US4713347A — Measurement of ligand/anti-ligand interactions us…

Claim 1

Independent Claim 89 words

A method for determining the presence of a ligand in a fluid sample comprising: (a) localizing antiligand which interacts with said ligand in at least one predetermined region, said predetermined region being at least partially contained within a test volume; (b) exposing said predetermined region to said fluid sample; and (c) measuring the bulk conductance of said test volume to determine the occurrence of ligand-antiligand interaction in said predetermined region by detecting changes in the bulk conductance of said test volume due to ligand-antiligand interaction in said predetermined region.

Claim 2

Independent Claim 89 words

A method for determining the presence of a ligand in a fluid sample comprising: (a) localizing antiligand which interacts with said ligand in at least one predetermined region, said predetermined region being at least partially contained within a test volume; (b) exposing said predetermined region to said fluid sample; and (c) measuring the bulk conductance of said test volume to determine the occurrence of ligand-antiligand interaction in said predetermined region by detecting changes in the bulk conductance of said test volume due to ligand-antiligand interaction in said predetermined region.

Claim 3

Dependent Claim 34 words

The method of claim 1, wherein said localization of said antiligand in said predetermined region comprises confining said antiligand within the boundaries of a membrane, said membrane being permeable at least to said ligand.

Claim 4

Dependent Claim 68 words

The method of claim 1, wherein said ligand is selected from the group consisting of antigens, cell surface antigens, antigenic determinants, haptens, antibodies, antibody fragments, monovalent antibody fragments, nucleic acid sequences, enzymes, cofactors, enzyme substrates, genetically or chemically altered proteins, receptor protein, permeases, other transport proteins, binding proteins, molecules bound by a permease, transport protein or binding protein, carbohydrates, lectins, metal ions, metal-binding proteins, and other metal-binding substances.

Claim 5

Dependent Claim 14 words

The method of claim 1, wherein said antiligand has high affinity for said ligand.

Claim 6

Dependent Claim 14 words

The method of claim 1, wherein said antiligand has low affinity for said ligand.

Claim 7

Dependent Claim 26 words

The method of claim 1, including the step of forming a complex between an antiligand specific for said ligand to be determined and an amplifying substance.

Claim 8

Dependent Claim 35 words

The method of claim 6, wherein said complex is preformed and said ligand in said fluid sample binds to said antiligand component of said complex and further binds to said antiligand in said predetermined region.

Claim 9

Dependent Claim 24 words

The method of claim 1, wherein the bulk conductance of said test volume is measured with electrodes which are in contact with an electrolyte.

Claim 10

Dependent Claim 49 words

The method of claim 8, wherein four electrodes are used to measure the bulk conductance of said test volume, two of said electrodes providing a current through said test volume whose bulk conductance is being measured and two of said electrodes measuring the voltage drop across said test volume.

Claim 11

Dependent Claim 14 words

The method of claim 9, wherein said voltage electrodes are recessed from said current.

Claim 12

Dependent Claim 15 words

The method of claim 9, wherein said current electrodes are recessed from said test volume.

Claim 13

Dependent Claim 22 words

The method of claim 1, wherein said localization of said antiligand in said predetermined region comprises immobilizing said antiligand on a matrix.

Claim 14

Dependent Claim 30 words

The method of claim 12, wherein said matrix is selected from the group consisting of gel beads, gel layers, glass beads, polymeric beads, microporous membranes, porous paper, and mixtures thereof.

Claim 15

Dependent Claim 18 words

The method of claim 12, wherein said matrix is contacted with a flowing stream of said fluid sample.

Claim 16

Dependent Claim 18 words

The method of claim 14, wherein said contacting comprises causing said fluid sample to flow through said matrix.

Claim 17

Dependent Claim 18 words

The method of claim 14, wherein said contacting comprises causing said fluid sample to flow past said matrix.

Claim 18

Dependent Claim 32 words

The method of claim 1, wherein said changes in bulk conductance are determined by comparing the bulk conductance of said test volume with the bulk conductance of at least one control volume.

Claim 19

Dependent Claim 16 words

The method of claim 17, wherein said control volume does not contain any localized substance therein.

Claim 20

Dependent Claim 38 words

The method of claim 17, wherein said fluid sample contains a known concentration of a control ligand, and control antiligand which interacts with said control ligand is localized in at least one predetermined region of said control volume.

Claim 21

Dependent Claim 18 words

The method of claim 17, wherein said control volume does not contain antiligand which reacts with said ligand.

Claim 22

Dependent Claim 21 words

The method of claim 20, wherein said control volume is substantially free of specific interactions which effect changes in bulk conductance.

Claim 23

Dependent Claim 45 words

The method of claim 21, said control volume at least partially containing therein at least one predetermined region, said predetermined region being exposed to said fluid sample and also having localized therein molecules whose physical properties are similar to the physical properties of said antiligand.

Claim 24

Dependent Claim 74 words

The method of claim 21, wherein at least one of (a) the bulk conductance of said test volume, and (b) the bulk conductance of a second control volume is further compared with the bulk conductance of said control volume, and wherein said fluid sample contains a known concentration of a control ligand, and control antiligand which interacts with said control ligand is localized in at least one predetermined region of said second control volume.

Claim 25

Dependent Claim 23 words

The method claim 1, including the step of forming a complex between a ligand which interacts with said antiligand and an amplifying substance.

Claim 26

Dependent Claim 28 words

The method of claim 24, wherein said complex is preformed and added to said fluid sample, said ligand component of said complex being present in a known amount.

Claim 27

Dependent Claim 19 words

The method of claim 24, wherein said complex is preformed and prebound to said antiligand in said predetermined region.

Claim 28

Dependent Claim 37 words

The method of claim 24, wherein said complex is preformed and compartmentalized in at least one region which is adjacent said predetermined region containing said localized antiligand, said compartment being at least partially within said test volume.

Claim 29

Dependent Claim 30 words

The method of claim 24, wherein said complex is formed in said fluid sample, said ligand component of said complex being derived from the ligand present in said fluid sample.

Claim 30

Dependent Claim 28 words

The method of claim 28, including the step of adding a known amount of ligand which interacts with said antiligand to said fluid sample containing said complex therein.

Claim 31

Dependent Claim 57 words

The method of claim 24, wherein said amplifying substance is a particle selected from the group consisting of antiligand which interacts with said ligand, antiligand which interacts with said ligand bound to a second amplifying substance, macromolecules, cells, small molecules, molecular complexes, latex beads, lipid vesicles, non-conducting polymer beads, other non-conducting particles, magnetic particles, and mixtures thereof.

Claim 32

Dependent Claim 21 words

The method of claim 30, wherein said particle is antiligand which interacts with said ligand bound to said second amplifying substance.

Claim 33

Dependent Claim 26 words

The method of claim 31, wherein said second amplifying substance can be further reacted to effect a change in the bulk conductance of said test volume.

Claim 34

Dependent Claim 14 words

The method of claim 32, wherein said further reaction comprises generation of a gas.

Claim 35

Independent Claim 90 words

A method for determining the presence of a ligand in a fluid sample comprising: (a) localizing ligand in at least one predetermined region, said predetermined region being at least partially contained within a test volume; (b) exposing said predetermined region to said fluid sample and to an antiligand which interacts both with said ligand in said fluid sample and said localized ligand; and (c) measuring the bulk conductance of said test volume to determine the occurrence of ligand-antiligand interaction by detecting changes in the bulk conductance of said test volume.

Claim 36

Independent Claim 90 words

A method for determining the presence of a ligand in a fluid sample comprising: (a) localizing ligand in at least one predetermined region, said predetermined region being at least partially contained within a test volume; (b) exposing said predetermined region to said fluid sample and to an antiligand which interacts both with said ligand in said fluid sample and said localized ligand; and (c) measuring the bulk conductance of said test volume to determine the occurrence of ligand-antiligand interaction by detecting changes in the bulk conductance of said test volume.

Claim 37

Dependent Claim 32 words

The method of claim 34, wherein said changes in bulk conductance are determined by comparing the bulk conductance of said test volume with the bulk conductance of at least one control volume.

Claim 38

Dependent Claim 39 words

The method of claim 34, wherein said localization of said localized ligand comprises confining said ligand within the boudaries of a membrane, said membrane being permeable to at least said antiligand and said ligand present in said fluid sample.

Claim 39

Dependent Claim 23 words

The method of claim 34, including the step of forming a complex between antiligand which interacts with said ligand and an amplifying substance.

Claim 40

Dependent Claim 24 words

The method of claim 34, including the step of forming a complex between a ligand which interacts with said antiligand and an amplifying substance.

Claim 41

Dependent Claim 16 words

The method of claim 34, wherein localization of said localized ligand comprises immobilization on a matrix.

Claim 42

Dependent Claim 18 words

The method of claim 39, wherein said matrix is contacted with a flowing stream of said fluid sample.

Claim 43

Independent Claim 86 words

A method for determining the presence of a ligand in a fluid sample comprising: (a) localizing antiligand which interacts with said ligand in at least one predetermined region, said predetermined region being separate from a test volume; (b) exposing said predetermined region to said fluid sample; and (c) measuring the bulk conductance of said test volume to determine the occurrence of ligand-antiligand interaction in said predetermined region by detecting changes in the bulk conductance of said test volume due to ligand-antiligand interaction in said predetermined region.

Claim 44

Independent Claim 86 words

A method for determining the presence of a ligand in a fluid sample comprising: (a) localizing antiligand which interacts with said ligand in at least one predetermined region, said predetermined region being separate from a test volume; (b) exposing said predetermined region to said fluid sample; and (c) measuring the bulk conductance of said test volume to determine the occurrence of ligand-antiligand interaction in said predetermined region by detecting changes in the bulk conductance of said test volume due to ligand-antiligand interaction in said predetermined region.

Claim 45

Dependent Claim 24 words

The method of claim 11, including the step of forming a complex between a ligand which interacts with said antiligand and an amplifying substance.

Claim 46

Dependent Claim 37 words

The method of claim 42, wherein said complex is preformed and compartmentalized in at least one region which is adjacent said predetermined region containing said localized antiligand, said compartment being at least partially within said test volume.

Claim 47

Dependent Claim 113 words

A sensor for determining the presence of a ligand in a fluid sample, said sensor comprising: (a) means for localizing (i) antiligand which interacts with said ligand or (ii) ligand which interacts with an antiligand, which antiligand also interacts with said ligand in said fluid sample, said localizing means selected from the group consisting of (a) a semipermeable membrane which is permeable at least to said ligand or (b) a matrix material selected from the group consisting of gel beads, gel layers, glass beads, polymeric beads, microporous membranes, porous paper, and mixtures thereof; and (b) means for measuring the bulk conductance of a test volume which at least partially includes said localizing means.

Claim 48

Dependent Claim 113 words

A sensor for determining the presence of a ligand in a fluid sample, said sensor comprising: (a) means for localizing (i) antiligand which interacts with said ligand or (ii) ligand which interacts with an antiligand, which antiligand also interacts with said ligand in said fluid sample, said localizing means selected from the group consisting of (a) a semipermeable membrane which is permeable at least to said ligand or (b) a matrix material selected from the group consisting of gel beads, gel layers, glass beads, polymeric beads, microporous membranes, porous paper, and mixtures thereof; and (b) means for measuring the bulk conductance of a test volume which at least partially includes said localizing means.

Claim 49

Independent Claim 133 words

An apparatus for determining the presence of a ligand in a fluid sample, said apparatus comprising: (a) in at least one predetermined region of said apparatus, means for localizing (i) antiligand which interacts with said ligand or (ii) ligand which interacts with an antiligand, which antiligand also interacts with said ligand in said fluid sample, said localizing means selected from the group consisting of (a) a semipermeable membrane which is permeable at least to said ligand or (b) a matrix material selected from the group consisting of gel beads, gel layers, glass beads, polymeric beads, microporous membranes, porous paper, and mixtures thereof; (b) means for contacting said fluid sample with said localizing means; and (c) means for measuring the bulk conductance of a test volume which at least partially contains said predetermined region.

Claim 50

Independent Claim 133 words

An apparatus for determining the presence of a ligand in a fluid sample, said apparatus comprising: (a) in at least one predetermined region of said apparatus, means for localizing (i) antiligand which interacts with said ligand or (ii) ligand which interacts with an antiligand, which antiligand also interacts with said ligand in said fluid sample, said localizing means selected from the group consisting of (a) a semipermeable membrane which is permeable at least to said ligand or (b) a matrix material selected from the group consisting of gel beads, gel layers, glass beads, polymeric beads, microporous membranes, porous paper, and mixtures thereof; (b) means for contacting said fluid sample with said localizing means; and (c) means for measuring the bulk conductance of a test volume which at least partially contains said predetermined region.

Claim 51

Dependent Claim 36 words

The apparatus of claim 45, wherein said region comprises at least one test region for determining the presence of said ligand and at least one control region to provide a comparative basis for making said measurement.

Claim 52

Dependent Claim 15 words

The apparatus of claim 45, wherein said test volume essentially entirely includes said localizing means.

Claim 53

Dependent Claim 40 words

The apparatus of claim 45, wherein said contacting means comprise a structure having: (a) an inlet channel for bringing said fluid sample to said localizing means; and (b) an outlet channel for removing said fluid sample from said localizing means.

Claim 54

Dependent Claim 12 words

The apparatus of claim 48, wherein said structure comprises a non-conductive material.

Claim 55

Dependent Claim 23 words

The apparatus of claim 45, wherein said non-conductive material is selected from the group consisting of plastic, ceramic, glass, quartz, and mixtures thereof.

Claim 56

Dependent Claim 69 words

The apparatus of claim 48, wherein said structure comprises at least three component parts, said first component part comprising said inlet channel, said second component part comprising said outlet channel, and said third component part comprising said localizing means, said first and second component parts being fastened in a sandwiched fashion about said third component part, said third component part being insertable between said first and second component parts.

Claim 57

Dependent Claim 24 words

The apparatus of claim 51, further comprising a fluid bath, wherein at least said inlet and outlet channels are submerged in said fluid bath.

Claim 58

Dependent Claim 19 words

The apparatus of claim 45, wherein said means for measuring the bulk conductance comprises a plurality of spaced-apart electrodes.

Claim 59

Dependent Claim 18 words

The apparatus of claim 53, wherein said apparatus additionally includes a current-modulating material adjacent to said localizing means.

Claim 60

Dependent Claim 15 words

The apparatus of claim 53, wherein said localizing means additionally comprises a current modulating material.

Claim 61

Dependent Claim 16 words

The apparatus of claim 53, wherein said plurality of electrodes are in contact with an electrolyte.

Claim 62

Dependent Claim 13 words

The apparatus of claim 56, wherein said plurality of electrodes comprises two electrodes.

Claim 63

Dependent Claim 13 words

The apparatus of claim 57, wherein said plurality of electrodes includes auxiliary electrodes.

Claim 64

Dependent Claim 12 words

The apparatus of claim 58, wherein said auxiliary electrodes comprise guard electrodes.

Claim 65

Dependent Claim 15 words

The apparatus of claim 56, wherein said plurality of electrodes comprises at least four electrodes.

Claim 66

Dependent Claim 28 words

The apparatus of claim 60, wherein two of said electrodes provide a current through said test volume and two measure the drop in voltage across said test volume.

Claim 67

Dependent Claim 14 words

The apparatus of claim 61, wherein said plurality of electrodes also includes auxiliary electrodes.

Claim 68

Dependent Claim 12 words

The apparatus of claim 62, wherein said auxiliary electrodes comprise guard electrodes.

Claim 69

Dependent Claim 26 words

The apparatus of claim 61, wherein each of said current electrodes and each of said voltage electrodes is placed on opposite sides of said localizing means.

Claim 70

Dependent Claim 15 words

The apparatus of claim 64, wherein said localizing means essentially entirely includes said test volume.

Claim 71

Dependent Claim 15 words

The apparatus of claim 61, wherein said current electrodes are recessed from said test volume.

Claim 72

Dependent Claim 28 words

The apparatus of claim 66, wherein said recession comprises placing said current electrodes in chambers, said chambers being in electrical contact with said test volume via conducting channels.

Claim 73

Dependent Claim 19 words

The apparatus of claim 67, wherein said conducting channels additionally contain a matrix which retards bubble formation and convection.

Claim 74

Dependent Claim 22 words

The apparatus of claim 61, wherein said current electrodes and said voltage electrodes are on the same side of said localizing means.

Claim 75

Dependent Claim 18 words

The apparatus of claim 69, wherein said current electrodes and said voltage electrodes lie in the same plane.

Claim 76

Dependent Claim 15 words

The apparatus of claim 70, wherein said voltage electrodes are recessed from said current flow.

Claim 77

Dependent Claim 15 words

The apparatus of claim 70, wherein said current electrodes are recessed from said test volume.

Claim 78

Dependent Claim 18 words

The apparatus of claim 70, wherein said apparatus additionally includes a current-modulating material adjacent to said localizing means.

Claim 79

Dependent Claim 14 words

The apparatus of claim 61, wherein said voltage electrodes are recessed from current flow.

Claim 80

Dependent Claim 28 words

The apparatus of claim 74, wherein said recession comprises placing said voltage electrodes in chambers, said chambers being in electrical contact with said test volume via conducting channels.

Claim 81

Dependent Claim 45 words

The apparatus of claim 75, wherein the width of the opening of said conducting channels adjacent to said test volume ranges from about 25 um to 100 um, and said openings are from about 40 um to 200 um apart with respect to each other.

Claim 82

Dependent Claim 19 words

The apparatus of claim 75, wherein said conducting channels additionally contain a matrix which retards bubble formation and convection.

Claim 83

Dependent Claim 18 words

The apparatus of claim 77, wherein said localizing means also comprises said matrix contained in said conducting channels.

Claim 84

Dependent Claim 42 words

The apparatus of claim 75, wherein the width of the opening of said conducting channels ranges from about 10 um to about 1 mm, and said openings are from about 10 um to about 1 mm apart with respect to each other.

Claim 85

Dependent Claim 15 words

The apparatus of claim 79, wherein said test volume ranges up to about 1 ul.

Claim 86

Dependent Claim 15 words

The apparatus of claim 79, wherein said test volume is less than about 0.1 ul.

Claim 87

Dependent Claim 18 words

The apparatus of claim 79, wherein the thickness of said test volume ranges up to about 1 mm.

Claim 88

Dependent Claim 18 words

The apparatus of claim 79, wherein the thickness of said test volume is less than about 0.1 mm.

Claim 89

Dependent Claim 18 words

The apparatus of claim 79, wherein the volume of said predetermined region is less than about 0.1 ul.

Claim 90

Dependent Claim 18 words

The apparatus of claim 79, wherein the thickness of said predetermined region is less than about 0.1 mm.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1990-12-14	1991-06-15	1991-12-15	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1994-12-14	1995-06-15	1995-12-15	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	1998-12-14	1999-06-15	1999-12-15	$7700.00	Window Closed / Lapsed

Measurement of ligand/anti-ligand interactions using bulk conductance

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