Archives of Razi Institute, Vol. 75, No.

4 (2020) 419-426 Copyright © 2020 by

Razi Vaccine & Serum Research Institute
DOI: 10.22092/ari.2018.123028.1236

Original Article
Development of Nano-ELISA Method for Serological
Diagnosis of Toxoplasmosis in Mice

Khodadadi, A. 1, Madani, R. 2, 3 *, Hoghooghi Rad, N. 1, Atyabi, N. 4

1. Department of Pathobiology, Faculty of Veterinary Medicine, Science and Research Branch, Islamic Azad University,
Tehran, Iran
2. Department of Proteomics, Biochemistry and Biotechnology, Razi Vaccine and Serum Research Institute, Agricultural
Research, Education and Extension Organization (AREEO) Karaj, Iran
3. Department of Microbiology, School of Specialized Science, Science and Research Branch, Islamic Azad University,
Tehran, Iran
4. Department of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

Received 22 August 2018; Accepted 18 November 2018

Corresponding Author: [email protected]

Abstract
Toxoplasmosis is a widespread parasitic disease caused by a protozoan parasite Toxoplasma gondii. Currently,
nanotechnology has been used for the diagnosis of many infectious diseases. It could be due to the fact that
nanoparticles play an important role in accurate and fast diagnosis. The purpose of this study was to design a
Nano-enzyme linked immunosorbent assay (Nano-ELISA) kit using excreted/secreted (E/S) antigens to have
higher sensitivity and specificity than those reported for the designed enzyme-linked immunosorbent assay
(ELISA) kit for the diagnosis of Toxoplasmosis in mice. Firstly, the serum samples were collected from 15
infected mice with T. gondii and 15 healthy ones. Then, E/S antigens were separated from parasite tachyzoites
and used for designing an ELISA kit. In addition, the mice sera were evaluated using the designed ELISA kit.
Finally, the serum samples were assessed by Nano-ELISA kits designed with E/S antigen and conjugate of gold
nanoparticles. The obtained results of the present study showed that the sensitivity and specificity of the
designed ELISA kit were reported as 80% and 86.66%, respectively, that both improved to 93.33% in these sera
with the designed Nano-ELISA kit. This finding revealed the significant improvement of sensitivity and
specificity using gold nanoparticles in designing the ELISA kit. Furthermore, according to the literature, the use
of E/S antigens in designing recognizable ELISA kits has been always highlighted considering the presence of
numerous antigens in T. gondii. The results of this study revealed that the use of E/S antigens in the preparation
of an ELISA kit was very effective. This is very important, especially in the lower titers of antibody requiring a
more accurate diagnosis. On the other hand, the Nano-ELISA method designed with E/S antigens can be more
sensitive and specific than ELISA for the diagnosis of Toxoplasmosis and can be the basis for further studies in
this regard.
Keywords: Nano-ELISA, Toxoplasmosis, Mouse

Développement d’un Nano-ELISA Pour le Diagnostic Sérologique de la Toxoplasmose Chez les Souris
Résumé: La toxoplasmose est une maladie parasitaire répandue causée par le parasite protozoaire Toxoplasma
gondii. Actuellement, la nanotechnologie est utilisée pour le diagnostic de nombreuses maladies infectieuses.
Cela peut être dû au fait que l’utilisation des nanoparticules peut aboutir à un diagnostic plus précis et rapide. Le
but de cette étude était de concevoir un kit de test d'immunoadsorbant lié aux nano-enzymes (Nano-ELISA)
utilisant des antigènes excrétés/sécrétés (E/S) pour obtenir une sensibilité et une spécificité plus élevées que
celles rapportées pour le test d'immunoadsorbant lié à des enzymes (ELISA) conçu pour le diagnostic de la
toxoplasmose chez la souris. Tout d’abord, les échantillons de sérum ont été collectés sur 15 souris infectées par
420 Khodadadi et al / Archives of Razi Institute, Vol. 75, No. 4 (2020) 419-426

T. gondii et 15 souris saines. Ensuite, les antigènes E/S ont été séparés des tachyzoïtes parasites et utilisés pour
concevoir un kit ELISA.Ensuite, les sérums de souris ont été évalués à l'aide du kit ELISA conçu. Enfin, les
échantillons de sérum ont été évalués par des kits Nano-ELISA conçus avec un antigène E/S et un conjugué de
nanoparticules d'or. Les résultats de cette étude ont montré que la sensibilité et la spécificité du kit ELISA conçu
étaient respectivement de 80% et 86,66%. L’utilisation du kit Nano-ELISA sur les mêmes sérums permettait
d’améliorer ces taux jusqu’à 93,33%. Ces résultats montrent que l’utilisation des nanoparticules d'or dans la
conception du kit ELISA améliore de façon significative la sensibilité et la spécificité du diagnostic e. De plus,
conformément à la littérature, l’intérêt de l'utilisation d'antigènes E/S dans la conception de kits ELISA est à
souligner, compte tenu de la présence de nombreux antigènes dans T. gondii. Les résultats de cette étude ont
également révélé que l'utilisation d'antigènes E/S dans la préparation d'un kit ELISA était très efficace. Ce point
estd’une importance majeure, en particulier dans les titres inférieurs d'anticorps nécessitant un diagnostic plus
précis.De plus, la méthode Nano-ELISA conçue avec des antigènes E/S peut être plus sensible et spécifique que
l'ELISA pour le diagnostic de la toxoplasmose et peut être le sujet d'études complémentaires.
Mots-clés: Nano-ELISA, Toxoplasmose, Souris

Introduction damages. The treatment of patients in the United States

Toxoplasmosis is a zoonotic parasitic disease in costs about 0.4-8.8 dollars, and this cost in England is
animals caused by a parasitic protozoan called estimated at 1.2 up to 12 million dollars (Markell et al.,
Toxoplasma gondii (Tavassoli et al., 2013). This 2006).
parasite has a high potential for the contamination of Rodents, ruminants, such as sheep and cattle, birds,
homoeothermic beings and can be hidden after and pigs are some of the hosts of this parasite. In
infecting many species. T. gondii is a parasite from the addition, cats are the final host of this parasite
Apicomplexa group that is responsible for the (Mosallanejad et al., 2012). The expansion of these
contamination of a wide range of vertebrates. Humans hosts and close relationship between these animals and
are affected by this pathogen in two ways, namely humans are the major causes of the prevalence of
acquired and congenital (Dubey, 2008). Toxoplasmosis in humans. Therefore, 500 to 1 million
As acquired immune deficiency syndrome (AIDS) individuals, who are about one-third of the world’s total
prevalence widespread, T. gondii as an opportunistic population, suffer from this disease (Shirbazou et al.,
pathogen has been identified as the leading agent of 2013).
mortality among AIDS patients. T. gondii-induced The provision of suitable sanitary conditions in the
encephalitis is the central complication of society, especially in high-risk communities, and
Toxoplasmosis in AIDS patients. About 20% up to prevention of mental disorders caused by feticide,
47% of individuals with antibodies against T. gondii stillbirth, preterm birth, and congenital difficulties,
suffer from Toxoplasmic encephalitis (Cox and show the importance of constant monitoring of this
Wakelin, 2010). common disease in urban and rural communities.
Toxoplasmosis leads to huge damages to both The diagnosis of Toxoplasmosis can be performed
humans and animals. Congenital Toxoplasmosis which using molecular methods, Immunoblotting techniques,
is the result of feticide, the birth of infants with tissue biopsy, and serology. These methods include the
complications, such as hydrocephalus, microcephaly, Sabin-Feldman dye test, indirect hemagglutination test,
low intelligence, severe abnormalities in organs, indirect fluorescent antibody test, and enzyme-linked
blindness, physical difficulties, and mental disorders, immunosorbent assay (ELISA) (Meganathan et al.,
such as Schizophrenia, annually leads to economic 2010). Another test named the modified agglutination
 Khodadadi et al / Archives of Razi Institute, Vol. 75, No. 4 (2020) 419-426 421

test with high sensitivity and specificity appears on that gold nanoparticles can easily conjugate with DNA,
tachyzoite (Gamble et al., 2005). antibodies, enzymes, and other biomolecules and
Polymerase chain reaction (PCR) is also one of the increase the number of biochemical detection signals
best methods for testing Toxoplasmosis, with high (Jia et al., 2009).
sensitivity and specificity (Koloren, 2013). Among the Since the serum levels of marker proteins in the early
above-mentioned methods, ELISA is reported with stages of most diseases are very low, and different
high sensitivity and specificity. In this case, such a antibody titers are observed at different stages of the
method with commercial kits can be widely applied in disease, it is not inside the diagnostic range of ELISA;
laboratories all around the world for the measurement therefore, many of the ELISA usual methods were not
of immunoglobulin M, immunoglobulin G, and reported with favorable results at different levels of the
immunoglobulin E antibodies (Montoya, 2002). disease (Jia et al., 2009). To date, no studies have been
Unacceptable positive and negative results can be the carried out to improve the diagnosis of Toxoplasmosis
most crucial issues in case of these kits. Multiple with designing Nano-enzyme linked immunosorbent
studies have been carried out for the identification of T. assay (Nano-ELISA). With this background in mind,
gondii compounds as antigens to be used in serologic the present study, in case of success, will be the first
diagnostic methods for the improvement of their study in the world leading to the development of a
diagnostic significance (Gamble et al., 2005; Glor et technique to increase and improve the sensitivity and
al., 2013). In this regard, high attention has been specificity of the methods for the diagnosis of
focused on the excreted/secreted (E/S) antigens of T. Toxoplasmosis.
gondii tachyzoite (Suzuki, 2002). Regarding the
reported characteristics of these antigens, it seems that Material and Methods
they are suitable for the detection of antibodies acting In this study, a total of 30 serum samples were
against the parasite in the serum (Nishikawa et al., gathered from mice. The mice were divided into two
2002). Several studies have shown that the ELISA groups. The positive group included 15 experimentally
method using the E/S antigens of T. gondii has high infected mice within the weight range of 22-25 g with
sensitivity and specificity for the diagnosis of approximately 7 weeks of age. The negative group
Toxoplasmosis in rats (Nguyen et al., 1996). consisted of 15 laboratory mice similar to those of the
Currently, nanotechnology has been used to diagnose positive group and free Toxoplasma parasite that
many infectious diseases (Hauck et al., 2010). On the provided from Pastor Institute that they were negative
other hand, gold nanoparticles are among the particles after serological tests and isolated for our research. It
that are useful for the diagnosis of some diseases. The should be mentioned that the commercial ELISA kit for
use of gold nanoparticles for protein and the diagnosis of Toxoplasmosis in mice with suitable
deoxyribonucleic acid (DNA) analysis is effective due conjugate was not available; accordingly, it was firstly
to the high absorption and optic refraction of this necessary to design an ELISA kit for the diagnosis of T.
particle at certain wavelengths, with fluorescence gondii with anti-rat conjugate and E/S antigens.
properties specific to optical detection techniques (e.g., Determination of excreted/secreted antigens for
spectroscopy) (Ambrosi et al., 2010). Furthermore, the ELISA. Studies have shown that the E/S antigens of T.
important characteristics of gold nanoparticles, with a gondii are well characterized to be used in ELISA kits
high surface to volume ratio and unique properties, lead (Nguyen et al., 1996; Nishikawa et al., 2002). For the
to the use of this nanoparticle as the basis of a isolation of these antigens, a procedure was performed
biomarker (Jia et al., 2009). Finally, it should be noted (Dubey, 2008). Firstly, six white laboratory mice free
422 Khodadadi et al / Archives of Razi Institute, Vol. 75, No. 4 (2020) 419-426

of T. gondii with an approximate age of 6-8 weeks and The protein content was reported as 30 mg/ml. After
weight range of about 22-25 g were selected. Then, 0.2 the determination of protein content, because the E/S
ml of a dilution, including 1 × 107 ml of tachyzoite T. antigens of T. gondii are proteins with a molecular
gondii, was intraperitoneally injected from the RH weight range of 6.5-200 kDa, sodium dodecyl sulfate-
strain. After 3 days, the animals were inhaled with polyacrylamide gel electrophoresis was used for the
carbon dioxide (with the consideration of all the ethical determination of the existing proteins. The samples
standards and animal protection rules) without bleeding containing the E/S antigens of T. gondii were evaluated
for the prevention of peritoneal fluid contamination by electrophoresis.
with blood. Design of ELISA method with E/S antigens. In
Phosphate-buffered saline (PBS) was injected into the order to design this procedure, the best serum dilution
peritoneum, and then the exudate was collected. and concentration of E/S antigen for coating should be
Subsequently, the obtained liquid was centrifuged for 5 determined in the first step. After diluting and checking
min at 500 g. The supernatant was suspended in a the cheek board for ELISA, the optimal concentration
Hanks’ Balanced Salt Solution. At this stage, the for ELISA is 2 mg/ml, and the proper dilution for
concentration of tachyzoite and contamination caused serum is 1/50. Then, the ELISA designing steps were
by the host cells were evaluated using the Neubauer performed with E/S antigen as follows:
counting (tachyzoite count with a dilution of 1/1000 Connecting the antigen to coagulation, blocking
and an infection with host cells with a dilution ratio of empty sites, conjugation of Sigma conjugated Anti Rat,
1.10). Afterward, washing operation was performed, adding a substrate (i.e., chromogenic substrate),
and the last step was repeated after each washing. stopping solution (stop solution), and reading samples
Finally, less than 0.5% of the infection with the with ELISA reader at 450 nm
mononuclear host and less than 0.25% of the infection All of the sera samples of 30 mice were evaluated for
with blood cells were detected. Up to reaching a final seroepidemiology the results of which are presented in
concentration of 1 × 107 ml, tachyzoite will be kept in the results section.
PBS. If there is no evidence of infectious agents, Design of Nano-ELISA with E/S Antigen. The
tachyzoite can be kept with no antibiotics (i.e., procedure was similar to designing an ELISA kit. Only
penicillin or streptomycin). the conjugation phase was prepared from a gold
Production of dilution antigen for ELISA. Firstly, nanoparticle colloid manufactured by PlasmaChem
5 ml of tachyzoite was centrifuged for 15 min by 2000 GmbH in Germany with a 1/500 dilution. Finally, the
g, and the supernatant was mixed with nine times samples were read with an ELISA reader at 450 nm.
distilled water. Three times freezing and watering The results of testing positive and negative samples are
process was performed to break the tachyzoite. Antigen presented in the results section in detail.
preparations were continued for 20 sec using a
sonication device at 4°C. Centrifuging for 30 min at the Results
same temperature by 10000 g leads to the separation of Positive and negative mice sera were evaluated using an
waste components and cellulose residues. The ELISA kit. The cut-off value was considered 1 indicating
supernatant was stored at a negative temperature of that the values higher and lower than 1 were regarded as
20°C until use. The prepared antigens were measured positive and negative, respectively. The results of the mice
two times for the determination of protein content using positive and negative serum test with an ELISA kit are
the Lowry protein assay and Bradford protein assay. presented in tables 1 and 2, respectively.
 Khodadadi et al / Archives of Razi Institute, Vol. 75, No. 4 (2020) 419-426 423

Table 1. Results of 15 positive mice sera with designed enzyme-linked immunosorbent assay kit
Test OD Test OD Test OD

1 1.364 6 1.250 11 2.185

2 1.902 7 1.508 12 1.054

3 1.190 8 0.579 α 13 1.722

4 2.135 9 1.183 14 1.483

5 0.882 α 10 1.645 15 0.874 α

(α): Samples no. 5, 8, and 15 have an optical density lower than the cut-off point and are negatively evaluated.

Table 2. Results of 15 negative mice sera with designed enzyme-linked immunosorbent assay kit
Test OD Test OD Test OD

1 0.480 6 0.459 11 0.245

2 0.539 7 1.132 α 12 0.331

3 0.281 8 0.770 13 0.381

4 0.775 9 1.180 α 14 0.409

5 0.138 10 0.157 15 0.525

(α): Samples no. 7 and 9 have higher optical density than the cut-off point and are positively evaluated.

The ability to correctly diagnose all patients is called Sensitivity: 100 ×

sensitivity. The ability to test for the correct diagnosis
of all those who are not sick is called specificity. Using Specificity: 100 ×
the obtained results containing sensitivity and
specificity formulas can lead to reaching the sensitivity
In this study, 15 sera from the infected laboratory
and specificity of the diagnostic kit calculated as
mice and 15 sera from the healthy laboratory mice were
follows:
tested using ELISA as previously described in Table 3.
True positive: those who are sick and tested positive
(TP). Table 3. Sensitivity and specificity of enzyme-linked
True negative: those who are not sick and whose tests immunosorbent assay kit in mice
True True False False Sensitivity Specificity
are negative (TN). positive negative positive negative (%) (%)
False positive: those who are not sick and whose tests
12 13 2 3 80.00 86.66
are positive (FP)
False negative: those who are sick and tested negative
(FN).
 424 Khodadadi et al / Archives of Razi Institute, Vol. 75, No. 4 (2020) 419-426

Results of Nano-ELISA method designed with E/S Discussion

antigen. The mice positive and negative sera designed Toxoplasmosis is a common parasitic disease caused
with the Nano-ELISA kit were prepared to be by a protozoan organism parasite called T. gondii. The
evaluated. The cut-off value was considered 1 prevalence of Toxoplasmosis is different according to
indicating that the absorbance values higher and lower location, cultural level, and health information.
than 1 were regarded as positive and negative, Accordingly, the prevalence of Toxoplasmosis in North
respectively. The results of the mice positive and America and United Kingdom is reported within the
negative serum test with the designed Nano-ELISA kit range of 16-40%, and it is estimated within the range of
are presented in tables 4 and 5, respectively. 50-80% in Central and South America and Europe
(Habibi et al., 2012; Glor et al., 2013).
Table 4. Results of 15 mice positive sera with designed Nano- According to the results of studies focusing on mice,
enzyme linked immunosorbent assay kit
Test OD Test OD Test OD the infection rate of T. gondii was reported as up to
1 1.481 6 1.533 11 2.182 70% in Montaña, Italy, in 1991 (Genchi et al., 1991).
2 2.175 7 1.489 12 1.579 Childs and Seegar showed the infection rate of 49.5%
3 1.225 8 0.540 α 13 1.919
in their studies in Baltimore, Maryland, in 1986 (Childs
4 1.884 9 1.646 14 1.510
5 1.249 10 2.039 15 1.043 and Seegar, 1986). According to studies carried out in
(α): Sample no. 8 has an optical density lower than the cutoff point England, it was reported that 42.2% of the infection
and is negatively evaluated.
was associated with T. gondii in rats according to PCR
(Hughes et al., 2006).
Table 5. Results of 15 mice negative sera with designed Nano-
enzyme linked immunosorbent assay kit The detection of antibodies produced against T.
Test OD Test OD Test OD gondii in the sera of individuals is a common method of
1 0.423 6 0.629 11 0.284 diagnosis in Toxoplasmosis (Fuentes et al., 1996). The
2 0.486 7 0.859 12 0.214 use of the ELISA method seems more appropriate
3 0.362 8 0.770 13 0.467
4 0.560 9 1.125 α 14 0.338 among the available techniques for the diagnosis of
5 0.125 10 0.123 15 0.418 Toxoplasmosis. The sensitivity of ELISA is relatively
(α): Sample no. 9 has an optical density higher than the cutoff
high; therefore, the reaction will also occur with 1-5 ng
point and is positively evaluated.
of antigen (Hassan et al., 1997).
The present study aimed to evaluate the Nano
According to the obtained results, the sensitivity and procedure in increasing the sensitivity and specificity of
specificity of the Nano-ELISA designed with E/S the serological methods of ELISA. As previously
antigen and gold nanoparticles are presented in Table 6. mentioned, a commercial ELISA kit was not available
for the diagnosis of Toxoplasmosis in mice; therefore,
Table 6. Sensitivity and specificity of designed Nano-enzyme it was first necessary to design an ELISA kit for mice
linked immunosorbent assay kit in mice with suitable conjugate and E/S antigens and then
True True False False Sensitivity Specificity
positive negative positive negative (%) (%) compare it to the newly designed Nano-ELISA kit with
14 14 1 1 93.33 93.33 gold nanoparticles.
The obtained results showed that the sensitivity and
specificity of the designed ELISA kit were 80.00% and
The results of the Nano-ELISA kit designed with the 86.66%, respectively; however, the sensitivity and
E/S antigens of T. gondii showed that the sensitivity specificity of the designed Nano-ELISA kit in the same
and specificity of this test was higher than those serum samples were 93.33% indicating a significant
reported for the ELISA designed kit with E/S antigens. improvement in the use of nanotechnology in the
 Khodadadi et al / Archives of Razi Institute, Vol. 75, No. 4 (2020) 419-426 425

ELISA method. The reason for the increased sensitivity Acknowledgment

and specificity of ELISA with nanoparticles in the
We wish to thank the Department of Proteomics,
current study is probably the high level of improvement Biochemistry and Biotechnology, Razi Vaccine and
in the volume of gold nanoparticles that causes more Serum Research Institute.
antibodies to enter the antigen-antibody complex with
the help of nanoparticles leading to a better dyeing. References
One of the advantages of this study was that in all of Ambrosi, A., Airo, F., Merkoci, A., 2010. Enhanced gold
nanoparticle based ELISA for a breast cancer biomarker. Anal
the ELISA methods, the serum dilution was much less Chem 82, 1151-1156.
studied than other methods. It should also be noted that Childs, J.E., Seegar, W.S., 1986. Epidemiologic observations on
the use of gold nanoparticles resulted in a lower infection with Toxoplasma gondii in three species of urban
mammals from Baltimore, Maryland, USA. Int J Zoonoses 13,
dilution of serum. Considering the emphasis of all 249-261.
government officials and authorities on using national Cox, F., Wakelin, D., 2010. Immunology and Immunopathology of
Human Parasitic Infections. Topley & Wilson's Microbiology and
products, another advantage of this study was designing Microbial Infections, pp. 422-442.
a native Nano-ELISA with gold nanoparticles. Dubey, J.P., 2008. Toxoplasmosis. OIE Terrestrial Manual, pp.
1284-1293.
Although this method has high sensitivity and Fuentes, I., Rodriguez, M., Domingo, C.J., del Castillo, F., Juncosa,
specificity, it requires a low cost of production of the T., Alvar, J., 1996. Urine sample used for congenital
toxoplasmosis diagnosis by PCR. J Clin Microbiol 34, 2368-2371.
masses. The present study also demonstrated that the Gamble, H.R., Dubey, J.P., Lambillotte, D.N., 2005. Comparison of
use of nanoparticles in the design of screening kits is a commercial ELISA with the modified agglutination test for
detection of Toxoplasma infection in the domestic pig. Vet
useful and could be very important, especially in some
Parasitol 128, 177-181.
diseases with fatal side effects. Genchi, G., Polidori, G.A., Zaghini, L., P.Lanfranchi, 1991. Aseppti
epidemiologici della toxoplasmosis nell’allevamento intensive del
suino. Arch Vet Ital 42, 105-111.
Authors' Contribution Glor, S.B., Edelhofer, R., Grimm, F., Deplazes, P., Basso, W., 2013.
Study concept and design: R. M. Evaluation of a commercial ELISA kit for detection of antibodies
against Toxoplasma gondii in serum, plasma and meat juice from
Acquisition of data: A. Kh. experimentally and naturally infected sheep. Parasit Vectors 6, 85.
Analysis and interpretation of data: N. H. R. Habibi, G., Imani, A., Gholami, M., Hablolvarid, M., Behroozikhah,
A., Lotfi, M., et al., 2012. Detection and Identification of
Drafting of the manuscript: N. A. Toxoplasma gondii Type One Infection in Sheep Aborted Fetuses
Critical revision of the manuscript for important in Qazvin Province of Iran. Iran J Parasitol 7, 64-72.
Hassan, M.M., Mansour, S.A., Atta, M., Shalaby, M.M., Seksaka,
intellectual content: R. M. M.A., Awad, A., 1997. The importance of detecting circulating
Statistical analysis: A. Kh. Toxoplasma antigens in human cases. J Egypt Soc Parasitol 27,
Administrative, technical, and material support: RVSRI 27-34.
Hauck, T.S., Giri, S., Gao, Y., Chan, W.C., 2010. Nanotechnology
diagnostics for infectious diseases prevalent in developing
Ethics countries. Adv Drug Deliv Rev 62, 438-448.
Hughes, J.M., Williams, R.H., Morley, E.K., Cook, D.A., Terry,
We hereby declare all ethical standards have been R.S., Murphy, R.G., et al., 2006. The prevalence of Neospora
respected in preparation of the submitted article. caninum and co-infection with Toxoplasma gondii by PCR
analysis in naturally occurring mammal populations. Parasitology
132, 29-36.
Conflict of Interest Jia, C.P., Zhong, X.Q., Hua, B., Liu, M.Y., Jing, F.X., Lou, X.H., et
al., 2009. Nano-ELISA for highly sensitive protein detection.
Biosens Bioelectron 24, 2836-2841.
The authors declare that there is no conflict of interest. Koloren, Z., 2013. Sensitive and Cost-Effective Detection of
Toxoplasma Gondii in Water Supplies of the Black Sea in Turkey
Grant Support by Loop-Mediated Isothermal Amplification (LAMP). Biotechnol
Biotechnol Equip 27, 3543-3546.
This study was supported by Razi Vaccine and Serum Markell, E., John, D., Krotoski, W., 2006. Medical Parasitology,
Research Institute. W.B. Saunders Co.
426 Khodadadi et al / Archives of Razi Institute, Vol. 75, No. 4 (2020) 419-426

Meganathan, P., Singh, S., Ling, L.Y., Singh, J., Subrayan, V., Nguyen, T.D., de Kesel, M., Bigaignon, G., Hoet, P., Pazzaglia, G.,
Nissapatorn, V., 2010. Detection of Toxoplasma gondii DNA by Lammens, M., et al., 1996. Detection of Toxoplasma gondii
PCR following microwave treatment of serum and whole blood. tachyzoites and bradyzoites in blood, urine, and brains of infected
Southeast Asian J Trop Med Public Health 41, 265-273. mice. Clin Diagn Lab Immunol 3, 635-639.
Montoya, J.G., 2002. Laboratory diagnosis of Toxoplasma gondii Toxoplasma gondii and Hammondia heydorni. J Vet Med Sci 64,
infection and toxoplasmosis. J Infect Dis 185 Suppl 1, 73-82. 161-164.
Mosallanejad, B., Avizeh, R., Razi Jalali, M.H., Hamidinejat, H., Shirbazou, S., Delpisheh, A., Mokhetari, R., Tavakoli, G., 2013.
2012. Seroprevalence of Toxoplasma gondii Among Wild Rats Serologic Detection of Anti Toxoplasma gondii Infection in
(Rattus rattus) in Ahvaz District, Southwestern Iran. Jundishapur J Diabetic Patients. Iran Red Crescent Med J 15, 701-703.
Microbiol. 5, 332-335. Suzuki, Y., 2002. Host resistance in the brain against Toxoplasma
Nishikawa, Y., Claveria, F.G., Fujisaki, K., Nagasawa, H., 2002. gondii. J Infect Dis 185, 58-65.
Studies on serological cross-reaction of Neospora caninum with Tavassoli, M., Ghorbanzadehghan, M., Esmaeilnejad, B., 2013. Foll
Toxoplasma gondii and Hammondia heydorni. J Vet Med Sci. 64, Detection of Toxoplasma gondii in sheep and goats blood samples
161-164. by PCR-RFLP in Urmia. Vet Res Forum 4, 43-47.