US5385822A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Methods for detection and quantification of cell subsets within subpopulations of a mixed cell population
Assignee: CREDITANSTALT BANKVEREIN
•
Filing: 1990-11-29
•
Granted: 1995-01-31
•
Expires: 2010-11-29
Abstract
Abstract The presence or quantity of a selected subset of cells, which is part of a subpopulation of a mixed cell population, is determined by a method in which a detectable reporter substance is uniformly incorporated into substantially all cells of the subpopulation containing the subset of interest. The subset of interest is then affinity-separated by incubating a test sample of the mixed cell population containing the labeled subpopulation with a specific binding substance which selectively binds to characteristic determinants of the cell subset of interest. Occurrence of the reporter substance in the separated fraction is then detected, and correlated to a predetermined standard to determine the presence or quantity of the subset of interest within the cell population. According to another aspect of the invention a method is provided for quantitating two or more selected subsets of cells within a subpopulation of a mixed cell population. After labeling, the entire subpopulation is affinity-separated from the mixed cell population, and occurrence of the reporter substance in the separated fraction is detected. Next, subsets of interest within the subpopulation are affinity-separated as described above, and the level of detected reporter substance in each subset is compared to the level detected in the entire subpopulation. According to further aspects of the invention test kits are provided for performing the above-described methods.
Inventors
- 👤 Meryle J. Melnicoff
- 👤 Bruce D. Jensen
- 👤 Katharine A. Muirhead
- 👤 Paul K. Horan
- 👤 Martin D. Summers
- 👤 William Wong
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-27 00:56
CPC Classifications
A; A61; A61K; A61K41/00; A61K41/0085; A61K47/00; A61K47/50; A61K47/51; A61K47/56; A61K47/59; A61K47/60; A61K49/00; A61K49/0002; A61K49/001; A61K49/0013; A61K49/0017; A61K49/0019; A61K49/0021; A61K49/0032; A61K49/06; A61K49/18; A61K49/1896; C; C07; C07C; C07C229/00; C07C229/02; C07C229/04; C07C229/06; C07C229/10; C07C229/16; C07D; C07D209/00; C07D209/02; C07D209/04; C07D209/08; C07D219/00; C07D219/04; C07D219/08; C07D263/00; C07D263/52; C07D263/62; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/569; G01N33/56966; G01N33/56972; Y; Y10; Y10S; Y10S435/00; Y10S435/975
Claims Analysis
Total Claims: 73
Claim 1
Independent Claim
269 words
A method for determining the presence or quantity of a selected subset of a subpopulation of cells within a mixed cell population containing said subset, said subpopulation of cells having at least one characteristic determinant and said subset of cells having at least one characteristic determinant, said method comprising: (i) selectively binding to the cells of said subpopulation a detectable reporter substance, either by a) directly binding a specific binding substance which specifically binds to said at least one characteristic determinant of said subpopulation and which is directly or indirectly conjugated to said detectable reporter substance or b) sequentially binding a first specific binding substance which specifically binds to said at least one characteristic determinant of said subpopulation, and a second specific binding substance which specifically binds to said first specific binding substance and which is directly or indirectly conjugated to said detectable reporter substance, such that a consistent amount of reporter substance is distributed in the subset of interest; (ii) contacting a test sample of said mixed cell population with a specific binding substance which specifically binds to said at least one characteristic determinant of said selected subset, resulting in the formation of a complex of cells and uncomplexed cells, said complex comprising said selected subset and the specific binding substance that binds to said subset; (iii) separating said complex from said uncomplexed cells; and (iv) following the completion of Steps (i), (ii), and (iii), detecting the occurrence of said detectable reporter substance in one of said complex or uncomplexed cells, from which the presence or quantity of said selected subset in said mixed cell population is determined.
Claim 2
Independent Claim
269 words
A method for determining the presence or quantity of a selected subset of a subpopulation of cells within a mixed cell population containing said subset, said subpopulation of cells having at least one characteristic determinant and said subset of cells having at least one characteristic determinant, said method comprising: (i) selectively binding to the cells of said subpopulation a detectable reporter substance, either by a) directly binding a specific binding substance which specifically binds to said at least one characteristic determinant of said subpopulation and which is directly or indirectly conjugated to said detectable reporter substance or b) sequentially binding a first specific binding substance which specifically binds to said at least one characteristic determinant of said subpopulation, and a second specific binding substance which specifically binds to said first specific binding substance and which is directly or indirectly conjugated to said detectable reporter substance, such that a consistent amount of reporter substance is distributed in the subset of interest; (ii) contacting a test sample of said mixed cell population with a specific binding substance which specifically binds to said at least one characteristic determinant of said selected subset, resulting in the formation of a complex of cells and uncomplexed cells, said complex comprising said selected subset and the specific binding substance that binds to said subset; (iii) separating said complex from said uncomplexed cells; and (iv) following the completion of Steps (i), (ii), and (iii), detecting the occurrence of said detectable reporter substance in one of said complex or uncomplexed cells, from which the presence or quantity of said selected subset in said mixed cell population is determined.
Claim 3
Dependent Claim
24 words
A method according to claim 1, wherein said subpopulation comprises lymphocytes and said subset comprises lymphocytes characterized by selected functions or stages of differentiation.
Claim 4
Dependent Claim
28 words
A method according to claim 1, wherein said subpopulation comprises T-lymphocytes and said subset is selected from the group consisting of helper T lymphocytes and suppressor/cytotoxic T lymphocytes.
Claim 5
Dependent Claim
25 words
A method according to claim 1, wherein said subpopulation comprises leukocytes and said subset is selected from the group consisting of lymphocytes, monocytes and granulocytes.
Claim 6
Dependent Claim
34 words
A method according to claim 1, which includes the step of relating the level of detected reporter substance to a predetermined standard to determine the quantity of said subset of cells in said sample.
Claim 7
Dependent Claim
39 words
A method according to claim 1, wherein the level of detected reporter substance is related to a standard containing a predetermined quantity of said reporter substance thereby to determine the number of said subset of cells in said sample.
Claim 8
Dependent Claim
22 words
A method according to claim 1, wherein said reporter substance is separated from said complex prior to detecting its presence or quantity.
Claim 9
Dependent Claim
25 words
A method according to claim 1, wherein said specific binding substance, which specifically binds to a determinant of said subpopulation, comprises at least one antibody.
Claim 10
Dependent Claim
56 words
A method according to claim 8, wherein said reporter substance is attached to one member of a specific binding pair, said antibody is attached to the other member of said specific binding pair, and said selective binding is achieved by the combined effect of immunological binding and binding between the members of said specific binding pair.
Claim 11
Dependent Claim
15 words
A method according to claim 9, wherein said specific binding pair comprises avidin and biotin.
Claim 12
Dependent Claim
18 words
A method according to claim 10, wherein said biotin is attached to said antibody by a cleavable linkage.
Claim 13
Dependent Claim
16 words
A method according to claim 8, wherein said at least one antibody comprises a monoclonal antibody.
Claim 14
Dependent Claim
15 words
A method according to claim 12, wherein said monoclonal antibody specifically binds to CD45 antigen.
Claim 15
Dependent Claim
32 words
A method according to claim 13 wherein the amount of said monoclonal antibody bound per cell to lymphocytes is at least 5 times greater than the amount bound per cell to monocytes.
Claim 16
Dependent Claim
21 words
A method according to claim 12, wherein said at least one antibody comprises a monoclonal antibody that specifically binds T lymphocytes.
Claim 17
Dependent Claim
39 words
A method according to claim 15, wherein said at least one antibody comprises a monoclonal antibody which specifically binds to an antigen selected from the group consisting of CD2, CD3, CD5, CD7 or any combination of said monoclonal antibodies.
Claim 18
Dependent Claim
13 words
A method according to claim 1, wherein said reporter substance comprises an enzyme.
Claim 19
Dependent Claim
11 words
A method according to claim 1, wherein said enzyme is beta-galactosidase.
Claim 20
Dependent Claim
72 words
A method according to claim 1, wherein the step of selectively binding said detectable reporter substance to said subpopulation of cells comprises sequentially binding to the surface of the cells, a first antibody which specifically binds to at least one determinant which is common to the cells of said subpopulation, and a second antibody which specifically binds to a determinant of said first antibody, and which is conjugated to said reporter substance.
Claim 21
Dependent Claim
26 words
A method according to claim 19, wherein said first antibody comprises a selected isotype and said second antibody specifically binds to a determinant of said isotype.
Claim 22
Dependent Claim
32 words
A method according to claim 19, wherein said first antibody comprises immunoglobulin obtained from a selected species and said second antibody specifically binds to a determinant of immunoglobulin from said selected species.
Claim 23
Dependent Claim
22 words
A method according to claim 1, wherein said specific binding substance that binds to said subsets is affixed to a solid phase.
Claim 24
Dependent Claim
23 words
A method according to claim 22, wherein said solid phase comprises magnetic or paramagnetic material and said complex is separated by magnetic separation.
Claim 25
Dependent Claim
54 words
A method according to claim 22, wherein said test sample is contacted in a container with said specific binding substance which specifically binds to said selected subset and said specific binding substance which specifically binds to said selected subset comprises antibody affixed to a surface of said container in contact with said test sample.
Claim 26
Dependent Claim
20 words
A method according to claim 1, wherein said specific binding substance to which said selected subset binds comprises an antibody.
Claim 27
Dependent Claim
13 words
A method according to claim 25, wherein said antibody comprises a monoclonal antibody.
Claim 28
Dependent Claim
15 words
A method according to claim 26, wherein said monoclonal antibody specifically binds the CD4 antigen.
Claim 29
Dependent Claim
64 words
A method according to claim 1, wherein the step of contacting said test sample with said specific binding substance which specifically binds to said selected subset additionally includes contacting said test sample with an auxiliary specific binding substance which specifically binds to said specific binding substance which specifically binds to said selected subset, said auxiliary specific binding substance being affixed to a solid phase.
Claim 30
Dependent Claim
30 words
A method according to claim 21, wherein said test sample is contacted with a biotinylated antibody as the specific binding substance and with avidin as the auxiliary specific binding substance.
Claim 31
Dependent Claim
18 words
A method according to claim 29, wherein said biotin is attached to said antibody by a cleavable linkage.
Claim 32
Dependent Claim
32 words
A method according to claim 28, wherein said test sample is contacted with a first antibody as the specific binding substance and with a second antibody as the auxiliary specific binding substance.
Claim 33
Dependent Claim
26 words
A method according to claim 31, wherein said first antibody comprises a selected isotype and said second antibody binds selectively to a determinant of said isotype.
Claim 34
Dependent Claim
32 words
A method according to claim 31, wherein said first antibody comprises immunoglobulin obtained from a selected species and said second antibody binds selectively to a determinant of said immunoglobulin from said species.
Claim 35
Independent Claim
372 words
A method for analyzing a subpopulation of cells, having at least one characteristic determinant, present within a mixed cell population, said subpopulation of cells including individual subsets of interest, each subset having at least one characteristic determinant, to determine the proportion of at least one selected subset of said subpopulation of cells, comprising: (i) selectively binding to a portion of cells of said mixed cell population a detectable reporter substance, either by a) directly binding a specific binding substance which specifically binds to a determinant of said portion of cells and which is directly or indirectly conjugated to said detectable reporter substance or b) sequentially binding a first specific binding substance which specifically binds to a determinant of said portion of cells, and a second specific binding substance which specifically binds to said first specific binding substance and which is directly or indirectly conjugated to said detectable reporter substance, whereby said detectable reporter substance uniformly labels substantially all cells of said subpopulation; (ii) contacting a first sample of said mixed cell population with a first reagent comprising at least one specific binding substance which specifically binds to said at least one characteristic determinant of said cell subpopulation, resulting in the formation of a first complex of cells and uncomplexed cells; (iii) separating said first complex from said uncomplexed cells; (iv) following the completion of steps (i), (ii), and (iii), detecting the occurrence of said detectable reporter substance in said first complex; (v) contacting a second sample of said cell population from step (i), of equivalent volume and cell concentration to said first sample, with a second reagent comprising a specific binding substance which specifically binds to said at least one characteristic determinant of said selected subset of interest included in said subpopulation of cells, resulting in the formation of a second complex of cells and uncomplexed cells; (vi) separating said second complex from said uncomplexed cells; (vii) detecting the occurrence of said detectable reporter substance in said second complex; and (viii) determining the proportion of said selected subset of interest in said cell subpopulation by quantitating the amount of said detectable reporter substance associated with said second complex relative to the amount of said detectable reporter substance associated with said first complex.
Claim 36
Independent Claim
372 words
A method for analyzing a subpopulation of cells, having at least one characteristic determinant, present within a mixed cell population, said subpopulation of cells including individual subsets of interest, each subset having at least one characteristic determinant, to determine the proportion of at least one selected subset of said subpopulation of cells, comprising: (i) selectively binding to a portion of cells of said mixed cell population a detectable reporter substance, either by a) directly binding a specific binding substance which specifically binds to a determinant of said portion of cells and which is directly or indirectly conjugated to said detectable reporter substance or b) sequentially binding a first specific binding substance which specifically binds to a determinant of said portion of cells, and a second specific binding substance which specifically binds to said first specific binding substance and which is directly or indirectly conjugated to said detectable reporter substance, whereby said detectable reporter substance uniformly labels substantially all cells of said subpopulation; (ii) contacting a first sample of said mixed cell population with a first reagent comprising at least one specific binding substance which specifically binds to said at least one characteristic determinant of said cell subpopulation, resulting in the formation of a first complex of cells and uncomplexed cells; (iii) separating said first complex from said uncomplexed cells; (iv) following the completion of steps (i), (ii), and (iii), detecting the occurrence of said detectable reporter substance in said first complex; (v) contacting a second sample of said cell population from step (i), of equivalent volume and cell concentration to said first sample, with a second reagent comprising a specific binding substance which specifically binds to said at least one characteristic determinant of said selected subset of interest included in said subpopulation of cells, resulting in the formation of a second complex of cells and uncomplexed cells; (vi) separating said second complex from said uncomplexed cells; (vii) detecting the occurrence of said detectable reporter substance in said second complex; and (viii) determining the proportion of said selected subset of interest in said cell subpopulation by quantitating the amount of said detectable reporter substance associated with said second complex relative to the amount of said detectable reporter substance associated with said first complex.
Claim 37
Dependent Claim
147 words
A method according to claim 34, which further comprises: (ix) contacting one or more additional samples of said mixed cell population from step (i), each sample being of equivalent volume and cell concentration to said first sample, with additional reagents, each said additional reagent comprising a specific binding substance which binds specifically to a characteristic determinant of an additional subset of interest included in said subpopulation of cells, resulting in the formation of additional complexes of cells and uncomplexed cells; (x) separating each of said additional complexes from said uncomplexed cells; (xi) detecting the occurrence of said detectable reporter substance in each of said additional complexes; and (xii) determining the proportion of each of said additional subsets in said cell population by quantitating the amount of reporter substance associated with each of said additional complexes relative to the amount of reporter substance associated with said first complex.
Claim 38
Dependent Claim
47 words
A method according to claim 35 which includes contacting at least one of said additional samples with an auxiliary specific binding substance which specifically binds to said additional reagent with which said additional sample is contacted, said auxiliary specific binding substance being affixed to a solid phase.
Claim 39
Dependent Claim
52 words
A method according to claim 34, wherein said first reagent comprises a mixture of specific binding substances, each said binding substance specifically binding to a characteristic determinant of individual subsets of interest comprising said subpopulation, whereby substantially all cells of said subpopulation are bound to one or another of said binding substances.
Claim 40
Dependent Claim
24 words
A method according to claim 34, wherein said subpopulation comprises lymphocytes and said subsets comprise lymphocytes characterized by selected functions or stages of differentiation.
Claim 41
Dependent Claim
28 words
A method according to claim 34, wherein said subpopulation comprises T-lymphocytes and said subsets are selected from the group consisting of helper T lymphocytes and suppressor/cytotoxic T lymphocytes.
Claim 42
Dependent Claim
25 words
A method according to claim 34, wherein said subpopulation comprises leukocytes and said subsets are selected from the group consisting of lymphocytes, monocytes and granulocytes.
Claim 43
Dependent Claim
23 words
A method according to claim 34, wherein said detectable reporter substance is removed from said complex prior to detecting its presence or quantity.
Claim 44
Dependent Claim
27 words
A method according to claim 34, wherein said specific binding substance, which specifically binds to a determinant of said portion of cells, comprises at least one antibody.
Claim 45
Dependent Claim
13 words
A method according to claim 42, wherein said reporter substance comprises an enzyme.
Claim 46
Dependent Claim
11 words
A method according to claim 43, wherein said enzyme is beta-galactosidase.
Claim 47
Dependent Claim
56 words
A method according to claim 42, wherein said reporter substance is attached to one member of a specific binding pair, said antibody is attached to the other member of said specific binding pair, and said selective binding is achieved by the combined effect of immunological binding and binding between the members of said specific binding pair.
Claim 48
Dependent Claim
15 words
A method according to claim 45, wherein said specific binding pair comprises avidin and biotin.
Claim 49
Dependent Claim
18 words
A method according to claim 46, wherein said biotin is attached to said antibody by a cleavable linkage.
Claim 50
Dependent Claim
16 words
A method according to claim 42, wherein said at least one antibody comprises a monoclonal antibody.
Claim 51
Dependent Claim
15 words
A method according to claim 48, wherein said monoclonal antibody specifically binds to CD45 antigen.
Claim 52
Dependent Claim
32 words
A method according to claim 49 wherein the amount of said monoclonal antibody bound per cell to lymphocytes is at least 5 time greater than the amount bound per cell to monocytes.
Claim 53
Dependent Claim
21 words
A method according to claim 48, wherein said at least one antibody comprises a monoclonal antibody that specifically binds T lymphocytes.
Claim 54
Dependent Claim
39 words
A method according to claim 57, wherein said at least one antibody comprises a monoclonal antibody which specifically binds to an antigen selected from the group consisting of CD2, CD3, CD5, CD7 or any combination of said monoclonal antibodies.
Claim 55
Dependent Claim
74 words
A method according to claim 34, wherein the step of selectively binding said detectable reporter substance to said subpopulation of cells comprises sequentially binding to the surface of the cells, a first antibody which specifically binds to at least one determinant which is common to the cells of said portion of cells, and a second antibody which specifically binds to a determinant of said first antibody, and which is conjugated to said reporter substance.
Claim 56
Dependent Claim
26 words
A method according to claim 53, wherein said first antibody comprises a selected isotype and said second antibody specifically binds to a determinant of said isotype.
Claim 57
Dependent Claim
32 words
A method according to claim 53 wherein said first antibody comprise immunoglobulin obtained from a selected species and said second antibody specifically binds to a determinant of immunoglobulin from said selected species.
Claim 58
Dependent Claim
31 words
A method according to claim 34, wherein said specific binding substance of said first reagent and said specific binding substance of said second reagent are each affixed to a solid phase.
Claim 59
Dependent Claim
54 words
A method according to claim 56 wherein said second sample is contacted in a sample container with said specific binding substance which specifically binds said selected subset and wherein said specific binding substance which specifically binds said selected subset comprises antibody affixed to a surface of said container in contact with said second sample.
Claim 60
Dependent Claim
55 words
A method according to claim 34, wherein said specific binding substance of said first reagent and said specific binding substance of said second reagent are each affixed to magnetic or paramagnetic particles, and said first complex including said subpopulation of interest and said second complex including said selected subset are magnetically separated from said samples.
Claim 61
Dependent Claim
26 words
A method according to claim 34, wherein the specific binding substance of each of said first reagent and said second reagent comprises at least one antibody.
Claim 62
Dependent Claim
27 words
A method according to claim 34, wherein the specific binding substance of each of said first reagent and said second reagent comprises at least one monoclonal antibody.
Claim 63
Dependent Claim
19 words
A method according to claim 60, wherein said monoclonal antibody of said second reagent specifically binds the CD4 antigen.
Claim 64
Dependent Claim
46 words
A method according to claim 34 which includes contacting said first sample or said second sample with an auxiliary specific binding substance capable of interacting selectively with said first reagent or said second reagent, respectively, said auxiliary specific binding substance being affixed to a solid phase.
Claim 65
Dependent Claim
24 words
A method according to claim 62 wherein said first sample or said second sample is contacted with antibody as the auxiliary specific binding substance.
Claim 66
Dependent Claim
38 words
A method according to claim 62 wherein said first reagent or said second reagent comprises antibodies of a selected isotype, and said auxiliary specific binding substance comprises an antibody which binds selectively to a determinant of said isotype.
Claim 67
Dependent Claim
42 words
A method according to claim 62 wherein said first reagent or said second reagent comprises immunoglobulin obtained from a selected species, and said auxiliary specific binding substances comprise an antibody which binds selectively to a determinant of said immunoglobulin from said species.
Claim 68
Dependent Claim
38 words
A method according to claim 62 wherein said first sample or said second sample is contacted with a biotinylated antibody as the first or second specific binding substance, respectively, and with avidin as the auxiliary specific binding substance.
Claim 69
Dependent Claim
18 words
A method according to claim 66 wherein said biotin is attached to said antibody by a cleavable linkage.
Claim 70
Dependent Claim
127 words
A test kit for determining the presence or quantity of one or more selected subsets of a subpopulation of cells in a test sample, said subset having at least one characteristic determinant, said test kit comprising: (i) a conjugate comprising a detectable reporter substance and a specific binding substance that specifically binds to at least one characteristic determinant of said subpopulation, said conjugate effecting uniform labelling of substantially all cells of said subpopulation by said detectable reporter substance, such that a consistent amount of reporter substance is distributed in the subset of interest; and (ii) a reagent comprising at least one specific binding substance affixed to a solid phase, said binding substance being capable of binding to at least one characteristic determinant of one of said subsets.
Claim 71
Dependent Claim
127 words
A test kit for determining the presence or quantity of one or more selected subsets of a subpopulation of cells in a test sample, said subset having at least one characteristic determinant, said test kit comprising: (i) a conjugate comprising a detectable reporter substance and a specific binding substance that specifically binds to at least one characteristic determinant of said subpopulation, said conjugate effecting uniform labelling of substantially all cells of said subpopulation by said detectable reporter substance, such that a consistent amount of reporter substance is distributed in the subset of interest; and (ii) a reagent comprising at least one specific binding substance affixed to a solid phase, said binding substance being capable of binding to at least one characteristic determinant of one of said subsets.
Claim 72
Dependent Claim
51 words
A test kit according to claim 68 which further comprises: i) a medium for uniformly labelling cells of said subpopulation with said reporter substance; ii) at least one pre-determined standard for determining the presence or quantity of said subsets in said test sample; and iii) reagent for detecting said reporter substance.
Claim 73
Dependent Claim
16 words
A test kit according to claim 68 wherein said solid phase comprises magnetic or paramagnetic material.
References Cited (Backward Citations)
US3970518A
Cited
US4157323A
Cited
US4248965A
Cited
US4219411A
Cited
US4230685A
Cited
US4284412A
Cited
US4582797A
Cited
US4532203A
Cited
US4677067A
Cited
US4748129A
Cited
US4727020A
Cited
US4666595A
Cited
US4710472A
Cited
US4876189A
Cited
AU2294888A
Cited
US4978625A
Cited
US5085985A
Cited
WO1990002334A1
Cited
CA2012939A1
Cited
CA2012934A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1998-01-30 | 1998-08-01 | 1999-01-31 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2002-01-30 | 2002-08-01 | 2003-01-31 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2006-01-30 | 2006-08-01 | 2007-01-31 | $7700.00 | Window Closed / Lapsed |