Enzyme-Linked Aptamer Assay (ELAA) for Detection of Toxoplasma ROP18 Protein in Human Serum.

Monica Vargas-Montes,Nestor Cardona,Jorge Enrique Gómez-Marín,Diego Mauricio Moncada,Diego Alejandro Molina,Yang Zhang

doi:10.3389/fcimb.2019.00386

Abstract

Toxoplasma gondii engenders the common parasitic disease toxoplasmosis in almost all warm-blooded animals. Being a critical secretory protein, ROP18 is a major virulence factor of Toxoplasma. There are no reports about ROP18 detection in human serum samples with different clinical manifestations. New aptamers against ROP18 protein were developed through Systematic Evolution of Ligands by Exponential enrichment (SELEX). An Enzyme-Linked Aptamer Assay (ELAA) platform was developed using SELEX-derived aptamers, namely AP001 and AP002. The ELAA was used to evaluate total antigen from T. gondii RH strain (RH Ag) and recombinant protein of ROP18 (rROP18). The results showed that the ELAA presented higher affinity and specificity to RH Ag and rROP18, compared to negative controls. Detection limit of rROP18 protein in serum samples was measured by standard addition method, achieving a lower concentration of 1.56 μg/mL. Moreover, 62 seropositive samples with different clinical manifestations of toxoplasmosis and 20 seronegative samples were tested. A significant association between ELAA test positive for human serum samples and severe congenital toxoplasmosis was found (p = 0.006). Development and testing of aptamers-based assays opens a window for low-cost and rapid tests looking for biomarkers and improves our understanding about the role of ROP18 protein on the pathogenesis of human toxoplasmosis.

Highlights

Toxoplasma gondii (T. gondii) is an intracellular parasite with cosmopolitan distribution that infects the majority of warm-blooded animals (Jones and Dubey, 2012)
We found that aptamer AP001 showed a higher affinity with a Kd value of 62.7 ± 17.27 nM; whereas the aptamer AP002 showed a Kd of 97.7 ± 22.20 nM (Figure 4C); these results suggested that it was feasible to continue working with AP001 aptamer in subsequent trials with human serum samples
Previous studies have reported that T. gondii produces some virulence factors that can modulate the host immune response and could explain the severe manifestations of toxoplasmosis, especially in South America (Bradley and Sibley, 2007; Etheridge et al, 2014; Petersen et al, 2017)

Summary

Introduction

Toxoplasma gondii (T. gondii) is an intracellular parasite with cosmopolitan distribution that infects the majority of warm-blooded animals (Jones and Dubey, 2012). Transmission of the parasite has been demonstrated in humans by the consumption of meat, vegetables and contaminated water (Lora-Suárez et al, 2007; Franco-Hernandez et al, 2016; Triviño-Valencia et al, 2016) For all these reasons, Food and Agriculture Organization (FAO) and World Health Organization (WHO) declared toxoplasmosis as a foodborne parasite infection disease of global concern (Robertson et al, 2013). The serological prevalence of toxoplasmosis is highly variable, ranging from 10 to 15% in the United States, to >60% in South and Central America (Gilbert et al, 2008) It Toxoplasma ROP18 Detection by ELAA has been reported that South America is the continent with the highest burden of the disease, with congenital and ocular toxoplasmosis frequently associated with more severe symptoms (de-la-Torre et al, 2007; De-la-Torre et al, 2009; Torgerson and Mastroiacovo, 2013). There are some indications that disease outcomes in humans can be influenced by the variability of the infecting T. gondii strain (Grigg et al, 2001; Reese et al, 2011; McLeod et al, 2012; Sánchez et al, 2014)

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Conclusion