Spatially-addressable immobilization of anti-ligands on surfaces
First Claim
1. A method for forming predefined regions on a surface of a solid support, the predefined regions capable of selectively immobilizing an anti-ligand, the method comprising:
- a) attaching to the surface a caged biotin analog having a low affinity for (i) the anti-ligand or (ii) a specific binding substance capable of binding an anti-ligand, said caged biotin analog comprising a photolabile chemical protecting group, which chemical protecting group is removable by irradiation to convert said caged biotin analog to a biotin analog capable of non-covalently immobilizing (i) the anti-ligand or (ii) the specific binding substance by an interaction having an affinity constant of 107 or stronger; and
b) selectively irradiating a predefined region of the surface to convert the caged biotin analogs in the predefined region to the biotin analog capable of non-covalently immobilizing (i) the anti-liqand or (ii) the specific binding substance.
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Accused Products
Abstract
Methods and compositions are described for immobilizing anti-ligands, such as antibodies or antigens, hormones or hormone receptors, oligonucleotides, and polysaccharides on surfaces of solid substrates for various uses. The methods provide surfaces covered with caged binding members which comprise protecting groups capable of being removed upon application of a suitable energy source. Spatially addressed irradiation of predefined regions on the surface permits immobilization of anti-ligands at the activated regions on the surface. Cycles of irradiation on different regions of the surface and immobilization of different anti-ligands allows formation of an immobilized matrix of anti-ligands at defined sites on the surface. The immobilized matrix of anti-ligands permits simultaneous screenings of a liquid sample for ligands having high affinities for certain anti-ligands of the matrix. A preferred embodiment of the invention involves attaching photoactivatable biotin derivatives to a surface. Photolytic activation of the biotin derivatives forms biotin analogs having strong binding affinity for avidin. Biotinylated anti-ligands can be immobilized on activated regions of the surface previously treated with avidin.
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Citations
16 Claims
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1. A method for forming predefined regions on a surface of a solid support, the predefined regions capable of selectively immobilizing an anti-ligand, the method comprising:
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a) attaching to the surface a caged biotin analog having a low affinity for (i) the anti-ligand or (ii) a specific binding substance capable of binding an anti-ligand, said caged biotin analog comprising a photolabile chemical protecting group, which chemical protecting group is removable by irradiation to convert said caged biotin analog to a biotin analog capable of non-covalently immobilizing (i) the anti-ligand or (ii) the specific binding substance by an interaction having an affinity constant of 107 or stronger; and b) selectively irradiating a predefined region of the surface to convert the caged biotin analogs in the predefined region to the biotin analog capable of non-covalently immobilizing (i) the anti-liqand or (ii) the specific binding substance. - View Dependent Claims (2, 3, 4)
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5. A method for attaching an anti-ligand to a surface comprising the steps of:
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a) linking to the surface caged biotin analogs having a relatively low affinity for avidin; b) irradiating caged biotin analogs on predefined regions of the surface to form biotin analogs having high binding affinities for avidin; c) reacting the biotin analogs with either an avidin analog and a biotinylated anti-ligand or an anti-ligand attached to an avidin analog. - View Dependent Claims (6, 7, 8, 9, 10, 11)
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12. A method for attaching a plurality of anti-ligands having different ligand binding specificities to predefined regions on a surface, said method comprising the steps of:
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a) attaching to the surface caged biotin analogs having a low affinity for the anti-ligand or specific binding substances, said caged biotin analogs comprising a photolabile chemical protecting group; b) irradiating the caged biotin analogs on a first predefined region of the surface, thereby forming biotin analogs on the first predefined region having an affinity constant of 107 or stronger for an anti-ligand or a specific binding substance; c) reacting the biotin analogs on the first predefined region with either (i)(a) the specific binding substance and a first anti-ligand or (b) a first anti-ligand attached to a specific binding substance, to form a biotin analog-specific binding substance-anti-ligand complex;
or (ii) a first anti-ligand to form a biotin analog-anti-ligand complex;d) washing the surface to remove unbound specific binding substance or anti-ligand; e) repeating steps (b-d) on a different region of the surface with a different anti-ligand. - View Dependent Claims (13)
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14. A method of screening a plurality of anti-ligands localized on predefined regions of a surface for affinity for ligands in solution comprising:
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a) attaching to the surface caged biotin analogs having a low affinity for the anti-ligand or specific binding substances, said caged biotin analogs comprising a photolabile chemical protecting group; b) irradiating the caged biotin analogs on a first predefined region of the surface, thereby forming biotin analogs on the first predefined region having an affinity constant of 107 or stronger for an anti-ligand or a specific binding substance; c) reacting the biotin analogs on the first predefined region with either (i)(a) the specific binding substance and a first anti-ligand or (b) a first anti-ligand attached to a specific binding substance, to form a biotin analog-specific binding substance-anti-ligand complex;
or (ii) a first anti-ligand to form a biotin analog-anti-ligand complex;d) washing the surface to remove unbound specific binding substance or anti-ligand; e) repeating steps (b-d) on a different region of the surface with a different anti-ligand to create a surface having a plurality of anti-ligands non-covalently bound on predefined regions of the surface; f) exposing the surface from step (e) to a solution containing at least one marked ligand; g) washing the surface free of unbound ligand; and h) identifying ligands binding to anti-ligands by determining regions on the surface where markers are located.
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15. A method of performing a competitive assay of a ligand in solution by measuring the ligand'"'"'s affinity for anti-ligands immobilized on predefined regions of a surface by the steps of:
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a) attaching to the surface caged biotin analogs having a low affinity for the anti-ligand or specific binding substances, said caged biotin analogs comprising a photolabile chemical protecting group; b) irradiating the caged biotin analogs on a first predefined region of the surface, thereby forming biotin analogs on the first predefined region having an affinity constant of 107 or stronger for an anti-ligand or a specific binding substance; c) reacting the biotin analogs on the first predefined region with either (i)(a) the specific binding substance and a first anti-ligand or (b) a first anti-ligand attached to a specific binding substance, to form a biotin analog-specific binding substance-anti-ligand complex;
or (ii) a first anti-ligand to form a biotin analog-anti-ligand complex;d) washing the surface to remove unbound specific binding substance or anti-ligand; e) repeating steps (b-d) on a different region of the surface with a different anti-ligand to create a surface having a plurality of anti-ligands non-covalently bound on predefined regions of the surface; f) incubating a solution containing at one or more marked ligands and unknown ligands with the surface of step (e); g) washing the surface free of unbound ligand; and h) measuring an amount of marked ligand remaining on the predefined regions of the surface. - View Dependent Claims (16)
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Specification