Evaluation of a Newly Designed Immunochromatographic Test using Gold Nanoparticles and Recombinant Antigen gra7 for Rapid Diagnosis of Human Toxoplasmosis

Abstract

Background: One of the most important complications of toxoplasmosis is its early diagnosis. It seems that GRA7 protein can be a good candidate for detection of the acute phase in Toxoplasmosis. Accordingly, the present study aimed to diagnose toxoplasmosis via a newly immunochromatographic test using recombinant antigen gra7. Methods: The parasite was cultured in mice and then were used for DNA extraction. The gra7 gene was amplified by PCR and cloned into the pET-32a (+) plasmid. Thereafter, the recombinant vector was transferred into the Escherichia coli Rosetta strain and gra7 was detected via SDS-PAGE and western blotting. The bacterial lysate was used to purify the protein by Ni-NTA affinity chromatography .Anti-human gold conjugated antibody, test line and control line were injected to conjugate pad and nitrocellulose membrane, respectively, and all the layer were assembled. By using serum of patients and healthy individuals, manufactured kits were evaluated. Results: Our results indicated that the selected gene was correctly cloned and the protein of interest was produced and purified. The test revealed sensitivity and specificity of 100 and 96.7 percent, respectively. The kit was also shown to be stable over 16 weeks in 37°C. Conclusion: The choice of antigen based on cellular and clinical features of the parasite, as well as the use of previous outcomes yielded to develop a rapid diagnostic test for toxoplasmosis.

Keywords

• toxoplasmosis
• immunochromatoga
• gra7 antigen
• rpd
• gold nanoparticles