There is a recognized need for development of protein capture tools that will reliably detect a variety of targets, over a broad range of concentrations, for use with human plasma and other complex sample mixtures for diagnostic and therapeutic applications. The products from our PEARL [Pathogen-Encoded Adaptable Receptor Library] technology will yield antibody-like molecules that can be engineered to demonstrate 100-10,000 fold higher affinity for their ligand than conventional antibodies. This will allow these novel capture reagents to be seamlessly incorporated into existing highthru- put assay procedures and platforms that currently use antibodies as the capture reagent;however because of the enhanced affinity and slower off-rate of the PEARL products and the sensitivity of assays will be improved. This is particularly important when dealing with low concentrations of target ligands in complex mixtures with a large molar excess of irrelevant proteins, a current bottleneck for proteomic studies in most medical subspecialties. In this proposal we address the last limit to full exploitation of our platform technology. Flow cytometry was selected to solve the bottleneck and thus evaluate the key tenet [hypothesis] of PEARL: When a sufficiently large pathogen library is screened, microorganisms will be found that bear unique antibody-like surface proteins that display high affinity toward one of hundreds of plasma/serum components found in either normal or disease-state subjects. We propose to evaluate this hypothesis using a subset of the PEARL collection, as bar coded bacteria, and immunoglobins [IgG1, IgG3, IgM, IgA] selected because of their medical and commercial significance as the target ligands. A double blind experiment will test this specific aim: Develop and validate flow-cytometry for evaluating members of a unique collection of bacterial pathogens known to bear surface proteins capable of specific [antibody-like] binding to many uniquely different physiologically significant plasma components. The key steps in that validation form the basis of the work scope of this proposal. Task #1 Develop and validate a multiparameter flow cytometric screening method to detect bacteria with binding potential for any targeted ligand. This will allow an initial screen to be completed in hours, not months. Task #2 Validate method(s) to distinguish between binding activities due to different gene products from those associated with multi functional binding of a single gene product. This will distinguish between potential starting candidates and identify the one(s) most closely resembling product specifications provided by the customers. Task #3 - Demonstrate that positive hits identified by flow cytometry are also observed using more traditional modes of analysis via ligand blot.
Virtually all clinical analyses are limited by one-or-more of these features: (a) insufficient sample for all needed tests;(b) insufficient sensitivity for use with limiting samples;and/or (c) difficulties in standardization across vendors and laboratories. These limits are an unavoidable downside of the most popular approach to assay design;i.e., use of antibodies which are limited by the affinity for antigen. The proposed PEARL approach retains the advantages of the current method while addressing virtually all of its problems. When successfully implemented, an enhanced method for assessing both new clinical paradigms [such as use of biotherapeutics] as well as improving old analyses [suboptimal diagnostic immunoassays] will be attained.
