# The Development of TIM-Barrel Based Multi-Epitope Protein for Toxoplasma gondii Serological Detection in Cats

## TL;DR

Researchers developed a multi-epitope protein using TIM-barrel structure to detect Toxoplasma gondii in cats, showing moderate effectiveness compared to existing methods.

## Contribution

A novel TIM-barrel-based multi-epitope protein for T. gondii detection in cats was engineered and evaluated.

## Key findings

- V4Z chimeric protein showed 86% sensitivity and 76% specificity in ELISA.
- V4Z had moderate agreement with IFAT (Kappa = 0.58) and strong negative predictive value (90%).
- Overall diagnostic performance of the protein remains limited and requires further refinement.

## Abstract

Toxoplasma gondii causes toxoplasmosis, which can develop into severe symptoms in pregnant and immunocompromised people. The T. gondii diagnosis relies on both direct and indirect methods with various specificities and sensitivities. The serodiagnosis has been introduced to T. gondii detection such as latex agglutination test (LAT), indirect immunofluorescent assay (IFAT), and enzyme-linked immunosorbent assays (ELISA). Recombinant antigens are commonly used in serodiagnosis and combinations of recombinant antigens have shown improved serodiagnosis efficacy. In this study, TIM-barrel-based proteins containing T. gondii B- and T-cell epitopes were used to detect T. gondii infection in cats via ELISA assay. Among 10 chimeric protein constructions, V4Z revealed 86% sensitivity and 76% specificity with moderate agreement with the reference IFAT. These findings suggest that TIM-barrel-based multi-epitope proteins are promising candidates for chimeric antigens in serological detection, which could be applied in pathogen detection by incorporating immunodominant epitopes.

Toxoplasma gondii, a pathogen of significant concern in animal production, companion animal health, and public health, particularly affects immunocompromised individuals and pregnant women. Current diagnostic techniques employ both direct and indirect methods, with serological assays widely used for detecting T. gondii infections in humans and animals. In this study, the TIM-barrel structure of Br2 β-glucosidase was engineered to create 10 chimeric multi-epitope proteins for T. gondii serological detection. Indirect ELISA screening identified three promising candidate proteins, V4Z, SFF, and S7V-V4Z-SFF, with sensitivities ranging from 71–86% and specificities ranging from 68–76%. Among these, ELISA-V4Z achieved the highest concordance with the reference IFAT method (Kappa = 0.58, 95% CI = 0.32–0.84) and demonstrated a moderate positive predictive value (PPV, 67%) and strong negative predictive value (NPV, 90%). These results suggest that the V4Z chimeric protein demonstrated the strongest performance among the tested candidates for T. gondii detection, exhibiting the highest sensitivity and specificity along with moderate agreement with the reference IFAT. However, its overall diagnostic performance remains limited. These findings highlight the need for further refinement and validation to enhance its diagnostic potential and assess its applicability for broader serological testing.