High throughput screening of libraries
First Claim
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1. A method for screening for a ligand binding protein of interest, comprising, a) encapsulating one or more members of a population of cells suspected of expressing a ligand binding protein of interest in a capsule comprising permeable walls, said walls containing a first capture reagent for said ligand binding protein of interest;
- b) incubating said encapsulated cells under conditions that allow for expression of said ligand binding protein of interest and capture of said ligand binding protein by said first capture reagent;
c) contacting said permeable capsule with a ligand specific for said captured ligand binding protein, to form a captured protein-ligand complex, wherein said ligand can be the same as or different from said capture reagent;
d) contacting said captured protein-ligand complex with a first detection molecule that binds said protein-ligand complex to form a protein-ligand-first detection molecule complex;
e) contacting said protein-ligand-first detection molecule complex with a second detection molecule that binds said first detection molecule to form a protein-ligand-first detection molecule-second detection molecule complex;
f) contacting said protein-ligand-first detection molecule-second detection molecule complex with a third detection molecule comprising a detectable label that binds to said protein-ligand-first detection molecule-second detection molecule complex to form a protein-ligand-first detection molecule-second detection molecule-third detection molecule complex; and
g) detecting the presence of said detectable label bound to said capsule, thereby identifying cells in said capsule expressing said ligand binding protein of interest.
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Abstract
The invention provides methods for screening libraries of cells for the production of ligand binding proteins. In one embodiment, libraries producing Fab antibody fragments are screened.
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Citations
40 Claims
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1. A method for screening for a ligand binding protein of interest, comprising,
a) encapsulating one or more members of a population of cells suspected of expressing a ligand binding protein of interest in a capsule comprising permeable walls, said walls containing a first capture reagent for said ligand binding protein of interest; -
b) incubating said encapsulated cells under conditions that allow for expression of said ligand binding protein of interest and capture of said ligand binding protein by said first capture reagent;
c) contacting said permeable capsule with a ligand specific for said captured ligand binding protein, to form a captured protein-ligand complex, wherein said ligand can be the same as or different from said capture reagent;
d) contacting said captured protein-ligand complex with a first detection molecule that binds said protein-ligand complex to form a protein-ligand-first detection molecule complex;
e) contacting said protein-ligand-first detection molecule complex with a second detection molecule that binds said first detection molecule to form a protein-ligand-first detection molecule-second detection molecule complex;
f) contacting said protein-ligand-first detection molecule-second detection molecule complex with a third detection molecule comprising a detectable label that binds to said protein-ligand-first detection molecule-second detection molecule complex to form a protein-ligand-first detection molecule-second detection molecule-third detection molecule complex; and
g) detecting the presence of said detectable label bound to said capsule, thereby identifying cells in said capsule expressing said ligand binding protein of interest. - 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)
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30. A method for screening for an Fab antibody fragment of interest, comprising,
a) encapsulating one or more members of a population of cells suspected of expressing an Fab fragment of interest in a capsule comprising permeable walls, said walls containing a first capture reagent for said Fab antibody fragment of interest, wherein said capture reagent does not prevent said Fab fragment from interacting with its antigen; -
b) incubating said encapsulated cells under conditions that allow for expression of said Fab fragment of interest and capture of said Fab fragment by said first capture reagent;
c) contacting said permeable capsule with a digoxigenin labeled antigen specific for said captured Fab fragment, to form a captured Fab-antigen complex;
d) contacting said captured Fab-antigen complex with an anti-digoxigenin IgG that binds said Fab-antigen complex to form an Fab-antigen-anti-digoxigenin IgG complex;
e) contacting the complex of d) with an digoxigenin labeled anti-IgG antibody to form an Fab-antigen-anti digoxigenin IgG-anti IgG antibody complex;
f) contacting the complex of e) with an anti digoxigenin antibody comprising a detectable label that specifically binds to said Fab-antigen-anti digoxigenin IgG-anti IgG antibody complex to form an Fab-antigen-anti digoxigenin IgG-anti IgG antibody-labeled anti digoxigenin antibody complex; and
g) detecting the presence of said detectable label bound to said capsule, thereby identifying cells expressing the Fab fragment of interest. h) isolating cells identified in g), placing cells from different capsules in different locations on a first permeable solid substrate, and growing said cells under conditions that allow expression of the Fab fragment of interest;
i) contacting said first solid substrate with a second permeable solid substrate for a time sufficient to allow said Fab fragment to diffuse from said first substrate to said second substrate, said second solid substrate comprising an anti Fab antibody that binds the Fab fragment of interest, wherein binding of said anti Fab antibody does not prevent the Fab fragment from interacting with its antigen;
j) contacting the second solid substrate with an antigen for the Fab fragment, said antigen comprising a detectable marker;
k) detecting the presence and location of said detectable marker on said second substrate; and
l) identifying the cells on said first substrate expressing Fab fragmentof interest using the location of said detectable marker on the second substrate;
m) repeating a) through
1) at least once; and
n) isolating the cells identified in the last repetition of
1);
growing said cells under conditions that allow for expression of the Fab fragment of interest; and
determining the expression of the Fab fragment of interest by an enzyme-linked immunosorbent assay (ELISA). - View Dependent Claims (31, 32, 33, 34, 35)
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36. A method for screening for a ligand binding protein of interest, comprising,
a) encapsulating one or more members of a population of cells suspected of expressing a ligand binding protein of interest in a capsule comprising permeable walls, said walls containing a first capture reagent for said ligand binding protein of interest; -
b) incubating said encapsulated cells under conditions that allow for expression of said ligand binding protein of interest and capture of said ligand binding protein by said first capture reagent;
c) contacting said permeable capsule with a ligand specific for said captured ligand binding protein, to form a captured protein-ligand complex, wherein said ligand can be the same as or different from said capture reagent;
d) contacting said captured protein-ligand complex with a first detection molecule that binds said protein-ligand complex to form a protein-ligand-first detection molecule complex said first detection molecule comprising an oligonucleotide;
e) contacting said oligonucleotide with a circular polynucleotide that hybridizes to said oligonucleotide;
f) extending said oligonucleotide by rolling circle amplification wherein said circular polynucleotide serves as a template to produce a linear concatemer. g) detecting the presence of said linear concatemer bound to said capsule, thereby identifying cells in said capsule expressing said ligand binding protein of interest. - View Dependent Claims (37, 38, 39, 40)
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Specification