Surrogate antibodies and methods of preparation and use thereof
First Claim
1. An isolated molecule comprising a specificity strand and a stabilization strand, said specificity strand comprising a nucleic acid sequence having a specificity region flanked by a first constant region and a second constant region;
- said stabilization strand comprises a first stabilization domain that interacts with said first constant region and a second stabilization domain that interacts with said second constant region; and
, said stabilization strand and said specificity strand comprise distinct molecules.
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Accused Products
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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Citations
47 Claims
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1. An isolated molecule comprising a specificity strand and a stabilization strand,
said specificity strand comprising a nucleic acid sequence having a specificity region flanked by a first constant region and a second constant region; -
said stabilization strand comprises a first stabilization domain that interacts with said first constant region and a second stabilization domain that interacts with said second constant region; and
,said stabilization strand and said specificity strand comprise distinct molecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A library of isolated molecules comprising:
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a population of molecules comprising a specificity strand and a stabilization strand, said specificity strand comprising a nucleic acid sequence having a specificity region flanked by a first constant region and a second constant region; and
, said stabilization strand comprises a first stabilization domain that interacts with said first constant region and a second stabilization domain that interacts with said second constant region; and
,wherein each of the first constant region of said specificity strands in said population are identical;
each of the second constant region of said specificity strands in said population are identical; and
, each of the specificity region of said specificity strands in said population are randomized; and
,wherein each of the stabilization strands in said population are identical. - View Dependent Claims (34)
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35. A library of isolated molecules produced by
a) providing a population of specificity strands wherein i) each of said specificity strand in said population comprises a nucleic acid sequence having a specificity region flanked by a first constant region and a second constant region; -
ii) each of the first constant region of said specificity strands in said population are identical;
iii) each of the second constant region of said specificity strands in said population are identical; and
,iv) each of the specificity region of said specificity strands in said population are randomized; and
,b) contacting said population of specificity strands with a stabilization strand;
wherein said stabilization strand comprises a first stabilization domain that interacts with said first constant region and a second stabilization domain that interacts with said second constant region; and
,said contacting occurs under conditions that allow for said first stabilization domain to interact with said first constant region and said second stabilization domain to interacts with said second constant region. - View Dependent Claims (36)
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37. A method for generating a surrogate antibody library comprising:
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a) providing a population of specificity strands wherein i) each of said specificity strand in said population comprises a nucleic acid sequence having a specificity region flanked by a first constant region and a second constant region;
ii) each of the first constant region of said specificity strands in said population are identical;
iii) each of the second constant region of said specificity strands in said population are identical; and
,iv) each of the specificity regions of said specificity strands in said population are randomized; and
,b) contacting said population of specificity strands with a stabilization strand;
wherein said stabilization strand comprises a first stabilization domain that interacts with said first constant region and a second stabilization domain that interacts with said second constant region; and
,said contacting occurs under conditions that allow for said first stabilization domain to interact with said first constant region and said second stabilization domain to interacts with said second constant region. - View Dependent Claims (38)
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39. A method for capturing a surrogate antibody comprising:
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a) contacting a ligand with a population of surrogate antibody molecules under conditions that permit formation of a population of ligand-bound surrogate antibody complexes, wherein each of the surrogate antibody molecules of the surrogate antibody population comprises a specificity strand and a stabilization strand, said specificity strand comprising a nucleic acid sequence having a specificity region flanked by a first constant region and a second constant region;
said stabilization strand comprises a first stabilization domain that interacts with said first constant region and a second stabilization domain that interacts with said second constant region;
b) partitioning said ligand and said population of surrogate antibody molecules from said population of ligand-bound surrogate antibody complexes; and
,c) amplifying the specificity strand of said population of ligand-bound surrogate antibody complexes. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46)
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47. A method of detecting a ligand comprising
a) contacting said ligand with a surrogate antibody molecule under conditions that permit formation of a population of ligand-bound surrogate antibody complexes, wherein said surrogate antibody molecule comprises a specificity strand and a stabilization strand, said specificity strand comprising a nucleic acid sequence having a variable region flanked by a first constant region and a second constant region; -
said stabilization strand comprises a first stabilization domain that interacts with said first constant region and a second stabilization domain that interacts with said second constant region; and
,b) detecting said ligand.
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Specification