US5482867A — Spatially-addressable immobilization of anti-liga…

Claim 1

Independent Claim 142 words

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.

Claim 2

Independent Claim 142 words

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.

Claim 3

Dependent Claim 306 words

The method of claim 1 wherein the caged biotin analog comprises the formula: ##STR32## wherein X and Z are selected from the group consisting of a) hydrogen, b) oxycarbonyls of lower alkyl aryl and benzyl groups, c) a nitroveratryloxycarbonyl group, d) a nitropiperonyloxycarbonyl group where Z is hydrogen and R is p-nitrophenol formate, e) ##STR33## wherein R 1 and R 2 are hydrogen, lower alkyl, aryl, benzyl, halogen, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, sulfonate, formamido or phosphido groups, f) ##STR34## wherein R 1 and R 2 are hydrogen, lower alkyl, aryl, benzyl, pyrenyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino nitro, carboxyl, formate, formamido, or phosphido groups and R 3 and R 4 are hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido or phosphido groups; g) ##STR35## wherein R 1 and R 2 are hydrogen, lower alkyl, aryl benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido or phosphido groups, and h) ##STR36## wherein R 1 , R 2 , R 3 and R 4 are hydrogen, lower alky, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido or phosphido groups; provided that when X is hydrogen, Z is not hydrogen or methyloxycarbonyl and provided that when Z is hydrogen, X is not hydrogen or methyloxycarbonyl; R is selected from the group consisting of hydrogen, lower alkyl, aryl, lower alkyl formate, aryl formate, methyl formate, p-nitrophenol formate, formamide, N-alkylformamide, N-succinimidyl, hydroxyl, alkoxyl, thiol, thioether, disulfide, hydrazide and amine groups and provided that when X or Z is methyloxycarbonyl, R is not methyl formate; U is O, S or NH; Y is selected from the group consisting of sulfur, oxygen, methylene, carbonyl, sulfinyl and sulfonyl groups, or Y represents two hydrogen atoms attached to the respective carbons; n=0-7; or acid salts of the compound.

Claim 4

Dependent Claim 19 words

A method as in claim 1 wherein the binding member is attached to the surface by a crosslinking group.

Claim 5

Dependent Claim 13 words

A method as in claim 1 wherein the surface is silica or glass.

Claim 6

Independent Claim 71 words

A method for attaching an anti-ligand to a surface comprising the steps of: 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.

Claim 7

Independent Claim 71 words

A method for attaching an anti-ligand to a surface comprising the steps of: 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.

Claim 8

Dependent Claim 153 words

The method of claim 5 wherein the caged biotin analog has the formula ##STR37## wherein X is selected from the group consisting of a) oxycarbonyls of lower alkyl, aryl and benzyl groups, provided that X is not methyl oxycarbonyl; b) a nitroveratryloxycarbonyl group, c) a nitropiperonyloxycarbonyl group, and R is p-nitrophenol d) ##STR38## wherein R 1 and R 2 are hydrogen, lower alkyl, aryl, benzyl, halogen, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, sulfonate, formamido or phosphido groups, e) ##STR39## wherein R 1 and R 2 are hydrogen, lower alkyl, aryl, benzyl, pyrenyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido, or phosphido groups and R 3 and R 4 are hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido or phosphido groups; R is selected from the group consisting of hydrogen, lower alkyl, aryl, benzyl and N-succinimidyl; or acid addition salts of the compound.

Claim 9

Dependent Claim 27 words

A method as in claim 6 wherein the biotin analog has the formula: ##STR40## wherein W is a surface or a crosslinking group attached to a surface.

Claim 10

Dependent Claim 26 words

The method of claim 5, wherein the biotin analog and avidin analog have an affinity constant (K a ) of about 10 15 M -1 .

Claim 11

Dependent Claim 19 words

The method of claim 5 wherein the anti-ligand is attached to the avidin analog by a bifunctional crosslinking group.

Claim 12

Dependent Claim 13 words

A method as in claim 5 wherein the anti-ligand is a monoclonal antibody.

Claim 13

Dependent Claim 13 words

A method as in claim 5 wherein the surface is glass or silica.

Claim 14

Independent Claim 172 words

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: 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 10 7 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.

Claim 15

Independent Claim 172 words

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: 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 10 7 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.

Claim 16

Dependent Claim 11 words

The method of claim 12 wherein the anti-ligand is an immunoglobulin.

Claim 17

Independent Claim 227 words

A method of screening a plurality of anti-ligands localized on predefined regions of a surface for affinity for ligands in solution comprising: 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 10 7 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.

Claim 18

Independent Claim 227 words

A method of screening a plurality of anti-ligands localized on predefined regions of a surface for affinity for ligands in solution comprising: 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 10 7 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.

Claim 19

Independent Claim 238 words

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: 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 10 7 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.

Claim 20

Independent Claim 238 words

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: 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 10 7 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.

Claim 21

Dependent Claim 32 words

The method of claim 15, wherein step (f) comprises first binding unknown ligands to anti-ligands on the surface and then incubating a solution containing one or more marked ligands with the surface.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1999-01-08	1999-07-10	2000-01-09	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2003-01-08	2003-07-10	2004-01-09	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2007-01-08	2007-07-10	2008-01-09	$7700.00	Window Closed / Lapsed

Spatially-addressable immobilization of anti-ligands on surfaces

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