Development of Yeast-displayed scFv and Monoclonal Antibodies for <i>Entamoeba histolytica</i> Cyst Detection

Thesis (Ph.D.)--University of Washington, 2015The aims of this dissertation research are two-fold: 1) develop an accelerated affinity reagent pipeline that overcomes the limitations seen in utilizing recombinant antibodies and in vitro display systems; and 2) validate a novel Entamoeba histolytica cyst biomarker in stool, utilizing reagents obtained in the accelerated pipeline as well as by classical means. As the human proteome remains in focus and as new and remerging disease threats continue to exact terrible burdens worldwide, there is an enormous need for antigen-specific affinity reagents that can be produced rapidly and with little cost. The most widely used affinity reagent, the monoclonal antibody, is generated through a months-long, laborious, and expensive methodology, with no guarantee of a successful output. In response, a variety `of antibody-derived molecules have been generated recombinantly and have been displayed on a wide number of cells and organisms as combinatorial libraries for rapid, easy isolations of desired affinity reagents. However, removing these reagents from their display environments, the environment in which they were selected to function, often results in inactivated and non-functional product. Here, we overcome this limitation by, first, stabilizing selected yeast-displayed recombinant antibody (called yeast-scFv) via lyophilization and by, secondly, creating a novel class of affinity reagent, called nanoyeast-scFv. Nanoyeast-scFv are simply generated by crudely fragmenting whole yeast-scFv into cell wall fragments that still display functional antibody. These reagents demonstrated superior sensitivity (in pg/mL to ng/mL range) and specificity to their antigens when tested in various solid support assay formats. Entamoeba histolytica is a protozoan pathogen that is difficult to detect, due to the lack of a sensitive and affordable diagnostic test and to the presence of morphologically identical commensal amoeba in endemic areas. Current stool antigen detection tests target the trophozoite form, which is typically not plentiful or stable in stool. Our strategy was to validate a novel biomarker of the organism’s cyst stage. About 50 individual E. histolytica proteins were rationally selected for further investigation by filtering through recent cyst proteomic and transcriptomic data and identifying E. histolytica-specific sequences. As E. histolytica cysts cannot be obtained in vitro, these targets were recombinantly expressed and subjected to yeast-scFv selection, as well as traditional monoclonal antibody production. A monoclonal antibody to a known cyst wall protein, the Jacob2 lectin, was found to bind fixed, E. histolytica cysts from xenic culture specifically. This is one of very few times that a monoclonal antibody to E. histolytica cysts has been identified, as well as the first time a monoclonal antibody specific to such cysts in a fixative has been reported. Supplemental Files (Excel): Table 2S1: Abundant E. histolytica Biomarkers (LC-MS/MS-identified in ≥3 of 5 Stool Samples) Table 2S2: LC-MS/MS-identified E. histolytica Biomarkers With Upregulated or Unchanged mRNA Transcript in Encysting Cultures Versus Axenic Culture