Overexpression of Schistosome-specific Antigens for Selection of scFv Antibody from Phage Display Libraries of Water Buffalo for Potential Use in Schistosomiasis Diagnosis

Abstract

Schistosomiasis is an acute and chronic parasitic disease caused by blood flukes of the genus Schistosoma. The Kato-Katz method is the standard for the diagnosis of intestinal schistosomiasis because of its specificity, simplicity, and low cost. However, Kato-Katz is less useful in light infections and other available detection methods are neither practical nor sensitive for routine or large-scale screening. Thus, improved tests are needed for the assessment of infection. The water buffalo is a natural host and reservoir of Schistosoma but is schistosomiasis-resistant, suggesting a robust immune repertoire, which can be tapped as a source of antibodies for diagnosis of schistosomiasis. Two schistosome antigens had been previously shown to be recognized by a phage display single-chain fragment of variation (scFv) library derived from a schistosome-infected water buffalo. However, specific antibodies have not been isolated using these antigens due to a lack of sufficient amounts and purified forms of the antigens. In this study, the schistosome antigens were cloned and overexpressed in vector with solubility tag, purified by immobilized metal affinity chromatography (IMAC), and used to interrogate the immune scFv library to isolate and identify specific schistosome antibody. An scFv specific to schistosome SAP domain-containing hypothetical protein was isolated but was only 105-bp long although certain regions align with the IgG VL, tetraglycylserine (Gly4Ser) linker, and IgG VH sequences of synthetic scFv constructs. Despite its length, scFv A63 can be potentially designed as a ligand in direct ELISA (enzyme-linked immunosorbent assay) by fusion with enzyme conjugates such as alkaline phosphatase to detect S. japonicum schistosomule and adult worm. The results in this study also suggest the need to construct a bigger and more diverse scFv library and to test a wider range of schistosome antigens, specifically those that will be present on the surface of the schistosome at all stages of S. japonicum life cycle.