Antigen sandwich capture assays form the basis of many diagnostic tests for disease and experimental assays for studying disease processes. Since recombinant antibodies, especially single domain antibodies (sdAb) can offer advantages over conventional hybridoma methods for generating affinity reagents especially towards toxic antigens, there is increasing interest in tapping this rich resource. However, the screening of non-competitive pairs of sdAb and their subsequent incorporation into a final sandwich assay is still a time- consuming bottleneck, especially when hundreds of clones are to be screened. We propose to accelerate the pairing process and produce an immediately useful assay from impure antibody preparations. Importantly, the solution will fit in to any existing antibody or alternative scaffold repertoire by "retrofitting" and create a rapid pipeline directly from diverse repertoire to antigen capture assay. We hypothesize that site specific in vivo haptenylation of recombinant antibodies, in a manner that is compatible with existing display technologies will enable rapid screening of clones as both captor and tracer from crude E. coli osmotic shockates to directly formulate sandwich assays. We have three specific aims to demonstrate this: 1. We will show that a single site specific biotin modification of sdAb enables distinction of captor fro tracer via conditional occlusion by neutravidin using pre-existing sdAb specific for polyvalent Marburgvirus nucleoprotein (NP) and non- competitive pairs of anti-botulinum neurotoxin (BoNT) sdAb. 2. We will engineer a host strain of E. coli that enables both low level expression for effective sdAb phage display, and high level expression for effective soluble sdAb production to enable immediate pairing of clones at any stage in a phage panning process. 3. We will combine the approaches and apply them to existing immune and non-immune sdAb repertoires, to compare and contrast sdAb selected on two model antigens BoNT A and Ebola virus Zaire with those isolated previously using conventional methods. Rapidly responding to emerging threats with diagnostic immunoassays in part depends on our ability to quickly identify pairs of antibodies that function in the desired assay format. Screening for function without protein purification accelerates this discovery process and expands the number of clones that can be analyzed. Though the strategy is geared towards simplicity to operate stand-alone in containment environments, the methodology will be compatible with high throughput automation. Indeed, proof of principle data would improve the likelihood of generating high quality diagnostic assays for any antigen of interest including cancer markers and other proteins indicative of disease.
Mining recombinant antibody libraries for binders that function well together in an immunoassay is a critical component of developing diagnostics for infectious disease agents and assays for host biomarkers to study disease processes. While identifying antibodies that bind the antigen of interest is reasonably straightforward, combining them in a sandwich assay demands protein purification and in vitro modification, processes that rapidly escalate costs and time when pairing hundreds of potential clones. Our application seeks to eliminate the need for protein purification and modification, enabling fully functional sandwich assays to be created from small scale crude extracts of E. coli. By streamlining the immunoassay development process we allow laboratories to focus resources on the highest performing clones and accelerate the development of diagnostics and assays to help guard human welfare.