Establishment and application of a sandwich ELISA method for measuring Toxoplasma gondii circulating fructose-1,6-bisphosphate aldolase (ALD) protein in cats

Toxoplasmosis is an important public health concern. Cats play a crucial role in increasing the risk of toxoplasmosis transmission to humans. Early diagnosis in cats is essential for the prevention and control of toxoplasmosis. In this study, we found that T. gondii aldolase (ALD) could be an effective diagnostic antigen, and then the recombinant ALD protein was expressed using the pET SUMO protein expression system, the mouse monoclonal antibody (MoAb) and rabbit polyclonal antibody (PoAb) of ALD were successfully produced, respectively. Furthermore, a reliable sandwich enzyme-linked immunosorbent assay (sELISA) was developed to detect circulating ALD in the sera of experimentally and naturally infected cats. rALD sELISA could detect T. gondii infection from 7DPI (post-infection day) to 14DPI with 100 % sensitivity and specificity, but could not detect T. gondii infection after 21DPI, indicating that it is a good early diagnosis tool. The detection limit was 7.8 ng/ml, the coefficients of variation (CV) of repeated tests within batches and between batches were confirmed to be less than 10 %. The results of 70 cat clinical serum samples detected by rALD sELISA were in almost perfect agreement beyond chance with those of a commercial ELISA kit (Cohen's kappa coefficient = 0.883). This sandwich ELISA method has high accuracy and can be used for early diagnosis of toxoplasmosis in cats.