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US5599668A
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Light scattering optical waveguide method for detecting specific binding events
Assignee: Abbott Laboratories
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Filing: 1994-09-22
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Granted: 1997-02-04
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Expires: 2014-09-22
Abstract
Abstract A waveguide binding assay method involves detecting the scattering of light directed into the waveguide, the scattering being the result of scattering labels specifically bound to the waveguide within the penetration depth of an evanescent wave. The waveguide may be transparent plastic or glass and the binding is typically by oligonucleotide hybridization or immunological capture. Light scattering labels include colloidal metals or non-metals, including gold, selenium and latex. A light absorbing member consisting of dye or concentrated particles may also be employed to enhance signal. Real-time binding and dissociation can be monitored visually or by video imaging, such as with a CCD camera and frame grabber software. Hybridization mismatches of as few as one base can be distinguished by real-time melting curves.
Inventors
- 👤 Donald I. Stimpson
- 👤 Julian Gordon
- 👤 Joanell V. Hoijer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-08 12:05
CPC Classifications
G; G01; G01N; G01N21/00; G01N21/17; G01N21/47; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6825; C12Q1/6834; C12Q1/6837; G01N21/55; G01N21/552; G01N21/75; G01N21/77; G01N21/7703; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/54366; G01N33/54373; B; B01; B01J; B01J2219/00; B01J2219/00274; B01J2219/00583; B01J2219/00603; B01J2219/00646; B01J2219/00648; B01J2219/00659; B01J2219/0068; B01J2219/00702; B01J2219/00707
Claims Analysis
Total Claims: 85
Claim 1
Independent Claim
311 words
A method for detecting the presence or amount of one or more specific binding analytes in a fluid sample, the method comprising: (a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample; (ii) a light receiving edge; and (iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized at a plurality of sites on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon; wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds at least one analyte; (b) contacting the reactive surface with a sample suspected to contain said one or more analytes and with a light scattering label attached to a specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said one or more analytes, in the case of a sandwich assay, or the immobilized first specific binding member of said first cognate binding pair, in the case of a competitive assay; thereby forming light scattering label complexes attached to the plurality of sites in proportion to the amount of analyte in the sample; (c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby simultaneously illuminating the entire reactive surface; (d) collecting visually detectable light scattered by said light scattering label; (e) comparing the degree of light scattering at each situs with either (i) the degree of light scattering at a non-situs portion, or (ii) the degree of light scattering at another situs, or both, whereby light scattering at each situs correlates to the presence or amount of the analyte for which the immobilized specific binding member at that situs is specific.
Claim 2
Independent Claim
311 words
A method for detecting the presence or amount of one or more specific binding analytes in a fluid sample, the method comprising: (a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample; (ii) a light receiving edge; and (iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized at a plurality of sites on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon; wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds at least one analyte; (b) contacting the reactive surface with a sample suspected to contain said one or more analytes and with a light scattering label attached to a specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said one or more analytes, in the case of a sandwich assay, or the immobilized first specific binding member of said first cognate binding pair, in the case of a competitive assay; thereby forming light scattering label complexes attached to the plurality of sites in proportion to the amount of analyte in the sample; (c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby simultaneously illuminating the entire reactive surface; (d) collecting visually detectable light scattered by said light scattering label; (e) comparing the degree of light scattering at each situs with either (i) the degree of light scattering at a non-situs portion, or (ii) the degree of light scattering at another situs, or both, whereby light scattering at each situs correlates to the presence or amount of the analyte for which the immobilized specific binding member at that situs is specific.
Claim 3
Dependent Claim
51 words
The method of claim 1 wherein said analyte is a sequence of nucleic acid and the immobilized first specific binding member of said first cognate binding pair is an oligonucleotide complementary to said sequence or complementary to one or more cognate binding oligonucleotides, one of which is complementary to said sequence.
Claim 4
Dependent Claim
32 words
The method of claim 2 wherein said analyte nucleic acid sequence includes a hapten reporter and the specific binding member attached to said light scattering label is specific for said hapten reporter.
Claim 5
Dependent Claim
27 words
The method of claim 1 wherein said analyte is an antigen and the immobilized first specific binding member of said first cognate binding pair is an antibody.
Claim 6
Dependent Claim
43 words
The method of claim 1 wherein said element contains multiple sites each containing different concentrations of the same immobilized first specific binding member, further comprising comparing the degree of light scattering at one situs with the degree of light scattering at another situs.
Claim 7
Dependent Claim
20 words
The method of claim 5 wherein said element contains multiple sites each containing a distinct immobilized first specific binding member.
Claim 8
Dependent Claim
18 words
The method of claim 1 wherein said element contains multiple sites each containing a distinct specific binding member.
Claim 9
Dependent Claim
36 words
The method of claim 1 wherein said method further comprises a second transparent element connected to said first element to form a two-dimensional capillary channel therebetween, such that the reactive surface is formed in the channel.
Claim 10
Dependent Claim
25 words
The method of claim 1 wherein said light scattering label is a particle selected from the group consisting of colloidal gold, colloidal selenium and latex.
Claim 11
Dependent Claim
19 words
The method of claim 1 wherein light is visually collected from a plurality of sites simultaneously by an observer.
Claim 12
Dependent Claim
33 words
The method of claim 1 wherein the collection of scattered light is accomplished by an array of photodetector devices selected from the group consisting of photodiodes, charge coupled devices, phototransistors, photoresistors and photomultipliers.
Claim 13
Dependent Claim
17 words
The method of claim 11 wherein the collection of scattered light is accomplished by a CCD camera.
Claim 14
Dependent Claim
50 words
The method of claim 1 wherein said light scattering label is attached to said first specific binding member of a second cognate binding pair via an intermediate cognate binding pair selected from the group consisting of: hapten and anti-hapten antibody; biotin and avidin or streptavidin; and complementary nucleic acid sequences.
Claim 15
Dependent Claim
48 words
The method of claim 1 comprising a further step, after step (e), of altering the conditions at the reactive surface of the waveguide device to initiate dissociation of the analyte from the immobilized first specific binding member, and repeating steps (c), (d) and (e) at the altered condition.
Claim 16
Dependent Claim
25 words
The method of claim 14 wherein the analyte is nucleic acid and said altering of conditions comprises increasing the stringency conditions at the reactive surface.
Claim 17
Dependent Claim
16 words
The method of claim 15 wherein stringency conditions are increased incrementally by stepwise increases in temperature.
Claim 18
Dependent Claim
26 words
The method of claim 15 wherein stringency conditions are increased incrementally by stepwise addition of a dilution agent that decreases the ionic strength of the solution.
Claim 19
Dependent Claim
34 words
The method of claim 14 wherein said altering of conditions is repeated in several increments and the repeating of steps (c), (d) and (e) is done without an intermediate washing away of dissociated analyte.
Claim 20
Dependent Claim
21 words
The method of claim 8 wherein said waveguide device is provided with a reaction surface having a coating of metasoluble protein.
Claim 21
Dependent Claim
21 words
The method of claim 1 wherein said waveguide device is provided with a reaction surface having a coating of metasoluble protein.
Claim 22
Dependent Claim
13 words
The method of claim 1 wherein said coating of metasoluble protein comprises casein.
Claim 23
Dependent Claim
32 words
The method of claim 1 wherein said contacting of step (b) further includes contacting the reactive surface with a light absorbing member effective to impart an effective O.D. of at least 15.
Claim 24
Independent Claim
284 words
A method for visually detecting the presence or approximate amount of at least one specific binding analyte in a fluid sample, the method comprising: (a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample; (ii) a light receiving edge; and (iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized on at least one test situs on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon; wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds said analyte; (b) contacting the reactive surface with the sample suspected to contain said analyte and with a light scattering label attached to a first specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said analyte, in the case of a sandwich assay, or the immobilized first specific binding member, in the case of a competitive assay; thereby forming light scattering label complexes attached to the situs in proportion to the amount of the analyte in the sample; (c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby illuminating the reactive surface; (d) visually examining the reactive surface for light scattering and comparing the degree of light scattering at the test situs with either (i) the degree of light scattering at a non-situs portion, or (ii) the degree of light scattering at another situs, or both, whereby scattering at the situs correlates to the presence or amount of said analyte.
Claim 25
Independent Claim
284 words
A method for visually detecting the presence or approximate amount of at least one specific binding analyte in a fluid sample, the method comprising: (a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample; (ii) a light receiving edge; and (iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized on at least one test situs on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon; wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds said analyte; (b) contacting the reactive surface with the sample suspected to contain said analyte and with a light scattering label attached to a first specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said analyte, in the case of a sandwich assay, or the immobilized first specific binding member, in the case of a competitive assay; thereby forming light scattering label complexes attached to the situs in proportion to the amount of the analyte in the sample; (c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby illuminating the reactive surface; (d) visually examining the reactive surface for light scattering and comparing the degree of light scattering at the test situs with either (i) the degree of light scattering at a non-situs portion, or (ii) the degree of light scattering at another situs, or both, whereby scattering at the situs correlates to the presence or amount of said analyte.
Claim 26
Dependent Claim
51 words
The method of claim 23 wherein said analyte is a sequence of nucleic acid and the immobilized first specific binding member of said first cognate binding pair is an oligonucleotide complementary to said sequence or complementary to one or more cognate binding oligonucleotides, one of which is complementary to said sequence.
Claim 27
Dependent Claim
32 words
The method of claim 23 wherein said analyte nucleic acid sequence includes a hapten reporter and the specific binding member attached to said light scattering label is specific for said hapten reporter.
Claim 28
Dependent Claim
27 words
The method of claim 23 wherein said analyte is an antigen and the immobilized first specific binding member of said first cognate binding pair is an antibody.
Claim 29
Dependent Claim
20 words
The method of claim 23 wherein said element contains a plurality of test sites, each containing immobilized specific binding member.
Claim 30
Dependent Claim
23 words
The method of claim 27 wherein said plurality of test sites each contain varying concentrations of the same immobilized first specific binding member.
Claim 31
Dependent Claim
20 words
The method of claim 27 wherein said plurality of test sites each contain a distinct immobilized first specific binding member.
Claim 32
Dependent Claim
36 words
The method of claim 23 wherein said method further comprises a second transparent element connected to said first element to form a two-dimensional capillary channel therebetween, such that the reactive surface is formed in the channel.
Claim 33
Dependent Claim
25 words
The method of claim 23 wherein said light scattering label is a particle selected from the group consisting of colloidal gold, colloidal selenium and latex.
Claim 34
Dependent Claim
47 words
The method of claim 23 comprising a further step, after step (d), of altering the conditions at the reactive surface of the waveguide device to initiate dissociation of the analyte from the immobilized first specific binding member, and repeating steps (c) and (d) at the altered condition.
Claim 35
Dependent Claim
21 words
The method of claim 23 wherein said waveguide device is provided with a reaction surface having a coating of metasoluble protein.
Claim 36
Dependent Claim
13 words
The method of claim 33 wherein said coating of metasoluble protein comprises casein.
Claim 37
Dependent Claim
32 words
The method of claim 23 wherein said contacting of step (b) further includes contacting the reactive surface with a light absorbing member sufficient to impart an effective O.D. of at least 15.
Claim 38
Dependent Claim
13 words
The method of claim 35 wherein the effective O.D. is at least 100.
Claim 39
Independent Claim
340 words
A method for detecting the presence or amount of at least one specific binding analyte in a fluid sample, the method comprising: (a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample; (ii) a light receiving edge; and (iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized at a situs on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon; wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds said analyte; (b) contacting the reactive surface with the sample suspected to contain said analyte and with a light scattering label attached to a first specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said analyte, in the case of a sandwich assay, or the immobilized first specific binding member, in the case of a competitive assay; thereby forming light scattering label complexes attached to said situs in proportion to the amount of analyte in the sample; (c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby simultaneously illuminating the entire reactive surface; (d) collecting visually detectable light scattered by said light scattering label at a first time, t 1 , using a photodetector device; (e) repeating steps (c) and (d) at least once to collect scattered light, if any, from said situs and non-situs portions at a second time, t 2 ; and (f) comparing the degree of light scattering at said situs at time t 1 with the degree of light scattering at said situs at time t 2 , whereby the light scattering at the situs correlates to the presence or amount of the specific analyte, and the difference over time in scattering of light provides kinetic information indicative of the amount of analyte present at said situs.
Claim 40
Independent Claim
340 words
A method for detecting the presence or amount of at least one specific binding analyte in a fluid sample, the method comprising: (a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample; (ii) a light receiving edge; and (iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized at a situs on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon; wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds said analyte; (b) contacting the reactive surface with the sample suspected to contain said analyte and with a light scattering label attached to a first specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said analyte, in the case of a sandwich assay, or the immobilized first specific binding member, in the case of a competitive assay; thereby forming light scattering label complexes attached to said situs in proportion to the amount of analyte in the sample; (c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby simultaneously illuminating the entire reactive surface; (d) collecting visually detectable light scattered by said light scattering label at a first time, t 1 , using a photodetector device; (e) repeating steps (c) and (d) at least once to collect scattered light, if any, from said situs and non-situs portions at a second time, t 2 ; and (f) comparing the degree of light scattering at said situs at time t 1 with the degree of light scattering at said situs at time t 2 , whereby the light scattering at the situs correlates to the presence or amount of the specific analyte, and the difference over time in scattering of light provides kinetic information indicative of the amount of analyte present at said situs.
Claim 41
Dependent Claim
51 words
The method of claim 37 wherein said analyte is a sequence of nucleic acid and the immobilized first specific binding member of said first cognate binding pair is an oligonucleotide complementary to said sequence or complementary to one or more cognate binding oligonucleotides, one of which is complementary to said sequence.
Claim 42
Dependent Claim
32 words
The method of claim 37 wherein said analyte nucleic acid sequence includes a hapten reporter and the specific binding member attached to said light scattering label is specific for said hapten reporter.
Claim 43
Dependent Claim
27 words
The method of claim 37 wherein said analyte is an antigen and the immobilized first specific binding member of said first cognate binding pair is an antibody.
Claim 44
Dependent Claim
35 words
The method of claim 37 wherein said element contains a plurality of test sites, each situs containing immobilized specific binding member which is the same or different from immobilized specific binding member at another situs.
Claim 45
Dependent Claim
36 words
The method of claim 37 wherein said method further comprises a second transparent element connected to said first element to form a two-dimensional capillary channel therebetween, such that the reactive surface is formed in the channel.
Claim 46
Dependent Claim
25 words
The method of claim 37 wherein said light scattering label is a particle selected from the group consisting of colloidal gold, colloidal selenium and latex.
Claim 47
Dependent Claim
18 words
The method of claim 37 wherein said step of collecting scattered light is performed using a CCD camera.
Claim 48
Dependent Claim
48 words
The method of claim 37 comprising a further step, after step (f), of altering the conditions at the reactive surface of the waveguide device to initiate dissociation of the analyte from the immobilized first specific binding member, and repeating steps (c), (d) and (f) at the altered condition.
Claim 49
Dependent Claim
21 words
The method of claim 37 wherein said waveguide device is provided with a reaction surface having a coating of metasoluble protein.
Claim 50
Dependent Claim
13 words
The method of claim 46 wherein said coating of metasoluble protein comprises casein.
Claim 51
Dependent Claim
32 words
The method of claim 37 wherein said contacting of step (b) further includes contacting the reactive surface with a light absorbing member sufficient to impart an effective O.D. of at least 15.
Claim 52
Independent Claim
291 words
A method for determining the nucleotide sequence of segment of unknown nucleic acid or for distinguishing two closely related nucleotide sequences, the method comprising: (a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample; (ii) a light receiving edge; and (iii) a reactive surface comprising a plurality of sites having oligonucleotide immobilized thereon, said sites defining an array of oligonucleotides having different sequences for hybridizing with the unknown nucleic acid, other non-situs portions of the surface of said element having no oligonucleotides immobilized thereon; (b) contacting the reactive surface under hybridizing conditions with said unknown nucleic acid wherein said unknown nucleic acid, either directly or through intermediate cognate binding pairs if desired, is labeled with a light scattering label; thereby forming light scattering label complexes attached at those sites of the reactive surface having an oligonucleotide, complementary to the sequence of the unknown nucleic acid, immobilized thereon; (c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby simultaneously illuminating the entire reactive surface; (d) collecting visually detectable light scattered by said light scattering label; (e) comparing the degree of light scattering at each situs with either (i) the degree of light scattering at a non-situs portion; or (ii) the degree of light scattering at another situs; and (f) further comprising incrementally increasing the stringency conditions at the reactive surface of the waveguide device to initiate dissociation of bound nucleic acid from the sites and repeating steps (d) and (e) at each increment; whereby single base pair differences between the oligonucleotides and the unknown nucleic acid can be distinguished from perfect matches by differences in dissociation properties.
Claim 53
Independent Claim
291 words
A method for determining the nucleotide sequence of segment of unknown nucleic acid or for distinguishing two closely related nucleotide sequences, the method comprising: (a) providing a waveguide device, the waveguide device comprising (i) a transparent element having a refractive index greater than that of the fluid sample; (ii) a light receiving edge; and (iii) a reactive surface comprising a plurality of sites having oligonucleotide immobilized thereon, said sites defining an array of oligonucleotides having different sequences for hybridizing with the unknown nucleic acid, other non-situs portions of the surface of said element having no oligonucleotides immobilized thereon; (b) contacting the reactive surface under hybridizing conditions with said unknown nucleic acid wherein said unknown nucleic acid, either directly or through intermediate cognate binding pairs if desired, is labeled with a light scattering label; thereby forming light scattering label complexes attached at those sites of the reactive surface having an oligonucleotide, complementary to the sequence of the unknown nucleic acid, immobilized thereon; (c) illuminating the light receiving edge of the waveguide with light effective to create total internal reflection within the waveguide, thereby simultaneously illuminating the entire reactive surface; (d) collecting visually detectable light scattered by said light scattering label; (e) comparing the degree of light scattering at each situs with either (i) the degree of light scattering at a non-situs portion; or (ii) the degree of light scattering at another situs; and (f) further comprising incrementally increasing the stringency conditions at the reactive surface of the waveguide device to initiate dissociation of bound nucleic acid from the sites and repeating steps (d) and (e) at each increment; whereby single base pair differences between the oligonucleotides and the unknown nucleic acid can be distinguished from perfect matches by differences in dissociation properties.
Claim 54
Dependent Claim
32 words
The method of claim 49 wherein said analyte nucleic acid sequence includes a hapten reporter and the specific binding member attached to said light scattering label is specific for said hapten reporter.
Claim 55
Dependent Claim
36 words
The method of claim 49 wherein said method further comprises a second transparent element connected to said first element to form a two-dimensional capillary channel therebetween, such that the reactive surface is formed in the channel.
Claim 56
Dependent Claim
25 words
The method of claim 49 wherein said light scattering label is a particle selected from the group consisting of colloidal gold, colloidal selenium and latex.
Claim 57
Dependent Claim
28 words
The method of claim 49 wherein the substantially simultaneous collection of scattered light from a plurality of sites is accomplished visually by eye and brain of an observer.
Claim 58
Dependent Claim
35 words
The method of claim 49 wherein the substantially simultaneous collection of scattered light is accomplished by an array of photodetector devices selected from the group consisting of photodiodes, charge coupled devices, phototransistors, photoresistors and photomultipliers.
Claim 59
Dependent Claim
19 words
The method of claim 54 wherein the substantially simultaneous collection of scattered light is accomplished by a CCD camera.
Claim 60
Dependent Claim
50 words
The method of claim 49 wherein said light scattering label is attached to said first specific binding member of a second cognate binding pair via an intermediate cognate binding pair selected from the group consisting of: hapten and anti-hapten antibody; biotin and avidin or streptavidin; and complementary nucleic acid sequences.
Claim 61
Dependent Claim
16 words
The method of claim 49 wherein stringency conditions are increased incrementally by stepwise increases in temperature.
Claim 62
Dependent Claim
26 words
The method of claim 49 wherein stringency conditions are increased incrementally by stepwise addition of a dilution agent that decreases the ionic strength of the solution.
Claim 63
Dependent Claim
25 words
The method of claim 49 wherein the repeating of steps (d) and (e) is done without an intermediate washing away of dissociated unknown nucleic acid.
Claim 64
Dependent Claim
21 words
The method of claim 49 wherein said waveguide device is provided with a reaction surface having a coating of metasoluble protein.
Claim 65
Dependent Claim
13 words
The method of claim 60 wherein said coating of metasoluble protein comprises casein.
Claim 66
Dependent Claim
32 words
The method of claim 49 wherein said contacting of step (b) further includes contacting the reactive surface with a light absorbing member effective to impart an effective O.D. of at least 15.
Claim 67
Independent Claim
283 words
A method for detecting the presence or mount of a specific binding analyte in a fluid sample, the method comprising: (a) providing a TIR device, the device comprising (i) a transparent TIR element having a refractive index greater than that of the fluid sample; (ii) a light receiving edge; and (iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized on at least one situs on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon; wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds said analyte; (b) contacting the reactive surface with (i) the sample suspected to contain said analyte; (ii) a light scattering label attached to a first specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said analyte, in the case of a sandwich assay, or the immobilized first specific binding member, in the case of a competitive assay, thereby forming light scattering label complexes attached to said situs in proportion to the amount of analyte in the sample; and (iii) a solution of a light absorbing member sufficient to impart an effective O.D. of at least 15; (c) illuminating the light receiving edge of the TIR element with light effective to create total internal reflection within the element, thereby illuminating the reactive surface; (d) detecting visually detectable light scattered by said light scattering label and comparing the degree of light scattering at the situs with the degree of light scattering at a non-situs portion, whereby background scattering is minimized by absorbance by the light absorbing material.
Claim 68
Independent Claim
283 words
A method for detecting the presence or mount of a specific binding analyte in a fluid sample, the method comprising: (a) providing a TIR device, the device comprising (i) a transparent TIR element having a refractive index greater than that of the fluid sample; (ii) a light receiving edge; and (iii) a reactive surface comprising a first specific binding member of at least one cognate binding pair immobilized on at least one situs on the surface of the element, other non-situs portions of the reactive surface having no specific binding member immobilized thereon; wherein said first specific binding member, through intermediate cognate binding pairs if desired, specifically binds said analyte; (b) contacting the reactive surface with (i) the sample suspected to contain said analyte; (ii) a light scattering label attached to a first specific binding member of a second cognate binding pair which, through intermediate cognate binding pairs if desired, specifically binds said analyte, in the case of a sandwich assay, or the immobilized first specific binding member, in the case of a competitive assay, thereby forming light scattering label complexes attached to said situs in proportion to the amount of analyte in the sample; and (iii) a solution of a light absorbing member sufficient to impart an effective O.D. of at least 15; (c) illuminating the light receiving edge of the TIR element with light effective to create total internal reflection within the element, thereby illuminating the reactive surface; (d) detecting visually detectable light scattered by said light scattering label and comparing the degree of light scattering at the situs with the degree of light scattering at a non-situs portion, whereby background scattering is minimized by absorbance by the light absorbing material.
Claim 69
Dependent Claim
51 words
The method of claim 63 wherein said analyte is a sequence of nucleic acid and the immobilized first specific binding member of said first cognate binding pair is an oligonucleotide complementary to said sequence or complementary to one or more cognate binding oligonucleotides, one of which is complementary to said sequence.
Claim 70
Dependent Claim
32 words
The method of claim 63 wherein said analyte nucleic acid sequence includes a hapten reporter and the specific binding member attached to said light scattering label is specific for said hapten reporter.
Claim 71
Dependent Claim
27 words
The method of claim 63 wherein said analyte is an antigen and the immobilized first specific binding member of said first cognate binding pair is an antibody.
Claim 72
Dependent Claim
35 words
The method of claim 63 wherein said element contains a plurality of test sites, each situs containing immobilized specific binding member which is the same or different from immobilized specific binding member at another situs.
Claim 73
Dependent Claim
44 words
The method of claim 63 wherein said TIR element is two-dimensional and said TIR device further comprises a second two-dimensional transparent element connected to said TIR element to form a two-dimensional capillary channel therebetween, such that the reactive surface is formed in the channel.
Claim 74
Dependent Claim
25 words
The method of claim 63 wherein said light scattering label is a particle selected from the group consisting of colloidal gold, colloidal selenium and latex.
Claim 75
Dependent Claim
19 words
The method of claim 63 wherein detecting scattered light is accomplished visually by eye and brain of an observer.
Claim 76
Dependent Claim
31 words
The method of claim 63 wherein detecting scattered light is accomplished by an array of photodetector devices selected from the group consisting of photodiodes, charge coupled devices, phototransistors, photoresistors and photomultipliers.
Claim 77
Dependent Claim
47 words
The method of claim 63 comprising a further step, after step (d), of altering the conditions at the reactive surface of the waveguide device to initiate dissociation of the analyte from the immobilized first specific binding member, and repeating steps (c) and (d) at the altered condition.
Claim 78
Dependent Claim
30 words
The method of claim 72 wherein the analyte is a nucleic acid and the step of altering conditions comprises increasing the stringency by increased heat or by decreased ionic strength.
Claim 79
Dependent Claim
21 words
The method of claim 63 wherein said waveguide device is provided with a reaction surface having a coating of metasoluble protein.
Claim 80
Dependent Claim
13 words
The method of claim 74 wherein said coating of metasoluble protein comprises casein.
Claim 81
Dependent Claim
26 words
The method of claim 63 wherein the light absorbing material is the light scattering label in a concentration sufficient to impart an effective O.D. of 15.
Claim 82
Dependent Claim
24 words
The method of claim 76 wherein the light absorbing material is selected from the group consisting of colloidal metals, colloidal non-metals, and latex particles.
Claim 83
Dependent Claim
30 words
The method of claim 63 wherein the light absorbing material is a dye selected from the group consisting of azo dyes, diazo dyes, triazine dyes, food colorings and biological stains.
Claim 84
Dependent Claim
13 words
The method of claim 63 wherein the effective O.D. is at least 100.
Claim 85
Dependent Claim
13 words
The method of claim 79 wherein the effective O.D. is at least 300.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2000-02-04 | 2000-08-05 | 2001-02-04 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-02-04 | 2004-08-05 | 2005-02-04 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-02-04 | 2008-08-05 | 2009-02-04 | $7700.00 | Window Closed / Lapsed |