A method of solid phase enzyme immunoassay and nucleic acid hybridization assay and dip-stick design and stabilized chromogenic substrate

Claim 2

A solid phase enzyme immunoassay wherein the presence of an analyte in a liquid sample is determined by contacting (i) a solid surface having attached thereto an immunoreactant specific for said analyte with (ii) said liquid sample and with (iii) an enzyme labeled material selected from the group consisting of material which selectively binds with said immunoreactant and material which selectively binds with said analyte, said presence of said analyte in said sample correlating to the amount of said enzyme labeled material bound to said solid surface resulting from said contacting; which comprises: (a) contacting said solid surface, having bound thereto said enzyme labeled material, with a liquid reagent containing a chromogenic material, said chromogenic material being capable of changing from a first color state to a second color state in correlation to the amount of said enzyme labeled material bound to said solid surface, and said chromogenic material being capable, when in said second color state, of substantially binding to said solid surface; and (b) analyzing the solid surface for the presence of the second color state.

Claim 3

A solid phase nucleic acid hybridization assay wherein the presence of single-stranded analyte nucleic acid in a liquid sample is determined by contacting (i) a solid surface-having attached thereto a single-stranded first fragment of nucleic acid having a nucleotide sequence of at least 15 base pairs and being capable of hybridizing with said analyte nucleic acid with (ii) said liquid sample and with (iii) an enzyme labeled single-stranded second fragment of nucleic having a nucleotide sequence of at least 15 base pairs, said second fragment of nucleic acid being selected from the group consisting of second fragment of nucleic acid which selectively hybridizes with said analyte nucleic acid and second fragment of nucleic acid which selectively hybridizes with said first fragment of nucleic acid, said presence of analyte nucleic acid in said liquid sample correlating to the amount of enzyme labeled second fragment of nucleic acid bound to said solid surface as a result of said contacting; which comprises: (a) contacting said solid surface, having bound thereto said enzyme labeled material, with a liquid reagent containing a chromogenic material, said chromogenic material being capable of changing from a first color state to a second color state in correlation to the amount of said enzyme labeled material bound to said solid surface, and said chromogenic material being capable, when in said second color state, of substantially binding to said solid surface; and b) analyzing the solid surface for the presence of the second color state.

Claim 4

A solid phase nucleic acid hybridization assay wherein the presence of single-stranded analyte nucleic acid in a liquid sample is determined by contacting (i) a solid surface-having attached thereto a single-stranded first fragment of nucleic acid having a nucleotide sequence of at least 15 base pairs and being capable of hybridizing with said analyte nucleic acid with (ii) said liquid sample and with (iii) an enzyme labeled single-stranded second fragment of nucleic having a nucleotide sequence of at least 15 base pairs, said second fragment of nucleic acid being selected from the group consisting of second fragment of nucleic acid which selectively hybridizes with said analyte nucleic acid and second fragment of nucleic acid which selectively hybridizes with said first fragment of nucleic acid, said presence of analyte nucleic acid in said liquid sample correlating to the amount of enzyme labeled second fragment of nucleic acid bound to said solid surface as a result of said contacting; which comprises: (a) contacting said solid surface, having bound thereto said enzyme labeled material, with a liquid reagent containing a chromogenic material, said chromogenic material being capable of changing from a first color state to a second color state in correlation to the amount of said enzyme labeled material bound to said solid surface, and said chromogenic material being capable, when in said second color state, of substantially binding to said solid surface; and b) analyzing the solid surface for the presence of the second color state.

Claim 5

The solid phase assay of claim 1 or 2, wherein the solid surface is separated from said liquid reagent subsequent to the contacting of step a) and prior to the analyzing of step b).

Claim 6

The solid phase assay of any of the claims 1 to 3, wherein said solid surface comprises a membrane.

Claim 7

The solid phase assay of claim 4, wherein said membrane comprises a material selected from the group consisting of cellulose, carboxymethylated cellulose, DEAE cellulose, cellulose phosphate, cellulose sulfate, nitrocellulose, carboxymethylated nitrocellulose, DEAE nitrocellulose, nitrocellulose phosphate, nitrocellulose sulfate, cellulose acetate/cellulose nitrate and a polyamide membrane.

Claim 8

The solid phase assay of any of the claims 1 to 3, wherein said solid surface is selected from the group consisting of a membrane contained in a dip-stick, a membrane contained in a microtiter well, and a treated or coated surface of a test tube.

Claim 9

The solid phase assay of any of the claims 1 to 3, wherein said solid surface comprises a plurality of water insoluble particles.

Claim 10

The solid phase assay pf any of the claims 1 to 7, wherein the enzyme is selected from the group consisting of horseradish peroxidase, alkaline phosphatase and beta-galactosidase.

Claim 11

The solid phase assay of any of the claims 1 to 8, wherein the liquid reagent further contains a redox reagent which reacts with said enzyme to cause said chromogenic material to change from said first color state to said second color state.

Claim 12

The solid phase assay of claim 9, wherein the redox reagent comprises hydrogen peroxide.

Claim 13

The solid phase assay of any of the claims 1 to 10, wherein the chromogenic material contains a benzidine moiety.

Claim 14

The solid phase assay of claim 11, wherein the chromogenic material containing a benzidine moiety is selected from the group consisting of 3,3'-dichlorobenzidine, 3,3',5,5'-tetramethylbenzidine, dianisidine, ortho-toluidine, 3,3'-diaminobenzidine, benzidine, 3-amino-9-ethylcarbozole and 4-chloro-l-naphthol.

Claim 15

An article of manufacture comprising: a porous membrane comprising a material selected from the group consisting of cellulose, carboxymethylated cellulose, DEAE cellulose, cellulose phosphate, cellulose sulfate, nitrocellulose, carboxymethylated nitrocellulose, DEAE nitrocellulose, nitrocellulose phosphate, nitrocellulose sulfate, cellulose acetate/cellulose nitrate and polyamide membrane, said membrane having opposed and parallel first and second planar surfaces, the distance between said first and second surfaces being no greater than 0.5 millimeters; said first surface having a first reagent access zone and a first binding zone which are substantially contiguous, said second surface having a second reagent access zone substantially opposed to said first reagent access zone; and carrier means fixedly coupled to said membrane at said first binding zone for rigidly supporting said first and second surfaces such that said first and second surfaces remain opposed, parallel and planar when immersed in a liquid or otherwise wetted.

Claim 16

An article of manufacture comprising: a porous membrane comprising a material selected from the group consisting of cellulose, carboxymethylated cellulose, DEAE cellulose, cellulose phosphate, cellulose sulfate, nitrocellulose, carboxymethylated nitrocellulose, DEAE nitrocellulose, nitrocellulose phosphate, nitrocellulose sulfate, cellulose acetate/cellulose nitrate and polyamide membrane, said membrane having opposed and parallel first and second planar surfaces, the distance between said first and second surfaces being no greater than 0.5 millimeters; said first surface having a first reagent access zone and a first binding zone which are substantially contiguous, said second surface having a second reagent access zone substantially opposed to said first reagent access zone; and carrier means fixedly coupled to said membrane at said first binding zone for rigidly supporting said first and second surfaces such that said first and second surfaces remain opposed, parallel and planar when immersed in a liquid or otherwise wetted.

Claim 17

The article of manufacture of claim 13 ,wherein said second surface further has a second binding zone which is contiguous to said second reagent access zone and which is substantially opposed to said first binding zone, and said carrier is,further.fixedly coupled to said membrane at said second binding zone.

Claim 18

15 . The article of manufacture of claim 13 or 14, wherein the carrier comprises opposed and parallel first and second leaflets, said membrane being sandwiched between said first and second leaflets with said first binding zone being coupled to said first leaflet and said second binding zone being coupled to said second leaflet.

Claim 19

16 The article of manufacture of claim 15, wherein said first and second leaflets have first and second apertures respectively, said first and second apertures being opposed respectively to said first and second reagent access zones.

Claim 20

The article of manufacture of claim 16, wherein said first and second apertures are respectively completely i circumscribed by said first and second leaflets.

Claim 21

18 . The article of manufacture of any of the claims 13 to 17, wherein a portion of said membrane has attached an immunoreactant useful in an enzyme immunoassay or a single-stranded fragment of nucleic acid of at least 15 base pairs useful i in an enzyme nucleic acid hybridization assay.

Claim 25

The method of claim 19, wherein the complementary redox agent is formic acid.

Claim 27

The method of claim 22, wherein the solubilizing agent is selected from the group consisting of dimethylsulfoxide, dimethylformamide, methanol and ethanol.

Claim 28

The method of claim 22 or 23, wherein the chromogenic material is 3,3',5,5'- tetramethylbenzidine and the complementary redox agent is formic acid.

Claim 29

As a composition of matter, a solution comprising: a chromogenic material containing a benzidine moiety, said chromogenic material being capable of changing from a first color state to a secqnd color state in correlation to an enzyme's reaction with a redox reagent; and a complementary redox agent in an amount (i) effective to retain the chromogenic material in its first color state during normal handling and storage conditions by inhibiting redox reaction causing said chromogenic material during said normal handling and storage conditions to change to said second color state; and (ii) effective to allow the chromogenic material to change from its first color state to the second color state as a result of redox reactions normally present for test positives in an enzyme immunoassay or an enzyme nucleic acid hybridization assay.

Claim 30

As a composition of matter, a solution comprising: a chromogenic material containing a benzidine moiety, said chromogenic material being capable of changing from a first color state to a secqnd color state in correlation to an enzyme's reaction with a redox reagent; and a complementary redox agent in an amount (i) effective to retain the chromogenic material in its first color state during normal handling and storage conditions by inhibiting redox reaction causing said chromogenic material during said normal handling and storage conditions to change to said second color state; and (ii) effective to allow the chromogenic material to change from its first color state to the second color state as a result of redox reactions normally present for test positives in an enzyme immunoassay or an enzyme nucleic acid hybridization assay.

Claim 31

The composition of matter of claim 25, wherein the chromogenic material containing a benzidine moiety is selected from the group consisting of 3 ,3'-dichlorobenzidine, 3,3',5,5'-tetramethylbenzidine, dianisidine, ortho-toluidine, 3,3'-diaminobenzidine, benzidine, 3-amino-9-ethylcarbazole and 4-chloro-l-naphthol.

Claim 32

The composition of matter of claim 25 or 26, wherein the complementary redox agent is formic acid.

Claim 33

The composition of matter of any of the claims 25 to 27, wherein the chromogenic material and the complementary redox reagent are solubilized by the presence of a solubilizing agent.

Claim 34

The composition of.matter of claim 28, wherein the solubilizing agent is selected from the group consisting of dimethylsulfoxide, dimethylformamide, methanol and ethanol.

Claim 35

The composition of matter of claim 29, wherein chromogenic material is 3,3',5,5'- tetramethylbenzidine and the complementary redox reagent is formic acid.