Development of a Lateral Flow Immunoassay Using Recombinant Dense Granular Antigen (GRA) 7 to Detect Anti-Toxoplasma gondii IgG Antibodies

AUTHORS

H. Morovati 1 , 2 , S. J. Seyyed Tabaei 3 , * , M. Gholamzad 4 , K. Omidfar 5 , A. Ahmadi 5 , Z. Arab Mazar 6 , A. Eshghi 2 , F. Sheikhsofla 7

1 Department of Parasitology and Mycology, Faculty Medicin, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Department of Quality Control, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization, Karaj, Iran

3 Department of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran

4 Department of Microbiology and Immunology, Islamic Azad University, Tehran Medical Branch, Tehran, Iran

5 Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran

6 Infectious Diseases and Tropical Medicine Research Center, ShahidBeheshti University of Medial Sciences, Tehran, Iran

7 Department of Technical and Research Lab, Arvin Pazhoohan Noor (APN), Tehran, Iran

How to Cite: Morovati H, Seyyed Tabaei S J, Gholamzad M, Omidfar K, Ahmadi A, et al. Development of a Lateral Flow Immunoassay Using Recombinant Dense Granular Antigen (GRA) 7 to Detect Anti-Toxoplasma gondii IgG Antibodies, Arch Razi Inst. 2019 ; 74(1):e92332. doi: 10.22092/ari.2018.115890.1155.

ARTICLE INFORMATION

Archives of Razi Institute: 74 (1); 39-49
Published Online: March 01, 2019
Article Type: Journal Article
Received: April 16, 2019
Accepted: April 26, 2018
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Abstract

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis and is of medical importance in pregnant women and immunosuppressed patients. In recent years, many methods have been developed for the detection of infection caused by this parasite; however, most of the developed methods are not adequately sensitive. The dense granular antigen (GRA) 7 is a highly immunogenic protein that is used as a specific antigen for the diagnosis of toxoplasmosis. This study was designed to produce recombinant GRA7 (rGRA7) antigen in bacterial system in order to be applied as an antigen for developing a simple and rapid lateral flow immunoassay test strip using a gold nanoparticle-pAb conjugate probe to detect Toxoplasma IgG-specific antibodies in human sera. After the extraction of genomic DNA from RH strain tachyzoites, polymerase chain reaction (PCR) was performed using specific primers considering restriction sites and BglII and XhoI enzymes. Subsequently, the GRA7 gene was cloned in pET-32a (+) expression vector, and then pET-32a(+)-GRA7 was transformed into E. coli Rosetta (DE3). The induction of protein production was accomplished by IPTG, and the product was finally purified by Ni-NTA affinity chromatography. In order to make the strip test, the anti-human gold nanoparticle conjugate was applied on conjugate pad, rGRA7 antigen was immobilized to a nitrocellulose membrane as the capture agent, and sample and absorbance pads were assembled on a backing card. For the analysis of the sensitivity and specificity of the assay, the selected patients’ sera samples were tested by standard chemiluminescence immunoassay (CLIA) method, and then compared with TOXO-IgG strip results. The findings showed that the use of rGRA7 is an accurate, sensitive, and inexpensive technique for the rapid detection of anti-Toxo­plasma IgG in human sera. Therefore, rGRA7 can be applied as a diagnostic agent in laboratories.

© 2019, Archives of Razi Institute. Razi Vaccine and Serum Research Institute.

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