Characterization of a Peptide Antibody Specific to the Adenylyl Cyclase-Associated Protein of Acanthamoeba castellanii

Abstract

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.

Detection of Acanthamoeba from Acanthamoeba Keratitis Mouse Model Using Acanthamoeba-Specific Antibodies

“…In the previous study, ACAP antibodies demonstrated A. castellanii-specificity to both trophozoite and cyst forms when these were co-cultured with HCE cells [24]. Here, we confirmed the specificity of ACAP antibodies to A. castellanii trophozoites and cysts cocultured with HCE cells, F. solani, S. aureus, and P. aeruginosa (Figure 1A), thus signifying their potential for differential AK diagnosis.…”

“…Therefore, it is difficult to accurately discern exactly how much Acanthamoeba antigen is contained within these samples. However, in our previous study, we reported that ACAP antibodies could detect the presence of Acanthamoeba from cell lysate at concentrations as low as 0.01 μg [24]. Combining these two results, our…”

“…Therefore, it is difficult to accurately discern exactly how much Acanthamoeba antigen is contained within these samples. However, in our previous study, we reported that ACAP antibodies could detect the presence of Acanthamoeba from cell lysate at concentrations as low as 0.01 µg [24]. Combining these two results, our findings suggest that the ACAP antibodies are highly sensitive to Acanthamoeba antigens and could be used to confirm the presence of Acanthamoeba even in trace amounts.…”

“…Given the current circumstances, developing highly sensitive Acanthamoeba-specific antibodies would enable rapid AK diagnosis. Recently, we have produced five specific antibodies to Acanthamoeba [21][22][23][24][25]. Among these antibodies, the potential application for AK diagnosis in vivo was demonstrated by ACAP and PBP antibodies and these were further evaluated to confirm our previous findings.…”

“…also demonstrated protective effects against A. castellanii infection [20]. In our previous study, we produced five polyclonal antibodies against the inosine-uridine preferring nucleoside hydrolase (IPNH), chorismite mutase (CM), carboxylesterase (CE), adenylyl cyclase-associated protein (ACAP), and periplasmic binding protein (PBP) of A. castellanii and showed their diagnostic potential for AK [21][22][23][24][25]. Although these Acanthamoeba-specific antibodies could be useful for improving AK diagnosis, one major limitation of the aforementioned studies is the fact that these antibodies were primarily tested in vitro and have not been applied to in vivo studies.…”