Generation of a Novel Bacteriophage Library Displaying scFv Antibody Fragments from the Natural Buffalo Host to Identify Antigens from Adult Schistosoma japonicum for Diagnostic Development

Abstract

The development of effective diagnostic tools will be essential in the continuing fight to reduce schistosome infection; however, the diagnostic tests available to date are generally laborious and difficult to implement in current parasite control strategies. We generated a series of single-chain antibody Fv domain (scFv) phage display libraries from the portal lymph node of field exposed water buffaloes, Bubalus bubalis, 11–12 days post challenge with Schistosoma japonicum cercariae. The selected scFv-phages showed clear enrichment towards adult schistosomes and excretory-secretory (ES) proteins by immunofluorescence, ELISA and western blot analysis. The enriched libraries were used to probe a schistosome specific protein microarray resulting in the recognition of a number of proteins, five of which were specific to schistosomes, with RNA expression predominantly in the adult life-stage based on interrogation of schistosome expressed sequence tags (EST). As the libraries were enriched by panning against ES products, these antigens may be excreted or secreted into the host vasculature and hence may make good targets for a diagnostic assay. Further selection of the scFv library against infected mouse sera identified five soluble scFv clones that could selectively recognise soluble whole adult preparations (SWAP) relative to an irrelevant protein control (ovalbumin). Furthermore, two of the identified scFv clones also selectively recognised SWAP proteins when spiked into naïve mouse sera. These host B-cell derived scFvs that specifically bind to schistosome protein preparations will be valuable reagents for further development of a cost effective point-of-care diagnostic test.

An overview of innovations and industrial solutions in Protein Microarray Technology

“…The most promising applications of protein microarrays have been in the area of clinical biomarker discovery, which is also the focus of many researchers utilizing this platform. Over the last 5 years, several studies have shown successful application of this technology in providing clues into the pathophysiology of many diseases and putative biomarkers for both infectious and noninfectious diseases [74][75][76][77][78][79][80][81][82].…”

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

“…Titer of the eluted scFv phage Titer of the input scFv phage = scFv-phage ELISA scFv-phage ELISA was conducted, as previously described (Hosking et al 2015a). The wells of microplates were coated with 100 μL of diluted purified antigen (0.01 μg/ μL in 0.1 M bicarbonate-carbonate coating buffer, pH 9.6) for 1 h at 37 °C, washed with 200 μL of PBST, and blocked with 200 μL of casein in PBS (1% w/v) for 2 h at 37 °C.…”

“…Bacteriophage libraries that display scFv fragments have proven to be useful reagents in the identification of antibodies against a specific target antigen. Hosking et al (2015a) previously generated an scFv-phage library encoding B-cell heavy chain variable region (V H ) and light chain variable region (V L ) gene families from the local draining lymph node of B. bubalis in China following experimental infection with S. japonicum cercariae. A pool of selected scFv-phage recognized stage-specific schistosome antigens on protein microarray, including two hypothetical proteins with NCBI GenBank accession numbers AY811797 and AY814150.…”

“…°C (or 2 h at 37 °C), as previously described(Hosking et al 2015a). The coating solution was removed gently by pipetting and the wells were washed thrice using 0.01 M PBS with 0.05% (v/v) Tween 20 (PBST).…”