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Abstract

A process is described for producing surrogate antibody molecules that mimic the structure, stability, and binding characteristics of a natural antibody. Surrogate antibody structure, composition of surrogate antibody libraries, methods of surrogate antibody preparation, and surrogate antibody applications are disclosed. Also disclosed are methods of surrogate antibody structural stabilization and resistance to nucleases. The surrogate antibodies comprise a specificity strand and a stabilization strand. The specificity strand comprises a nucleic acid sequence having a specificity region flanked by a first constant region and a second constant region. The stabilization strand comprises a first stabilization region that interacts with the first constant region and a second stabilization region that interacts with the second constant region. In further embodiments, the stabilization strand and the specificity strand comprise distinct molecules. In other embodiments, the surrogate antibody molecules may comprise polyoligonucleotides that have at least one nucleotide sequence that forms a loop with specific ligand-binding properties. Surrogate antibody libraries containing a large population of random binding molecules are pre-assembled and used in a process that captures and amplifies those molecules having prerequisite binding characteristics. The amplified surrogate antibody molecule produced by the process has identical structure and binding characteristics to the parent molecule captured from the initially assembled library. Surrogate antibody molecules contain binding loop(s) that are formed and stabilized by the hybridization of at least two adjacent and juxtaposed strands, one strand having a greater number of nucleotides than the other. The preparation of a polyclonal surrogate antibody reagent proceeds through phases of capture/enrichment and amplification, specificity enhancement, and affinity enhancement. Depending upon the intended application, polyclonal surrogate antibody reagents can be processed to monoclonality. These molecules expand upon the binding characteristics of natural immunoglobulins, and do not require animals, animal facilities, cell culture or the stimulation of an immune response, in their development. They can be used as an effective replacement for natural antibody molecules, and therefore can be used in testing methods like immunoassay, as therapeutic agents, for specific labeling, and for research purposes. Targets ligands compatible with the development of surrogate antibodies include compounds, organisms, and cells that when complexed to a surrogate antibody in solution attain characteristics that can be physically or chemically differentiated from uncomplexed surrogate antibody.

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First Claim