Production of a polyclonal antibody against inosine-uridine preferring nucleoside hydrolase of Acanthamoeba castellanii and its access to diagnosis of Acanthamoeba keratitis

Abstract

Acanthamoeba keratitis (AK) is a rare disease but its prevalence throughout the globe continues to grow, primarily due to increased contact lens usage. Since early-stage symptoms associated with AK closely resemble those from other corneal infections, accurate diagnosis is difficult and this often results in delayed treatment and exacerbation of the disease, which can lead to permanent visual impairment. Accordingly, developing a rapid Acanthamoebaspecific diagnostic method is highly desired. In the present study, a rapid and differential method for AK diagnosis was developed using the secretory proteins derived from the pathogenic Acanthamoeba. Among the vast quantities of proteins secreted by the pathogenic Acanthamoeba, an open reading frame of the inosine-uridine preferring nucleoside hydrolase (IPNH) gene was obtained. After expressing and purifying the IPNH protein using the pGEX 4T-3 vector system, mice were immunized with the purified proteins for polyclonal antibody generation. Western blot was performed using protein lysates of the human corneal cell, non-pathogenic amoeba, pathogenic amoeba, and clinical amoeba isolate along with lysates from other causes of keratitis such as Staphylococcus aureus, Pseudomonas aeruginosa, and Fusarium solani to confirm Acanthamoeba-specificity. Western blot using the polyclonal IPNH antibody revealed that IPNH was Acanthamoeba-specific since these proteins were only observed in lysates of Acanthamoeba origin or its culture media. Our findings indicate that the IPNH antibody of Acanthamoeba may serve as a potential agent for rapid and differential AK diagnosis.

Detection of Acanthamoeba spp. using carboxylesterase antibody and its usage for diagnosing Acanthamoeba-keratitis

“…We have previously reported the diagnostic potential of Acanthamoeba -specific antibodies targeting the inosine-uridine preferring nucleoside hydrolase and chorismate mutase [ 22 , 23 ]. While these studies revealed that antibody-based detection method can specifically identify A .…”

“…successfully detected both trophozoites and cysts of A . castellanii [ 22 , 23 ]. Five monoclonal antibodies (AMEC1, AMEC2, AMEC3, MTAC1, and MTAC3) were generated from hybridoma cell lines of mice immunized with live mixtures of A .…”

Chorismate mutase peptide antibody enables specific detection of Acanthamoeba

“…Acanthamoeba , human corneal epithelial (HCE) cells, Fusarium solani , Pseudomonas aeruginosa , and Staphylococcus aureus were prepared as previously described [ 21 ]. Briefly, both of the non-pathogenic (ATCC 30011; yeast culture isolate) and pathogenic (ATCC 30868; human corneal isolate) strains of Acanthamoeba castellanii Castellani were purchased from the American Type Culture Collection (ATCC).…”

“…Acanthamoeba-specificity of the chorismate mutase was confirmed using western blot as previously described [21]. Protein samples from the whole cell lysates and the culture media of Acanthamoeba spp., HCE cells, F. solani, P. aeruginosa, and S. aureus were prepared using a lysis buffer.…”

Commensals Serve as Natural Barriers to Mammalian Cells during Acanthamoeba castellanii Invasion

“…The blots were then incubated with horseradish peroxidase (HRP)-conjugated IgG for 1 h at 25°C. Proteins were detected using Immobilon Western HRP substrate ( 50 ). The optical densities of the bands were measured using ImageJ software ( 51 ).…”