Evaluation of a cystatin-like protein of Trichinella spiralis for serodiagnosis and identification of immunodominant epitopes using monoclonal antibodies

An antigenic cystatin-like protein (Ts-CLP) selected from cDNA library of intestinal infective larvae at 6 h post-infection, was expressed by prokaryotes in the form of a histidine-tagged protein (rTs-CLP). The fusion protein was purified by an on-column refolding procedure using Ni-NTA affinity chromatography. An indirect rTs-CLP ELISA was developed using 270 known negative serum samples from commercial swine maintained under non-special pathogen free conditions. Based on the distribution of the signal-to-positive (S/P) ratio, a cut-off value was set at 0.30. Using this cut-off value, rTs-CLP ELISA was evaluated using sera from swine experimentally infected with 1000 and 50,000 muscle larvae of Trichinella spiralis. Specific IgG antibodies were detectable by rTs-CLP ELISA as soon as 17 days post-infection (dpi), but the commercial ELISA kit based on excretory-secretory (ES) antigens did not permit detection before 21 dpi. Three monoclonal antibodies (McAbs) against Ts-CLP (designated 1H9, 6B5 and 7F8) were obtained by screening with both rTs-CLP ELISA and ES ELISA methods. Two dominant epitopes recognized by McAbs were determined by analysis with overlapping fusion peptides and synthetic peptides. One epitope(39) HEALFSSDLKQESGV(53 )was recognized by 1H9 and 6B5, and the other epitope(178) REALFSSDSKEQSGV(192) was recognized by 7F8. The generation of McAbs against TsCLP and the characterization of the two dominant epitopes provide a foundation for the development of a specific early semdiagnostic strategy for T. spiralis infection.

Automated summary

An antigenic cystatin-like protein Ts-CLP was selected from intestinal infective larvae cDNA library and expressed as a histidine-tagged protein. A specific ELISA method based on rTs-CLP was developed for early detection of Trichinella spiralis infection, providing a foundation for specific semi-diagnostic strategy. Monoclonal antibodies against Ts-CLP were obtained, and two dominant epitopes were identified, contributing to potential diagnostic advancements.