Improvement
of Higher Education Personnel [CAPES grant # 88882.385457/
2007-01 2019–2023] and by the U.S. Department of
Agriculture, Agricultural Research Service, under Agreement
No. 59-8072-6-001. Any opinions, findings, conclusions, or
recommendations expressed in this publication are those of
the author(s) and do not necessarily reflect the view of the
U.S. Department of Agriculture.
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