Development and characterization of Dot Blot detection assay for the diagnosis of acanthamoeba keratitis

Abstract

Acanthamoeba keratitis (AK), is a rare, vision threatening infection caused by Acanthamoeba castellanii. Management of this disease is based on accurate diagnosis which is easily misdiagnosed due to the similarity of its clinical symptoms with other infections. Till date no quick and accurate diagnostic assay is available. Acanthamoeba mannose binding protein (MBP1) is a crucial antigen for developing effective diagnostic strategies. This study aimed to generate and characterize a polyclonal antibody (pAb) against the MBP1 protein of Acanthamoeba. rMBP of Acanthamoeba castellani was prepared in pQE-30 expression vector and was purified by using Ni2+ NTA ion-affinity chromatography. Four rabbits were immunized subcutaneously with 50 µg rMBP antigen + 50 µl Freund's incomplete adjuvant to generate the polyclonal Ab. After complete dose, blood serum was collected. pAb was purified and confirmed by SDS-PAGE and western blot. The pAb was tested against the Acanthamoeba antigen by Dot Blot assay. The results confirmed that the pAb could specifically bind to the rMBP antigen, with no binding observed to the negative control. This study demonstrates that the polyclonal antibody against rMBP can be used as a sensitive and specific diagnostic tool. The MBP1 antigen, therefore, has potential as a diagnostic marker for AK, offering a rapid, sensitive, and easy-to-use assay, particularly beneficial in resource-limited settings.

Keywords: Acanthamoeba Keratitis; Diagnosis; Dot Blot assay; Mannose binding protein; Polyclonal Antibody.