Early diagnosis of acute experimental toxoplasmosis via SAG1 detection using nitrogen-doped carbon dot-based sandwich ELISA.

Toxoplasmosis, caused by protozoan parasite Toxoplasma gondii, represents a serious opportunistic infection in immunocompromised individuals and pregnant women. Early diagnosis is necessary, yet current serological assays often cannot discriminate between acute and chronic infections. This work aimed to achieve early diagnosis of acute toxoplasmosis by detecting tachyzoite-specific surface antigen 1 (SAG1) using novel nitrogen-doped carbon dot (CDNs)-based sandwich ELISA. Truncated recombinant SAG1 was expressed in Escherichia coli and used for immunization of mice and rabbits to produce polyclonal antibodies, and those produced in rabbits were conjugated to the synthesized CDNs (AbCDNs). Characterization of CDNs and AbCDNs conjugates was done for their optical, morphological, and chemical properties. The diagnostic performance of AbCDNs-based ELISA was evaluated using sera from mice infected with virulent T. gondii RH strain across different infection timelines and compared to unconjugated antibody-based ELISA. Interestingly, AbCDNs-based assay demonstrated a markedly improved limit of SAG1 detection (2.38 ng/mL) and quantification (7.23 ng/mL) compared to unconjugated antibodies (11.64 ng/mL and 35.29 ng/mL, respectively). This platform enabled significant detection of circulating SAG1 from day one post-infection (PI), peaking on day three and continuing until day seven, whereas the conventional assay detected SAG1 only from day three till seventh day PI. Thereby, incorporation of CDNs doubled the diagnostic sensitivity from 40% to 80%. This study represents the first application of AbCDNs-based ELISA as a cost-effective and scalable tool for early diagnosis of acute toxoplasmosis via SAG1 detection. Hence, it addresses critical gaps in toxoplasmosis diagnosis, especially for high-risk populations.