Targeted Identification of Acanthamoeba in Clinical Corneal Specimens Using a Monoclonal Antibody: Proof of Concept for a New Diagnostic Tool

Purpose: To evaluate the diagnostic potential of direct immunofluorescence using a monoclonal antibody and its derived recombinant antibody fragments for specific detection of Acanthamoeba in human clinical corneal samples. Methods: A previously characterized monoclonal antibody and 2 recombinant fragments derived from it were fluorescently labeled and tested in direct immunofluorescence assays against Acanthamoeba trophozoites and cysts in axenic culture, followed by testing in formalin-fixed, paraffin-embedded corneal sections. Specificity was assessed by examining reactivity against fungi, bacteria, and human corneal tissue. Results: All fluorescently labeled antibodies recognized Acanthamoeba trophozoites and cysts in culture with no observed cross-reactivity against bacterial or fungal cultures or human keratinocytes. In corneal sections, the antibodies clearly detected Acanthamoeba in samples from patients with amoebic keratitis, with labeling correlating to parasite morphology. No specific signal was observed in corneal sections from fungal keratitis, bacterial keratitis, or noninfectious disease controls, although varying levels of background autofluorescence were noted in some non- Acanthamoeba specimens. Findings were consistent across monoclonal and recombinant antibody formats. Conclusions: Direct immunofluorescence using monoclonal and recombinant antibodies is a promising diagnostic strategy for specific detection of Acanthamoeba in human corneal samples. These proof-of-concept results support further validation in larger cohorts and extension to corneal scraping samples to determine diagnostic performance for early detection of amoebic keratitis.