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<i>Acanthamoeba</i> spp. is the causative agent of <i>Acanthamoeba</i> keratitis (AK), a vision-threatening parasitic disease whose primary risk factor has been attributed to poor contact lens hygiene. Unfortunately, differential diagnosis of AK is challenging as the clinical manifestations for AK are similar to those of bacterial, fungal, or even viral keratitis. Since delayed AK diagnosis can incur permanent vision impairment, a rapid and sensitive diagnostic method is urgently needed. Here, the diagnostic potential of polyclonal antibodies targeting the chorismate mutase (CM) of <i>Acanthamoeba</i> spp. was evaluated in AK animal models. CM antibody specificity against <i>Acanthamoeba</i> trophozoites and cysts was confirmed by immunocytochemistry after co-culturing <i>Acanthamoeba</i> with <i>Fusarium solani</i>, <i>Pseudomonas aeruginosa</i>, and <i>Staphylococcus aureus</i>, and human corneal epithelial (HCE) cells. Enzyme-linked immunosorbent assay (ELISA) was performed using CM-specific immune sera raised in rabbits, which demonstrated that the antibodies specifically interacted with the <i>Acanthamoeba</i> trophozoites and cysts in a dose-dependent manner. To evaluate the diagnostic potential of the CM antibody, AK animal models were established by incubating contact lenses with an inoculum containing <i>A. castellanii</i> trophozoites and subsequently overlaying these lenses onto the corneas of BALB/c mice for 7 and 21 days. The CM antibody specifically detected <i>Acanthamoeba</i> antigens in the murine lacrimal and eyeball tissue lysates at both time points. Our findings underscore the importance of antibody-based AK diagnosis, which could enable early and differential AK diagnosis in clinical settings.