International Journal of Biological Macromolecules 107 (2018) 1732–1737

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Designing a direct ELISA kit for the detection of Staphylococcus aureus

enterotoxin A in raw milk samples
Arezoo Nouri a,1 , Hamed Ahari b,∗,1 , Delavar Shahbazzadeh c
a
Food Science and Technology, Department of Food Science and Technology, Islamic Azad University, Science and Research Branch, Tehran, Iran
b
Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran
c
Biotechnology Research Center, Pasteur Institute of Iran, Pasteur sq., Pasteur st., Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Under favorable growth conditions, Staphylococcus aureus infects and causes illnesses in humans and
Received 13 July 2017 animals. Staphylococcal enterotoxins are considered the most important factor for food poisoning, with
Received in revised form 8 October 2017 these toxins usually found in milk and its byproducts. This study investigated the presence of a classical
Accepted 9 October 2017
enterotoxin of S. aureus (enterotoxin A) in raw cow milk samples obtained from Region 3 of Tehran. Sixty
Available online 10 October 2017
cow milk samples were collected from traditional dairy sales centers in the study site. Electrophoresis,
high-performance liquid chromatography, sandwich ELISA, and direct ELISA were evaluated as methods
Keywords:
for detecting toxins in the milk. A direct ELISA kit was designed, with the best factors, the most suitable
Staphylococcus
Enterotoxin A
densities, and the most optimal temperatures and temporal incubation conditions incorporated into the
ELISA design. Among the evaluated methods, direct ELISA exhibited the highest accuracy in toxin detection.
Raw milk The designed kit achieved a correctness coefficient of 0.98 and detected enterotoxin A in 23% of the
samples. It also exhibited a relative sensitivity and other features that are comparable to those of other
bacterial detection techniques. Finally, the kit achieved good matching and repetition results, and no
cross-reactions occurred between enterotoxins during the procedure.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction
Staphylococcus aureus is regarded as the third most important
driver of the transmission of foodborne diseases globally [1]. This
bacterium can grow and produce different enterotoxins in various
regions and food products under different conditions, making it
the most common cause of food poisoning [2]. Under ideal condi-
tions, S. aureus proliferates in milk and its byproducts, which are
thus significant sources of S. aureus toxins [3–5]. S. aureus growth
in milk and its byproducts may occur at the onset of or during pro-
duction and preservation processes [6]. During pasteurization, S.
aureus cells develop high resistance and maintain biological activ-
ity [7,8]. Staphylococcal poisoning results from the consumption
of food infected with enterotoxins produced by the bacterium [9].
Classical staphylococcal enterotoxins are referred to as enterotox-
∗ Corresponding author at: Department of Food Science and Technology, Islamic
Azad University, Science and Research Branch, Tehran, 14658-36443, Iran.
E-mail address: Dr.h.ahari@gmail.com (H. Ahari).
1
Arezoo Nouri and Hamed Ahari are first authors and contributed equally to this
work.
ins A, B, E, and D, with type A being the most commonly detected
enterotoxin in food contaminated by S. aureus [10].
Sadeghi investigated the spread of enterotoxigenic S. aureus
in 92 and 86 cow and sheep milk samples, respectively. The
author tested 76 samples by converse agglutination, latex agglu-
tination, and multiplex polymerase chain reaction for the presence
of enterotoxins and related genes. The results showed that out of
44 and 32 cow and sheep milk samples, 19 (43%) and 13 (40%)
were positive for enterotoxins, respectively [11]. Kuo and Silverma
compared the effectiveness of ELISA and radioimmunoassay (RIA)
in detecting staphylococcal enterotoxins and examined the sensi-
tivities of the methods by spectrophotometry and specific enzyme
assay. The authors reported that both methods were sensitive at
the nanogram level for every milliliter of food product [12].
Nagaraj et al. [13] detected enterotoxin G with sandwich ELISA
methods [13]. Wu et al. [14] comprehensively reviewed the meth-
ods for detecting staphylococcus toxins and found that techniques
based on ELISA are fast-acting and highly precise approaches [14].
Nia et al. [15] compared different bacterial detection methods to
detect staphylococcus enterotoxin B in milk and buffer solutions.
The authors reported that ELISA determined the concentration of
the enterotoxin in model and food samples more precisely than
other methods [15].

https://doi.org/10.1016/j.ijbiomac.2017.10.052
0141-8130/© 2017 Elsevier B.V. All rights reserved.
 A. Nouri et al. / International Journal of Biological Macromolecules 107 (2018) 1732–1737
Table 1
Solutions and concentrations used in electrophoresis.
Solutions 10 percent 12 percent
1.5 m buffer tris in pH 2.5 mL 2.5 mL
against 8.8
30% of Acril amid 3.3 mL 5 mL
Water 4 mL 2.3 mL
10% SDS 0.1 mL 0.1 mL
10% APS 0.1 mL 0.1 mL
TEMED 0.004 mL 0.004 mL
As can be seen, numerous studies have been conducted around
the world to determine the reasons of enterotoxin proliferation
and detect their presence in food products. The important role of
staphylococcal enterotoxins in public health is equally recognized
in the present research. Accordingly, a direct ELISA kit was designed
and used to detect enterotoxin A in raw cow milk samples obtained
from Region 3 of Tehran and to precisely evaluate milk quality.
2. Materials and methods
2.1. Sample collection
A total of 60 cow milk samples were collected from traditional
dairy sales centers in Tehran and transferred at a suitable time to
the quality control laboratory of the Pasteur Institute of Iran. The
samples were then tested by ELISA for the presence of a classical S.
aureus enterotoxins (enterotoxin A).
2.2. Sample preparation
2.2.1. Standard staphylococcus samples
The S. aureus strain that produces enterotoxin A (ATCC 29213)
was supplied frozen by the Pasteur Institute of Iran and was
thawed and homogenized completely using a centrifuge. Subse-
quently, 10 mL of the enterotoxin was cultured in Mueller-Hinton
broth at 37 ◦ C for 24 h. After the cultivation period, disturbance
was observed in the culture, indicating bacterial activation. The
culture was then transferred to four microtubes and centrifuged
at 7000 rpm. To detect toxins, liquid bacteria containing the
enterotoxin were injected into a high-performance liquid chro-
matography (HPLC) system. The HPLC preparations were as follows.
Every sample (1 g) with 1 mL of 1% phosphate potassium solution
was homogenized for 2 min. The homogenized solution, together
with 10 mL of acetonitrile, was centrifuged for 15 min at 4000 rpm.
The surface of the solution was mixed with 10 mL hexane, after
which the solution was dissolved in 0.5 mL water for 5 min and
then placed in an ultrasonic bath for 10 min. The solution was then
passed through a 0.2 plastic filter, and 50 ␮L of the solution was
injected into the HPLC system.
2.2.2. Food samples
The samples were centrifuged for 5 min at 3500 rpm and kept
at a maximum temperature of 10 ◦ C. Subsequently, the fatty layers
of the sample surfaces were removed. The aquatic phase at a pro-
portion of 1–20 was diluted with sterile distilled water and then
filtered using 22% micron syringe filters.
2.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)
Gel materials and buffers were prepared in accordance with the
procedure presented in Table 1. The solutions for examination were
mixed in a falcon tube and immediately poured into the spaces
between glass plates. To remove the bubbles that formed in the
solutions, 1 mL of 96% ethanol was poured onto the liquid surface
1733
with a sampler. The ethanol penetrated into the spaces between
the glass plates.
15 percent Given that pouring liquids in order is important, the final sub-
2.5 mL stance added into the mixture was TEMED. After 10–20 min, the
substance poured between the glass plates was stiffened and poly-
5 mL merized. If the speed at which the mixture is poured between the
2.3 mL glass plates increases, the amount of TEMED and the speed of poly-
0.1 mL
merization reaction will correspondingly increase. This will also
0.1 mL
0.004 mL lead to the formation of stacked gels. All the derived solutions were
mixed in a falcon tube and immediately poured into the spaces
between the glass plates. The combs were then carefully placed
between the glass plates. After 10–20 min, the mixture was poured
between the glass plates and was stiffened and polymerized.
A glass cassette was placed into a tank, and a running buffer was
added up to a level higher than that reached by the gel. To every
microtube, 25 ␮L of a sample and 5 ␮L of loading buffer solution
were added. The lids of the microtubes were closed using a parafilm,
and then the microtubes were placed in a bain-marie to boil the
samples for 10 min.
A total of 20 ␮L of every sample was extracted using a Hamilton
syringe, and 3 ␮L of the marker protein was poured into the wells.
Electronic flow was run. Voltage was maintained at 100 V until color
disappeared from the stacking gel domain, after which the voltage
was increased to 200 V.
The gel was slowly extracted from the glass sheets and placed
in a color solution. If color is newly supplied, the process will last
for about 20 min. After the gel was painted, it was placed in a
de-staining solution to remove the color. Note that the gel used
in this work can separate proteins that contain acrylamide and
bis-acrylamide. These agents are harmful and should therefore be
handled safely.
2.4. Bovine serum albumin
To prepare bovine serum albumin, 0.4, crystallized bovine serum
albumin was used, with the number of samples counted by the
researchers. The amount of bovine serum albumin desired was cal-
culated on the basis of a simple proportion.
®
2.5. Phosphate buffered saline with tween 20 (PBST)
About 8 g of NaCl, 0.2 g of KCL, 0.24 g of KH2 PO4 , and 1.44 g of
Na2 HPO4 with a volume of 1000 mL were used to prepare PBST.
When the pH reached the desired value (i.e., 7.4), the solution was
autoclaved at 121.2 ◦ C.
2.6. Sandwich ELISA
For testing with sandwich ELISA, 96-well plates (NUNC) were
used. First, a microplate was covered with the initial antibody (10 g
in every sink), the covered buffer (with different densities, with
an initial density of 1:2000) was dissolved, and a microplate col-
umn was poured into every sink. Then, the sample in the microplate
was preserved for 1 h at 37 ◦ C. Every sink was washed with PBST and
dried several times, each involving the addition of 100 ␮L of PBST to
each well. The sinks were dried by slight shaking and wiping with a
cloth. Second, empty spaces on the floors of the sinks were blocked
to prevent infusion with the antibody. Blocking was performed by
milk powder after 0.05 burred, then 200 ␮L of the powder was
poured into the sinks, which were preserved for 30 min at 37 ◦ C.
The plate was then cleaned and washed three times with PBST, and
the antigen (milk with toxin, each time with different densities)
was added into a 100 ␮L sink and preserved in an incubator for 1 h.
Afterwards, the plate was re-washed three times, and 100 ␮L of
another antibody was added (1:4000). A 1 h incubation was carried
out at 37 ◦ C, and the plate was once again washed three times by
1734 A. Nouri et al. / International Journal of Biological Macromolecules 107 (2018) 1732–1737

Table 2
Selection of buffer amount (microliter) in the repetitions intended for the design of sandwich ELISA.

sample Repetition 1 absorption Repetition 2 absorption Repetition3 absorption

1 Lacking floor antibody 1–384 1.902 1–6400 2.182 1–1568 1.933

2 Lacking floor antibody 1–768 1.329 1–12,800 1.849 1–3 2.21
3 Lacking antigen 2.491 1–24,600 2.630 1–6 1.803
4 Lacking antigen 2.55 1–49,600 3.017 1–12 2.969
5 Lacking antigen 1–400 2.583 1–98 2.935 1–24 2.896
6 1–800 2.981 1–196 2.891 1–48 3.083
7 1–1600 2.876 1–393 2.697 1–96 2.706
8 1–3200 3.178 1–784 3.015 1–192 2.829

sample Repetition 1 absorption Repetition 2 absorption Repetition3 absorption

1 Lacking floor antibody 1–5 2.468 2–4 3.084 2–6 2.548

2 Lacking floor antibody 3–5 1.905 3–4 3.935 2–6 2.355
3 Lacking antigen OVER 2–4 2.689 2–6 2.363
4 Lacking antigen 2.818 2–4 2.797 2–6 2.398
5 Lacking antigen –4 3.088 2–5 2.576 3–4 2.091
6 1–5 2.736 2–5 2.490 3–4 3.749
7 1–5 2.808 2–5 2.581 5–3 2.124
8 1–6 2.629 2–5 2.756 6–3 2.462

using PBST. Two normal sulfuric acid was poured into every sink. performed to compare mean values. MS Excel (2013) was used to
Finally, optical density (OD) was determined with an ELISA reader construct graphs. Error type I was considered equal to a significance
and Gen5 software at a wavelength of 450 nm. Selection of buffer level of 0.05.
amount for ELSIA Sandwich is presented in Table 2.

3. Results and discussion

2.7. Direct ELISA
The 100 ␮L raw milk samples were added to an ELISA plate sink,
to which different proportions of the enterotoxin were injected.
The microplates were placed in an incubator at 37 ◦ C for 1 day. The
plates containing the samples were then washed with PBST three
times. Each time the samples were removed from the microplates,
tissue was dabbed onto the plates for the complete removal of the
washing liquid. No reactions between the substances occurred in
every washing.
Blanking was conducted by injecting 200 ␮L of 4% bovine serum
albumin into the ELISA plate sinks, covering the surfaces of the
plates with foil, and placing the plates in an incubator at 37 ◦ C for
50 min. While the plates were being incubated, the antibody was
prepared by mixing 1 ␮L of the antibody with 1000 ␮L of PBST in a
microtube. After the pre-set period passed, the plates were taken
out of the incubator and washed three times with PBST. To every
sink, about 100 ␮L of the antibody was added, and the surface of
the plates were covered with aluminum foil. The plates were again
placed in the incubator for 1 h at 37 ◦ C. The contents of the plates
were removed, and the plates were washed with PBST. The antibody
at a density of 1:4000 was poured into the tubes and homogenized
completely. From this solution, 100 ␮L of the prepared antibody
was added to every sink, whose surface was covered with alu-
minum foil. Each sink was then kept at 37 ◦ C. After 1 h, the contents
of the plate were removed and 100 ␮L of TMB was added into the
plate. The surface of the microplate was covered with aluminum
foil, and the microplate was placed in an incubator at 37 ◦ C for
10 min. To stop the testing, 100 ␮L of two normal sulfuric acid was
added. Thus, the content of the plates turned yellow, indicating that
testing can be concluded.
Finally, the reactions of the samples under direct ELISA at
a wavelength of 450 nm were determined, and the information
related to the reactions of the pores (OD) was recorded separately
using Gen5.
2.8. Statistical analysis
Data were analyzed by random factorial analysis with three rep-
etitions in SPSS (version 22). Duncan multi domain testing was
3.1. Investigating different methods of enterotoxin detection
As previously indicated, different methods were evaluated to
design a new technique for detecting S. aureus enterotoxin A in
raw milk samples. The first method evaluated was electrophoresis,
which is a technique in which samples with electrical loads move on
the basis of an electrical square from an abnormal interconnection.
Under this condition, the movement speed of molecules is affected
not only by their electrical loads but also by other factors, such as
molecular size and shape. Electrophoresis is typically conducted to
separate large molecules, such as proteins and nucleic acid, but in
some cases, it is employed to separate bearing molecules, such as
sweets, amino acid peptides, and simple ions. Because no consti-
tutive bonds were observed, electrophoresis was an inapplicable
basis for the design of the ELISA kit. Because the molecular weight
of S. aureus enterotoxins is about 23–29, such weight and the shape
of enterotoxins prevent passing through gel holes. The passage of
molecules is necessitated by constitutive bonds. Therefore, bond
shape presents problems, thus requiring the evaluation of another
method. The detection results in the current research (i.e., pres-
ence in 23% of the samples) accords with those of other researchers
e.g., Hall et al. [16]. In microbiologically examining samples from
six hygienic and treatment centers, found a contamination rate of
55.6% [17]. The second method evaluated was sandwich ELISA, in
which testing is based on reactions between antibodies and anti-
gens. A specific antibody reacts with a pre-determined antigen. The
antibody is linked to an enzyme as an indicator that testing can
proceed. Sandwich ELISA testing concludes with the addition of an
enzyme substrate and a charging substrate to a colorful product. In
the sandwich ELISA conducted in this work, some buffers were used
in the sinks. Given that no suitable reactions were observed with
this method, this technique was disregarded as a basis for designing
the ELISA kit.
The inefficiency of sandwich ELISA may be attributed to the fact
that the surface and floor antibodies were of the same type and
that the molecular weight of the enterotoxin was low (about 23–29
kDA). The floor antibody or the sample used should have a high
molecular weight to enable the connection of desired antibodies
from different angles. This enables reactions to be detected by an
 A. Nouri et al. / International Journal of Biological Macromolecules 107 (2018) 1732–1737 1735

Table 3
Determination of the best antibody and anti-antibody densities.

The amount of absorption The amount of antibody and antigen The amount of absorption The amount of antibody and antigen

0.783 1.3000v 2.102 1.1000

0.606 1.4000 2.215 1.1000
0.543 1.4000 1.189 1.2000
0.210 1.1000 no antigen 1.043 1.2000
0.184 1.4000 no antigen 0.752 1.3000

ELISA reader. Additionally, the antigen used should have at least

two different domains to enable linking to two antibodies. This is
probably another reason why the sandwich ELISA could not detect
the enterotoxin of interest.
The third method that was assessed was HPLC, which is a
technique for detecting components at the nanometer scale and
precisely determining molecular size. This method had been used
for detection purposes even before the invention of nanotechnol-
ogy. In this technique, mixed components are separated at different
speeds of various molecular movements in the same environ-
ments and similar initial energy levels. Despite the advantages of
this method, however, it could not exhibit a suitable detection of
responses in relation to enterotoxin A. The HPLC results indicated
that all the S. aureus enterotoxin suspensions were equal in proper-
ties with the uncontaminated sample, thus reflecting the absence Fig. 1. Standard graph for ELISA kit.
of toxins in the suspensions. To ensure that the composition of
the suspensions was correct, the procedure was repeated more
than 10 times. After suspension injection, the same results were
obtaining suitable reagents, the density of desired reagents was
obtained at each run. Because the sensitivity of this method is very
determined, after which the 60 cow milk samples were tested with
high and the sample injected into the machine was highly precise,
the developed method. The results showed that 23% of the samples
detection for this sample may not be possible with conventional
carried S. aureus enterotoxin A.
methods. Furthermore, given that S. aureus is very sensitive and
Still for the purpose of the design, the kit was run several times
may be removed under inappropriate conditions, HPLC failed to
to determine the repetitions that generated the best results. A graph
recognize the enterotoxin in the sample. However, Williams et al.
was then drawn, and a line equation was formulated. The compo-
conducted a study on detection of S. aureus SEB by HPLC and found
nents that effectively contributed to the design of the kit were the
SEB different samples [18].
selection of suitable factors, heating durations, and heating tem-
Different methods are available for detecting S. aureus. Some
peratures. For precise bacterial detection, the designed ELISA kit
examples are latex agglutination, ELISA, immunodiffusion, and RIA,
testing was conducted three times. The repetitions registered an
which all require conditions that are conducive to the expression
error percentage of ±1.3, indicating the repeatability of the devel-
of enterotoxin genes. Given this requirement, a possibility is that
oped testing method.
in unusual conditions, these methods may fail to detect the pres-
The results of the present study are in line with those of other
ence of staphylococcal enterotoxins and register a false negative.
researchers, such as [16].
Thus, researchers replaced molecular detection methods with bio-
Tavakoli and Riazipour [17] microbiologically investigated food
chemical and serological methods. With molecular methods, which
samples from six hygienic centers in Tehran and found an S. aureus
detect poison-coding genes, it is possible to detect S. aureus strains
contamination rate of 55.1%.
that contain known coded genes of staphylococcal enterotoxins.
They can also detect low concentrations that cannot be determined
by immunological methods. To design a new method for detecting 3.2. Investigating different parameters
S. aureus enterotoxins, sandwich ELISA was used as basis, and the
table presenting different antibody concentrations was referred to 3.2.1. Antibody and anti-antibody densities
in determining the optimal antibody concentration. As indicated in As shown in Table 3, different antibody and anti-antibody den-
Table 1, different antibody and antigen concentrations were used sities were used to determine the optimal densities for the design
to ascertain the best antibody density. of the direct ELISA kit. The table also indicates that the best den-
The results showed that the best densities achieved with sand- sity was the one that exhibited the highest level of absorption; that
wich ELISA and other ELISA-based methods of ELISA are those that is, the last two samples to which no antigen was added had the
reflected the highest attraction or absorption in sample 4 at repeti- highest difference. The first two samples, in which antibody con-
tion 2; sample 6 at repetition 3, sample 8 at repetition 2, sample 2 at centration was 1.1000 and absorption rates were 2.102 and 2.215,
repetition 5, and sample 6 at repetition 6 had the highest absorp- exhibited the highest numerical difference from the samples that
tion levels among the samples. These samples also exhibited the had absorption rates of 0.210 and 0.184. The density of these sam-
highest difference compared with the samples to which no antigen ples was selected as the optimum density. S. aureus enterotoxin
was added. After testing and error identification for the different A was injected into all the samples used as bases for designing
detection methods, direct ELISA was used as the final basis for the the standard kit, and a buffer was used to detect the enterotoxin.
design of the new direct ELISA kit. The best absorption rate was Based on the results regarding absorption and toxin amounts, the
achieved at a wavelength of 450 nm, and a standard equation was procedure was carried out three times. The concentrations of the
formulated. With this equation and the replacement of absorption detected enterotoxin exhibited meaningful statistical differences
amounts of the raw milk samples, the concentration of entero- (P < 0.05). The standard graph depicting the designed ELISA kit is
toxin A in the milk was determined. In designing the ELISA kit and shown in Fig. 1. On the basis of the graph, the obtained curved
1736 A. Nouri et al. / International Journal of Biological Macromolecules 107 (2018) 1732–1737
Table 4
Toxin adsorption in Elisa kit.
No. Adsorption in 450 nm Toxin amount (ng)
1 1.354 ± 0.58 500
2 1.354 ± 0.71 250
3 0.986 ± 0.12 125
4 0.783 ± 0.95 62.5
5 0.583 ± 0.37 31.2
equation with an R2 equal to 0.9864 was expressed in the following
form:
Y = 0.2708|n(x) − 0.3074,
where Y denotes the amount of toxin absorption at a wavelength
of 450 nm in the standard sample, and x represents the amount of
added toxin for the design of the standard kit. To detect enterotoxin
A in the raw milk samples, absorption amount was incorporated
into the above-mentioned equation, and the ration of absorption
was determined. Because the graph of the standard kit is of curved
form, the best equation is a regression equation.
3.2.2. Determination of most suitable reagents
The most suitable covering buffer for the antigen, PBST with a
pH equal to 7.4, and the most suitable blocking buffer, TMB, along
with 0.4 cow albumin, were selected. The best dilution medium for
the samples was the distilled water that showed less scheme. The
most suitable antigen connected to the solid phase for the detection
of enterotoxin A concentration had a weight of 1.5 ␮g in milliliter
and a volume of 50 ␮L in every sink.
The best bovine serum albumin concentration was also selected
in determining the presence of 2 ␮L of the enterotoxin. The best
antibody with a conjugation of 1:4000 and the least scheme was
selected.
3.2.3. Determination of optimal incubation conditions
To ascertain the best coverage time and temperature, one com-
plete day (20 h) and 37 ◦ C were considered. The highest sensitivity
and difference in diffusion absorption between positive and neg-
ative samples was observed at 1 h and 37 ◦ C for the samples
containing the toxin and the conjugated antibody (Table 4).
3.2.4. Sensitivity of the designed kit
According to table when there were no toxin in the samples and
when the amount of toxin in the samples were 7.8 ng, adsorption
does not differ. However when toxin amount is 15.6 ng, adsorp-
tion changes. Thus, sensitivity of the designed kit is approximately
15.6 ng toxin.
3.3. Determination of raw cow milk samples with enterotoxin A
As indicated by the company that designs ELISA kits, at a wave-
length of 450 nm, positive samples have a high absorption rate (0.3).
The results in Table 1 show that out of the 60 raw milk samples, 15
carried the enterotoxin, whereas 45 did not. In this work, the ELISA
kit was designed by a direct method. As previously stated, suit-
able reagents were obtained, their densities were measured, and
the designed kit was used to detect enterotoxin A in the 60 raw
cow milk samples. The kit detected the enterotoxin in 23% of the
samples.
The design also involved the repeated running of the designed
kit to select the repetition that generated the best results. On this
basis, a graph was drawn, and a line equation was formulated. The
selection of suitable cases, heating durations, and heating temper-
atures contributed to the design of the kit.
Filleron et al. [19] conducted a study on different S. aureus equal-
ities in the swollen breasts of cows from two husbandry farms. On
31 S. aureus equalities, the authors used a molecular template to
determine polymorphism in the protein coding gene method. In
investigating 250 raw milk samples Bianchi et al. [20] found that
46 equalities of positive coagulase staphylococcus aureus with a
mean value of 3.5 × 10 cfu/mL. Among these 46 separate equalities,
28 had at least one gene-coding enterotoxin A or C. Out of the 28, 21
equalities (75%) were classified as SEC genes and 3 (25%) were cat-
egorized as SEA genes. Furthermore, 3 qualities (9.09%) had both
SEA and SEC genes. Considering the presence of enterotoxigenic
staphylococcus in raw milk as a potential danger to public health,
the recognition and monitoring of milk contamination resources
should be incorporated as part of quality control standards for milk
and other dairy products [20].
4. Conclusion
Several methods were evaluated for the design of a standard
ELISA kit that can be used to detect S. aureus enterotoxin A; these
methods were electrophoresis, HPLC, y sandwich ELISA, and direct
ELISA. The findings confirmed that direct ELISA generated the best
detection outcomes. The new direct ELISA kit designed in this work
was compared with the other detection methods. The designed kit
had a relative sensitivity that was comparable to those of the other
methods and high repeatability. Sensitivity was approximately
15.6 ng toxin and detection time of 15 min. No cross-reactions
occurred during the detection, and the enterotoxin was detected
in 23% of the samples.
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