Food Control 19 (2008) 1–8

www.elsevier.com/locate/foodcont

Review

Determination of food authenticity by enzyme-linked

immunosorbent assay (ELISA)
a,*
Luis Asensio , Isabel González b, Teresa Garcı́a b, Rosario Martı́n b

a
Departamento de Nutrición, Bromatologı́a y Tecnologı́a de los Alimentos, Facultad de Farmacia, CEU – Universidad San Pablo,
28668 Boadilla del Monte, Madrid, Spain
b
Departamento de Nutrición, Bromatologı́a y Tecnologı́a de los Alimentos, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain

Received 16 June 2006; received in revised form 6 February 2007; accepted 16 February 2007

Abstract

This work intends to provide an updated and extensive overview on the applications of ELISA techniques for meat, fish and milk
species discrimination; fruit juice labeling authentication; genetically modified and irradiated food detection; feedstuffs origin and aller-
gen ingredients identification. These methods have been widely used because they reduce the use of costly, sophisticated equipment and
time of analysis and are suitable for routine analysis of a large number of samples. Therefore, ELISA could allow, together with other
analytical methods such as DNA-based methods, consumer protection and confidence, and an accurate implementation of the traceabil-
ity for successful regulatory food controls.
Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Food authentication; ELISA; Polyclonal and monoclonal antibodies

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Meat and meat-based products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Fish and fishery products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
4. Milk and dairy products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
5. Fruit juice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
6. Genetically modified foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
7. Irradiated food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
8. Feedstuffs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
9. Allergen ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
10. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1. Introduction

Authenticity testing of food products, such as meat, milk

or fish, is important for labeling and assessment of value
*
Corresponding author. Tel.: +34 91 372 64 49; fax: +34 91 351 04 75. and is therefore necessary to avoid unfair competition and
E-mail address: lasen.fcex@ceu.es (L. Asensio). assure consumers protection against fraudulent practices

0956-7135/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2007.02.010
2 L. Asensio et al. / Food Control 19 (2008) 1–8
commonly observed in the food industry. Additionally,
fraudulent adulteration of food products may be objection-
able for health reasons, since consumption of products con-
taining, undeclared constituents may cause problems such
as allergy in sensitized individuals (Mackie, 1996).
In this context, food components identification has been
mostly performed in the last few years by different tech-
niques. Chromatographic and electrophoretic techniques
have proved to be useful in food components identification
(Berrini, Tepedino, Borromeo, & Secchi, 2006; Mackie
et al., 2000; Mayer, 2005). However, although they are con-
siderable value in certain instances, these methods are not
convenient for routine sample analyses because they are
relatively costly, time consuming, and complex to perform.
Consequently, in the last years the identification of meat
and meat-based products, fish and seafood, milk and dairy
products, and other foods has been performed primarily by
genetic (Bottero, Civera, Anastasio, Turi, & Rosati, 2002;
Matsunaga et al., 1999; Terzy et al., 2005) and immunolog-
ical techniques (Carrera et al., 1997; Liu, Chen, Dorsey, &
Hsieh, 2006).
Genetic methods are the most specific and sensitive
methods for food components authentication. However,
they require expensive laboratory equipment and a certain
degree of expertise. As an alternative, immunological
assays can be used to reduce the test time and cost. Among
these last methods, the Enzyme-Linked ImmunoSorbent
Assay (ELISA for short) has been the most widely used
technique for regulatory purposes in detecting food
authenticity because of its specificity, simplicity and sensi-
tivity, among other advantages (Mackie, 1996). On the
whole, ELISA is an immunological technique that involves
an enzyme (a protein that catalyzes a biochemical reaction)
to detect the presence of an antibody or an antigen in a
sample. The two most used variants of ELISA for food
authentication are the indirect and the sandwich ELISA.
The indirect ELISA utilizes two antibodies, one of which
is specific to the antigen and the other of which is coupled
to an enzyme. This second antibody gives the assay its
‘‘enzyme-linked’’ name, and will cause a chromogenic or
fluorogenic substrate to produce a signal. Sometimes this
second antibody may be linked to a protein such as avidin
or streptavidin if the primary antibody is biotin labeled. In
the sandwich ELISA the antigen is bound between two
antibodies: the capture antibody and the detection anti-
body. The detection antibody can be coupled to an enzyme
or can bind the conjugate (enzyme-linked antibody) that
will produce the biochemical reaction (Goldsby, Kindt,
Osborne, & Kuby, 2003).
ELISA tests may be run in a qualitative or quantitative
format. Qualitative results provide a simple positive or neg-
ative result for a sample. The cutoff between positive and
negative is determined by the analyst and may be statisti-
cal. In quantitative ELISA, the optical density or fluores-
cent units of the sample is interpolated into a standard
curve which is typically a serial dilution of the target (Gold-
sby et al., 2003).
Nevertheless, the prerequisite for an ELISA is the avail-
ability of amounts of antibodies sufficient to detect ana-
lytes. Both polyclonal and monoclonal antibodies
(MAbs) can be used in ELISA methods for food compo-
nents identification. Polyclonal antibodies offer a number
of benefits such as recognition of a mixture of different epi-
topes of the antigens, more tolerance to small changes in
the nature of antigen, like polymerization or slight denatur-
ation and they are a preferred choice for detection of dena-
turated proteins. However, they present limitations such as
variable affinity, limited production and a requirement for
extensive purification procedures to eliminate cross-reactiv-
ity for a particular species identification (Harlow & Lane,
1999). In contrast, MAbs are a homogeneous population
of antibodies produced by hybridoma technology that have
defined biological activity, consistent specificity and their
production is not limited (Goding, 1996). Both polyclonal
antibodies and MAbs are used in the ELISA variants for
food authentication that have been previously described
(Harlow & Lane, 1999).
Furthermore, although the great limitation of ELISA
methodology is that the target proteins sometimes are
denatured during food processing and therefore the target
protein epitope may not be present in the condition detect-
able by the antibodies, this limitation has been solved
because of the development of antibodies against thermo-
stable proteins (Ansfield, 1994; Ansfield, Reaney, & Jack-
man, 2000; Berger, Mageau, Schwab, & Johnston, 1988).
In the last years, advances in ELISA technology have
led to rapid development of different commercial immuno-
assays kits for use in the food and feed industry. The most
used immunoassays kits for food components identification
are ELISA and lateral flow tests. ELISA test kits may be
performed in microtiter plates and immunosticks formats.
Lateral flow tests or ‘‘dipsticks’’ employ the same immuno-
assay principles as the ELISA tests but coat the antibodies
and other reagents on a nitrocellulose membrane rather
than the inside of test wells or paddles and they use colloi-
dal gold, dye, or latex bead conjugates to generate signal
rather than enzymes bead conjugates. The simplicity of
both type of tests and the short time required for the anal-
ysis make them suitable for food screening tests of a large
number of samples (Bonwick & Smith, 2004).
On the basis of this information, we report in the present
review the ELISA applicability on food authenticity in the
last few years. We have described the use of ELISA tech-
niques for meat, fish and milk species discrimination; fruit
juice labeling authentication; genetically modified and irra-
diated food detection; feedstuffs origin and allergen ingre-
dients identification.
2. Meat and meat-based products
Legislative authority establishes that meat products
must be accurately labeled regarding species content. Meat
species adulteration in ground and comminuted products
has been a widespread problem in retail markets. Identifi-
 L. Asensio et al. / Food Control 19 (2008) 1–8
cation of the species of origin in meat samples is relevant to
consumers for several reasons: (i) possible economic loss
from fraudulent substitution or adulteration, (ii) medical
requirements of individuals who might have specific food
allergies, and (iii) religious reasons. Thus, reliable and sen-
sitive analytical tools are required for detection and identi-
fication of animal food ingredients. DNA-based methods
and ELISA techniques have been the most widely used
for meat authentication. As it has been shown in the intro-
duction, DNA-based methods are the most specific and
sensitive methods for meat species identification, although
they require some expensive laboratory equipment and a
certain degree of knowledge. On the contrary, ELISA,
being sensitive and specific, are quicker than genetic meth-
ods for routine analysis of large sample numbers.
Consequently, ELISA methods have been used in the
last years for identifying meats of different animal species
using antibodies against muscular and serum animal pro-
teins or thermostable proteins (Berger et al., 1988; Martı́n,
Azcona, Casas, Hernández, & Sanz, 1988; Rencova,
Svoboda, & Necidova, 2000).
Additionally, qualitative studies have been reported to
identify the origin of cooked mammalian meats products
(Ayaz, Ayaz, & Erol, 2006; Macedo-Silva et al., 2000).
Also, quantitative methods have been shown. For example,
Martin, Chan, and Chiu (1998) carried out successfully a
quantitative evaluation of pork adulteration in raw ground
beef by ELISA. Furthermore, Chen and Hsieh (2000) and
Liu et al. (2006) developed MAbs against porcine thermal-
stable muscle protein and quantified pork meat in raw and
heat-processed meat and feed products by this technique.
The detection limit was determined as 0.5–0.05% (w/w)
pork in heterologous meat mixtures.
Some companies such as Strategic Diagnostics, Inc.,
Tepnel, Neogen Corporation, Eurofins and ELISA Tech-
nologies, Inc., among others, have developed a variety of
meat species test kits (ELISA tests and lateral flow devices)
that detect and identify species content in cooked, raw and
thermally processed meat, meat products and animal feed.
Lateral flow tests are faster than ELISA tests, although
they are qualitative tests. On the contrary, ELISA tests
are more suitable for quantitative analysis. Nevertheless,
both type of tests provide rapid, reliable and cost-effective
screening in the meat species identification (Bonwick &
Smith, 2004; Giovannacci et al., 2004). In fact, some of
these test kits are being used by regulatory agencies to
detect meat species adulteration and to enforce national
and trans-national laws and regulations.
In other meat products, non-meat proteins, such as
soyabean proteins, are added due to their interesting nutri-
tional and functional properties. Nevertheless, one form of
adulteration for economic gain is the addition of these
cheaper proteins as undeclared ingredients in meat prod-
ucts. This fraud often occurs causing unfair competition
and it is a potential health hazard for individuals with aller-
gies. Thus, ELISA techniques have been used to identify
these substitutions preventing willful or unintentional
3
substitution of high priced proteins with lower priced
proteins.
For example, ‘‘chorizo,’’ a Spanish style sausage, is
often substituted with soy because the meat product’s
strong flavor easily masks foreign flavors. Cota, Vallejo,
Calderón, and González (1998) produced polyclonal anti-
sera against soy flour proteins to quantify this adulteration
by ELISA. Another example of adulteration in meat prod-
ucts has been shown by Macedo-Silva, Shimokomaki, Vaz,
Yamamoto, and Tenuta-Filho (2001). These authors deter-
mined soy proteins in hamburgers prepared from beef,
chicken and swine meat. Consequently, they obtained poly-
clonal antibodies against these proteins and quantified soy
protein in these meat products by ELISA. Recently, Kopp-
elman, Lakemond, Vlooswijk, and Hefle (2004) developed
a sandwich ELISA test applicable for the quantification
of soy ingredients and soy-containing foods that were pro-
cessed in different ways. They reached a limit of quantita-
tion of 1 ppm using polyclonal antibodies by this
technique.
Different companies (Neogen Corporation, Zeu Inmu-
notec, ELISA Systems and Tepnel, among others) com-
mercialize immunochemical diagnostic kits for the rapid
detection and quantification of soy proteins in foods. These
kits contain all the necessary reagents, controls and acces-
sories for on-site rapid testing.
3. Fish and fishery products
The ability to identify fish species following the removal
of external characteristics by processing such as canning,
smoking or filleting, although problematic, is of great com-
mercial importance. Once the morphological characters
have been removed during processing, the identification
becomes difficult and there is a risk for fraudulent substitu-
tion of lower-valued fish species for high-valued fish in sea-
food products. To prevent fraudulent fish substitutions,
food laboratories need to have available techniques to
ascertain the fish species used in the manufacture of fish
products. Fish species identification has been mostly per-
formed in the last few years by PCR-based and ELISA
methods that are suitable for routine analysis of a large
number of samples (Céspedes et al., 1999; Rehbein et al.,
2002). However, ELISA techniques are less expensive and
simpler to develop than DNA-based methods.
Generally, work relating to fish species identification is
scarce, partly due to the variety of fish species that are com-
mercialized. Nevertheless, in the last years polyclonal anti-
bodies have been produced against the muscular protein of
sardines (Taylor & Jones, 1992), different flat fish species
(sole, Greenland halibut, European plaice and flounder)
(Céspedes et al., 1999) and clam species (Fernández et al.,
2002). These antibodies have been used in an indirect
ELISA and allowed the unequivocal identification of these
species.
Furthermore, MAbs have been produced against fish
muscle proteins being possible the identification of the
4 L. Asensio et al. / Food Control 19 (2008) 1–8
red snapper (Huang, Marshall, Kao, Otwell, & Wei, 1995)
or the discrimination of raw and cooked grouper among
other less valued fish species (Asensio et al., 2003a,
2003b).
For fish species identification, no ELISA test kit has
been developed yet. It could be due to the great variety
of fish species that are commercialized. Therefore, the
future development of rapid diagnostic kits in this area
could be very useful for field screening purposes in inspec-
tion programs.
4. Milk and dairy products
Fraudulent incorporation of cheaper bovine milk during
cheese making is a common practice that can become a
problem for reasons related to allergy, religious, ethical
or cultural objections, and legal requirements. Accurate
evaluation of the milk species used in dairy products is
therefore needed, especially for high-grade cheeses made
exclusively with sheep’s or goats’ milk, many of which
are registered by European law with a Protected Designa-
tion of Origin (PDO) (Bottero et al., 2002). Nevertheless,
mixtures of bovine milk with ovine or caprine milk can also
be used to make cheeses that may lack the PDO quality
indication. In these cases, it is necessary to indicate on
the label the species used in the manufacture of cheeses.
Therefore, for legal reasons and for consumer protection
and confidence, cheeses should be authentic and correctly
labeled using reliable and sensitive analytical tools such
as ELISA.
This methodology has been one of the most widely used
form of milk species identification techniques because it is
easy to use, rapid and readily automated (Hurley, Cole-
man, Ireland, & Williams, 2004). To date, a great number
of enzyme immunoassays have been reported for species
authentication in milk and cheese. For example, Hurley
et al. (2004, 2006) developed an indirect competitive and
sandwich ELISA for the detection of low percentages of
undeclared bovine milk in goats’, ewes’ and buffalo’s milk.
In other study, López-Calleja et al. (2006) applied an indi-
rect ELISA using a MAb against bovine b-casein (Anguita
et al., 1995) that provided to detect adulteration in non-
declared cows’ cheeses.
In the market there are also immunoassays kits for spe-
cies authentication in milk and cheese. These kits that are
commercialized by companies such as Zeu Inmunotec or
Euro-Diagnostica, among others, require a minimal
amount of training and equipment and are used for the
rapid identification and quantification of the milk species
used in dairy products.
5. Fruit juice
The quality control of citrus juice beverages is of crucial
importance as these products can be easily falsified.
Increasing number of papers related to the adulteration
of fruit juice beverages have been published, indicating
the significance of this relevant problem (Jezek & Suhaj,
2001; Tzouros & Arvanitoyannis, 2001). Adulteration can
be based on a simple dilution with water or substitution
of cheaper artificial ingredients (sugars, acids, and
colorants).
Furthermore, due to increasing information on the
chemical composition of fruits, more refined methods of
adulteration are now used. Most of these methods are
based on supplementation of citrus juice with pulp and peel
extracts or with some cheaper fruit juice (Mears & Shen-
ton, 1973). To assure consumers protection reliable and
sensitive analytical tools are required for detection the
adulteration in juices.
For this purpose, Sass-Kiss and Sass (2000, 2002)
obtained polyclonal antibodies developed against charac-
teristic juice and peel peptides of grapefruit and orange.
These antibodies were used in an immunoassay and
allowed the detection of fruit juice products adulterated.
6. Genetically modified foods
Traceability systems document the history of a product
and may serve the purpose of both marketing and health
protection. In this framework, segregation and identity
preservation systems allow for the separation of genetically
modified and non-modified products from ‘‘farm to fork’’.
Implementation of these systems comes with specific tech-
nical requirements for each particular step of the food pro-
cessing chain (Miraglia et al., 2004).
Additionally, according to Regulation (EC) 1830/2003
of the European Parliament and of the Council, traceabil-
ity requirements for food and feed produced from geneti-
cally modified organisms (GMOs) should be established
to facilitate accurate labeling of such products, in accor-
dance with the requirements of Regulation (EC) 1829/
2003 on genetically modified food and feed. Therefore,
foods and food ingredients that are to be delivered to the
final consumer in which either protein or DNA resulting
from genetic modification is present, are subjected to addi-
tional specific labeling requirements.
On the basis of this information, to regulate the presence
of GMOs in crops, foods, feeds and ingredients, the devel-
opment of reliable and sensitive methods for GMO detec-
tion are necessitated. Current methodologies for the
analysis of GMO are focused on either one of two targets,
the transgenic DNA inserted or the novel protein(s)
expressed in a genetically modified product. For most
DNA-based detection methods, the PCR is employed and
for most protein-based methods, ELISA methods with
antibodies binding the novel protein are used (Lüthy,
1999).
On the whole, ELISA is the method of choice to screen
for a particular GMO in raw material, semiprocessed foods
and processed ingredients, provided the expressed protein
is not degraded and can be detected. However, because
ELISA has lower detection power than PCR methods, it
is less sensitive for testing finished food products with
 L. Asensio et al. / Food Control 19 (2008) 1–8
many ingredients, especially if the threshold for detection is
low. Although protein-based tests are practical and effec-
tive, some GM products do not express a detectable of pro-
tein (Ahmed, 2002; Hemmer, 1997).
ELISA methods have been used for detection and quan-
titation of proteins expressed by most biotechnology-
derived crops in commercial production. They have been
demonstrated useful for analysis of processed fractions of
agricultural commodities such as soybean toasted meal or
dried soybean powder (Lipp, Anklam, & Stave, 2000;
Stave, 2002). However, Terry, Harris, and Parkes (2002),
that worked in the detection of genetically modified crops
in foodstuff, did not detect the protein expressed by the
ELISA test because this protein was highly degraded under
the harsh conditions during food elaboration. Similar
results were obtained by Margarit, Reggiardo, Vallejos,
and Permingeat (2006). These authors allowed the quanti-
fication of the CryIA(b) protein present in the transgenic Bt
maize in low processed foods but no protein was detected
in highly processed food and feedstuffs.
In addition, some companies (Neogen Corporation,
Strategic Diagnostics, Inc., and Eurofins, for example)
have developed rapid immunochemical screening tests for
the detection of genetically modified organisms in foods.
These test kits are best used for raw agricultural or slightly
processed products but have limitations to be used in
highly processed foods (Ahmed, 2002).
7. Irradiated food
Food irradiation is a process where food is exposed to
electron beams, X-rays or gamma rays. Irradiation can
be used to reduce the number of pathogenic micro-
organisms in food ingredients, such as Salmonella, Cam-
pylobacter and E. coli, in order to increase the storage life
of the end product and/or to reduce the health risks for
consumers. It can also delay fruit ripening, help stop vege-
tables such as potatoes and onions from sprouting and be
used as a disinfestations method to protect food from
insects and other organisms (Olson, 1998).
In February 1999, framework Directive 1999/2/CE, con-
cerning irradiated food was issued. It states that the words
‘‘irradiated’’ or ‘‘treated with ionising radiation’’ must
appear on the label or packaging, which must be suitable
in every case, and on the documents which accompany irra-
diated foodstuffs or foodstuffs containing irradiated ingre-
dients. Additionally, implementing Directive 1999/3/CE
contains an initial Community list of foods and food ingre-
dients authorized for irradiation treatment namely dried
aromatic herbs, spices and vegetable seasonings, and sets
a maximum overall average absorbed radiation dose of
10 kGy for this purpose. Therefore, both directives allow
the European Union (EU) harmonization in food
ionization.
On the basis of this information, any irradiated product
purchased by consumers must be properly labeled. So that,
to establish whether irradiated products had been labeled
5
development of reliable and rapid methods for detection
of irradiated food is desirable not only to help administra-
tive control of the facilities licensed for the food irradiation
and compulsory certification of treated foods, but also to
build consumer confidence on irradiated food.
In the last decade, a number of analytical methods have
been investigated for the detection of radiation treatment
of foods: electron spin resonance spectroscopy, gas chro-
matography, thermoluminiscence, DNA comet assay and
enzyme-linked immunosorbent assay (Khan, Khan, &
Delincée, 2005). The DNA comet assay offers great poten-
tial as a rapid tool to detect whether a wide variety of food-
stuffs have been radiation processed. Nevertheless, ELISA
methods have demonstrated to be also rapid and simpler
and can be carried out using low-cost instrumentation.
In the last years, different works have demonstrated the
application of ELISA techniques on irradiated food detec-
tion. Deeble et al. (1994) developed an immunoassay
method to identify this treated food. This study was based
in modified DNA bases in irradiated food. When food is
treated, DNA thymidines are transformed into dihydrot-
hymidines (DiHT). These authors obtained antibodies
against these modified bases and quantified successfully
these changes by ELISA using ranges of 40 Gy. Also,
Tyreman et al. (2004) developed a fast test to identify these
DiHT in irradiated food. These researchers produced
MAbs against DiHT and detected treated food with a
working range of 0.5–2 kGy by a competitive ELISA.
8. Feedstuffs
Bovine Spongiform Encephalopathy (BSE), a fatal neu-
rodegenerative disease commonly referred to as ‘‘Mad Cow
Disease’’, has had a significant impact on the livestock
industry. Regulatory controls to prevent the spread of
BSE have prohibited the use of proteins derived from
mammalian tissues in feed in several countries. Analytical
methods capable of determining the constituents of animal
origin in feedstuffs under different regulations are vital for
successful regulatory controls. PCR-based methods are
specific and sensitive nature, but generally are not able to
distinguish between different tissues of the same species.
Most of the immunoassays for meat speciation are not
applicable to feed detection because of the denaturation
of protein antigen by the high-temperature rendering
process. However, different studies have demonstrated the
potential application of ELISA techniques to solve these
limitations. Ansfield et al. (2000), produced a sensitive
immunoassay capable of detecting ruminant and porcine
heat stable proteins in compound animal feedstuffs. Chen
and Hsieh (2000) worked with troponin I (TnI), a regula-
tory muofibrillar protein, as a suitable thermostable
marker protein for species identification in severely heated
meats. Specific MAbs to porcine muscle were successfully
developed in this work for the detection of pork in
heat-processed meat products. Also, Chen and Hsieh
(2002) developed several MAbs against TnI enable the
6 L. Asensio et al. / Food Control 19 (2008) 1–8
identification of muscle tissues without cross-reaction to
blood, milk and plant proteins. These MAbs, used in an
indirect ELISA, detected successfully pork, beef, sheep,
turkey, chicken, deer and ostrich muscle tissues in com-
pound feeds and have been used by two companies (Neo-
gen Corporation and ELISA Technologies, Inc.) to
design rapid test kits. Furthermore, Kim et al. (2005) devel-
oped an immunoassay system using MAbs against auto-
claved bovine smooth muscles and bovine meat and bone
meal (MBM). These researchers could differentiate bovine
MBM from other species of MBM and ingredients used
for commercial animal feeds, and detect down to 0.05%
MBM mixed in animal feed.
In other recent works, Yesilbag and Kalkan (2005)
worked with central nervous system tissues. These are
sometimes mixed to meat products and could transfer
BSE to human. Therefore, these researchers developed a
semi-quantitative ELISA to detect glial fibrillary acidic
protein as marker. They successfully quantified this risky
material with a detection limit of <0.2%.
Additionally, ScheBo Biotech company developed an
ELISA diagnostic kit that allows the determination of risk
material (brain and spinal cord) in extracts from any meat
products or sausages (raw, cooked or requiring boiling in
water). This test has been evaluated in an international col-
laborative study (Agazzi, Barrero-Moreno, Lücker, von
Holst, & Anklam, 2002). Other companies such as Enfer
Group, Bio-Rad Laboratories, Inc., Prionics, Cedi
Diagnostics and Roche Diagnostics Corporation have
developed immunoassay rapid tests for the monitoring of
BSE in bovine animals that have been approved by the
Commission of the European Communities (Commission
Regulation No. 260/2005 of 16 February 2005). Other
immunoassay kits, developed by Neogen Corporation,
Strategic Diagnostics, Inc., and ELISA Technologies,
Inc., allow the rapid detection of meat and bone meal in
animal feeds. Some of these immunoassays also have been
evaluated to allow regulatory agencies and food and feed
processors to quickly and easily identify the species content
of processed foods and feeds (Myers, Yancy, Farrell,
Washington, & Frobish, 2005; von Holst, Boix, Baeten,
Vancutsem, & Berben, 2006).
9. Allergen ingredients
People suffering from food allergies are dependent on
accurate food labeling, as an avoidance diet is the only
effective countermeasure. Even a small amount of aller-
genic protein can trigger severe reactions in highly
sensitised patients. Therefore, sensitive and reliable tests
such as ELISA are needed to detect potential cross-
contamination.
For example, coeliac patients present a broad range of
sensitivity to gluten intake, with clinical manifestations to
minimal amounts of gliadin. Consequently, ELISA meth-
ods have been used to certify gluten-free products because
of their specificity and sensitivity. Valdes, Garcia, Llorente,
and Méndez (2003), developed a highly sensitive and spe-
cific sandwich ELISA to quantify low levels of wheat
(gliadins), barley (hordeins) and rye (secalins) prolamins,
which have been seen as the celiac-active components of
gluten, in foods for coeliacs. This method has recently been
validated by Codex Alimentarius Commission as a tech-
nique for the quantification of hydrolysed gluten. Other
research groups have developed polyclonal or monoclonal
antibodies against these prolamins and some of the assays
developed have been commercialized (Bermudo-Redondo
et al., 2005; Denery-Papini, Nicolas, & Popineau, 1999;
Van Eckert et al., 2006).
Also, trace levels of peanuts, hazelnuts, almonds, etc,
can elicit an adverse reaction and an unintentional expo-
sure to the allergens may have devastating consequences
to sensitive individuals. Some researchers have developed
successfully ELISA techniques (using polyclonal antibodies
and MAbs) to detect them into ice creams, chocolates or
sauces (Kiening et al., 2005; Yeung & Collins, 1996).
Furthermore, as it is known that egg is one of the five
major allergenic foods that are responsible for more than
3/4 of food allergies in children, researchers have worked
to develop polyclonal antibodies and MAbs against egg
proteins using ELISA techniques. They have detected suc-
cessfully egg contained into ice creams, noodles, pasta and
breads (Williams, Westphal, & Shriver-Lake, 2004; Yeung,
Newsome, & Abbott, 2000).
In the market, a great range of test kits (Neogen,
R-Biopharm and Tepnel kits, for example) have been
developed for allergen ingredients detection using ELISA
test kits or ‘on-site’ rapid tests that employ lateral flow
methods. Some of these kits have been evaluated by differ-
ent inter-laboratory studies to be used for regulatory pur-
poses (Park et al., 2005; Poms et al., 2005; Van Hengel,
Capelletti, Brohee, & Anklam, 2006).
10. Conclusions
Of the wide range of analytical methods available,
ELISA is well suited for the determination of food authen-
ticity because it is sensitive and specific; fast and cheap;
easy to perform and the investment in equipment is much
less than other techniques. Although ELISA limitations
for genetically modified food detection have been shown
in this review, this methodology could allow, together with
other analytical methods such as DNA-based methods,
consumers protection against fraudulent practices in the
food industry.
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