Direct enzyme-linked aptamer assay (DELAA) for diagnosis of toxoplasmosis by detection of SAG1 protein in mice and humans

• Published in: Acta tropica 2022-02, Vol.226, p.106255-106255, Article 106255
• Main Authors: Cui, Wen, Shen, Xuhang, Wang, Cong, Bibi, Asma, Cudjoe, Obed, Zhao, Liang, Yu, Li, Du, Jian, Xu, Yuanhong, Chen, Xi, Shen, Jilong, Wang, Wei
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Protozoan; Antigens, Protozoan - genetics; Aptamer; Diagnosis; Enzyme-Linked Immunosorbent Assay; Humans; Mice; Protozoan Proteins - genetics; SAG1; Toxoplasma - genetics; Toxoplasmosis; Toxoplasmosis - diagnosis
• ISSN: 0001-706X; 1873-6254
• DOI: 10.1016/j.actatropica.2021.106255

•As a major surface antigen of Toxoplasma tachyzoites, SAG1 is a key marker for laboratory diagnosis. We screened out the aptamer from the synthetic oligonucleotide library which targets Toxoplasma SAG1 and developed a novel method of direct enzyme-linked aptamer assay (DELAA). Mouse sera of acute infection and human sera, that had been verified to be positive or negative for Toxoplasma DNAs by PCR, were subjected to DELAA and circulating antigen detection. The results showed that DELAA has high sensitivity, specificity and an better efficacy than ELISA-based circulating antigen(s) determination in diagnosis of active and reactivated toxoplasmosis. Toxoplasma gondii is a single-celled parasite commonly found in mammals and birds. Diagnosis of toxoplasmosis largely depends on measurements of the antibody and/or antigen and Toxoplasma DNAs due to the presence of tissue dwelling duplicating tachyzoites, or quiescent cysts in latent infection of the parasite. As a major surface antigen of T.gondii tachyzoites, SAG1 is a key molecule for laboratory diagnosis. However, there are no methods available yet for SAG1 detection using aptamer-based technology. Recombinant SAG1 (r-SAG1) of Toxoplasma WH3 strain (type Chinese 1) was expressed in E.coli and subjected to the synthetic oligonucleotide library for selection of nucleic acid aptamers which target the r-SAG1 antigen, with systematic evolution of ligands by exponential enrichment (SELEX) strategy. The specific aptamers were screened out and used in direct enzyme-linked aptamer assay (DELAA) for detection of native SAG1 (n-SAG1) obtained from tachyzoite lysates, mouse sera of acute infection, and human sera that had been verified for Toxoplasma DNAs by PCR amplification. As results, the soluble r-SAG1 protein was obtained from E.coli lysates by purification and identification with immunoblotting, followed by biotinylation. The selected aptamers were amplified by PCR and DNA sequencing. The results showed that the aptamer-2, with the highest affinity to n-SAG1 in the sera of animals with minimal difference in the four aptamer candidates, has a high specificity and sensitivity when used in detection of n-SAG1 in the sera of humans when compared with the commercial kit of ELISA for T.gondii circulating antigen test. We concluded that a new direct enzyme-linked aptamer assay (DELAA) was developed for the detection of the n-SAG1 protein of T. gondii. With increased sensitivity and specificity, stability, easy and chea