Development of a serological assay based on a recombinant protein containing polyantigenic epitope peptides derived from Toxoplasma gondii

The development of a sensitive and specific detection method for feline toxoplasmosis infection is important, as cats, being the definitive host of Toxoplasma gondii , play a crucial role in the transmission of toxoplasmosis. In this study, six T. gondii antigens (SAG1, SAG2, ROP1, GRA1, LEA870, and LEA880) were selected for analysis. Dominant B-cell epitopes were predicted using bioinformatics and were subsequently linked and expressed as a recombinant multiepitope protein designated Polyantigenic Epitope Peptide 1 (PEP1). This protein served as the coating antigen to establish an indirect enzyme-linked immunosorbent assay (iELISA) for the detection of T. gondii -specific antibodies in cat sera. The optimal assay conditions were determined by checkerboard titration and subsequent validation: antigen coating at 5 μg/mL (4 °C, 12 h), blocking with 0.5% BSA (37 °C, 60 min), a serum dilution of 1:1,000 (37 °C, 45 min), a secondary antibody dilution of 1:2,500 (37 °C, 60 min), and incubation with 3,3',5,5'-Tetramethylbenzidine (TMB) substrate for 25 min at room temperature in the dark. In the preliminary evaluation, the assay showed no cross-reactivity with sera positive for feline panleukopenia virus (FPV), feline herpesvirus (FHV), or feline calicivirus (FCV). The detection limit was achieved at a serum dilution of 1:6,400, and both intra- and inter-assay coefficients of variation were below 10%, indicating high specificity, sensitivity, and reproducibility. Parallel testing of 78 feline serum samples with a T. gondii lysate antigen iELISA (TLA-iELISA) yielded a 97% overall agreement. The results indicate that the PEP1-based indirect ELISA is a reliable tool for the serodiagnosis of feline toxoplasmosis and provides a solid technical platform for future studies of vaccine efficacy.