Development of an Immunoassay Test System for the Diagnosis of Trypanosomiasis in Horses

Equine trypanosomiasis continues to be a significant infectious disease resulting in economic losses and diminished performance in horses.Diagnosis is difficult because to the variability of clinical symptoms and the constraints of existing laboratory techniques.There is a necessity for cost-effective, dependable, and highly sensitive immunoassays tailored for field application.The study aimed to develop and validate a highly specific immunoassay system for the serological diagnosis of trypanosomiasis in farm animals (n = 86 samples were used for validation).Key analytical techniques, including spectrophotometry, SDS-PAGE, and ELISA, were applied to confirm protein purity, molecular weight, immunoreactivity, and storage stability.Optimisation showed that antigen concentration of 0.8-1.0μg/well, serum dilution 1:80, and conjugate dilution 1:6000 ensured optimal performance, with signal-to-noise ratio 6.0-6.2,background OD 0.26-0.32,and CV ≤ 10 %.Sensitivity analysis demonstrated a minimum titre of 1:160, average 1:860, with most antibodies detected at 1:640-1:1280 and stable reproducibility (CV ≤ 8.4 %, up to 12 % at higher dilutions).No cross-reactivity was observed with other infectious diseases (signal-to-noise 1.1-1.3),while sera from cattle and goats showed low values (0.20-0.22), in contrast to positive samples (6.0-6.2).These results confirm high specificity, sensitivity, and reproducibility, supporting practical application of the test system.