US5646001A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Affinity-binding separation and release of one or more selected subset of biological entities from a mixed population thereof
Assignee: Immunivest Corp
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Filing: 1995-02-28
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Granted: 1997-07-08
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Expires: 2015-02-28
Abstract
Abstract A method for separation of a mixture of biological entities into at least three distinct, subpopulations. Different antibodies are provided, with each antibody bound to a solid support in a unique manner such that by a manipulation of the physical or chemical environment, the bonds between the antibodies and the solid supports can be selectively broken. The mixed population of cells is incubated with the antibodies. The cells are magnetically separated from a test medium and collected in a monolayer upon a collection surface. Then by manipulation of the physicochemical environment, specific linkages can be broken and desired cell subpopulations released from the collection surface. This method has medically significant diagnostic and therapeutic applications, as entire cell types can be separated from non-malignant medically vital cell types. Cancer can be diagnosed, staged, and monitored. Genetic analysis from maternal blood, CVS, or amniocentesis samples is possible. Diseases such as AIDS, tuberculosis or hepatitis can be monitored. This invention also has utility in the fields of bone marrow transplantation, fetal cell research, in vitro fertilization, and gene therapy.
Inventors
- 👤 Leon W. M. M. Terstappen
- 👤 Galla C. Rao
- 👤 Dhanesh I. Gohel
- 👤 Brian P. Feeley
- 👤 Steven Gross
- 👤 Ellen S. Church
- 👤 Paul A. Liberti
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 22:59
CPC Classifications
A; A23; A23B; A23B2/00; A23B2/60; B; B03; B03C; B03C1/00; B03C1/02; B03C1/035; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/54313; G01N33/54326; G01N33/569; G01N35/00; G01N35/0098; Y; Y10; Y10S; Y10S435/00; Y10S435/962; Y10S435/973; Y10S436/00; Y10S436/824; Y10S436/825; Y10T; Y10T436/00; Y10T436/25
Claims Analysis
Total Claims: 40
Claim 1
Independent Claim
314 words
A method for isolating a selected subset of biological entities from among a plurality of subsets in a mixed population of said biological entities, said biological entities being selected from the group consisting of cells, bacteria, viruses, or components thereof and toxins, said plurality of subsets comprising a first subset having a first characteristic determinant and at least one other subset having at least a second characteristic determinant, one of said first subset and said at least one other subset being said selected subset, said method comprising the steps of: (a) providing a plurality of capture agents, each capture agent comprising a receptor attached to a solid support, said plurality of capture agents including a first capture agent comprising a receptor which specifically binds, either directly or indirectly, to said first characteristic determinant and at least one other capture agent comprising a receptor which specifically binds, either directly or indirectly, to said second characteristic determinant, the subset to which each said capture agent binds being its target subset; (b) contacting a test sample comprising said mixed population of biological entities with said plurality of capture agents, whereby the receptor of said first capture agent specifically binds to said first subset and the receptor of said at least one other capture agent specifically binds to said at least one other subset, at least one of said first and said second capture agents being dissociably linked to its target subset; (c) collecting on a collection surface the bound subsets from said test sample and separating from said bound subsets any subset of said population than is not bound to a capture agent; (d) selectively releasing one dissociably linked target subset, among said bound subsets, from said collection surface by dissociating said one dissociably linked subset from all or part of the capture agent to which it is bound; and (e) isolating said selectively released target subset.
Claim 2
Independent Claim
314 words
A method for isolating a selected subset of biological entities from among a plurality of subsets in a mixed population of said biological entities, said biological entities being selected from the group consisting of cells, bacteria, viruses, or components thereof and toxins, said plurality of subsets comprising a first subset having a first characteristic determinant and at least one other subset having at least a second characteristic determinant, one of said first subset and said at least one other subset being said selected subset, said method comprising the steps of: (a) providing a plurality of capture agents, each capture agent comprising a receptor attached to a solid support, said plurality of capture agents including a first capture agent comprising a receptor which specifically binds, either directly or indirectly, to said first characteristic determinant and at least one other capture agent comprising a receptor which specifically binds, either directly or indirectly, to said second characteristic determinant, the subset to which each said capture agent binds being its target subset; (b) contacting a test sample comprising said mixed population of biological entities with said plurality of capture agents, whereby the receptor of said first capture agent specifically binds to said first subset and the receptor of said at least one other capture agent specifically binds to said at least one other subset, at least one of said first and said second capture agents being dissociably linked to its target subset; (c) collecting on a collection surface the bound subsets from said test sample and separating from said bound subsets any subset of said population than is not bound to a capture agent; (d) selectively releasing one dissociably linked target subset, among said bound subsets, from said collection surface by dissociating said one dissociably linked subset from all or part of the capture agent to which it is bound; and (e) isolating said selectively released target subset.
Claim 3
Dependent Claim
16 words
A method as claimed in claim 1, including the further step of recovering said isolated subset.
Claim 4
Dependent Claim
64 words
A method as claimed in claim 1, wherein said first or said second capture agent, bearing its target subset and remaining attached to its solid support after performing steps (a)-(e), is dissociably bound to its target subset, and said method further includes the steps of dissociating said remaining capture agent from its target subset and isolating the subset dissociated from said remaining capture agent.
Claim 5
Dependent Claim
22 words
A method as claimed in claim 3, including the further step of recovering said isolated subset dissociated from said remaining capture agent.
Claim 6
Dependent Claim
53 words
A method as claimed in claim 1, wherein the receptor of at least one of said first capture agent and said at least one other capture agent is selected from the group consisting of antibodies, anti-haptens, anti-lectins, peptides, peptide-polynucleotide conjugates, polynucleotides, Protein A, Protein G, concanavilin A, soybean agglutinin, hormones and growth factors.
Claim 7
Dependent Claim
18 words
A method as claimed in claim 1, wherein said capture agents are attached to a stationary solid support.
Claim 8
Dependent Claim
27 words
A method as claimed in claim 6, wherein said solid support is selected from the group consisting of a microtiter plate, test tube, membrane and filter medium.
Claim 9
Dependent Claim
18 words
A method as claimed in claim 1, wherein said capture agents are attached to a mobile solid support.
Claim 10
Dependent Claim
26 words
A method as claimed in claim 8, wherein said solid support is selected from the group consisting of inorganic particulate material and organic-inorganic composite particulate materials.
Claim 11
Dependent Claim
14 words
A method as claimed in claim 8, wherein said solid support is magnetically responsive.
Claim 12
Dependent Claim
63 words
A method as claimed in claim 10, wherein said bound subsets are collected in a substantially uniform thickness on said collection surface under the influence of a magnetic field, said magnetic field being substantially uniform at said collection surface and being stronger in said test sample adjacent to said collection surface than in the test sample which is distant from said collection surface.
Claim 13
Dependent Claim
31 words
A method as claimed in claim 11, wherein said bound subsets are deposited on said collection surface in a thickness which is about the size of said subset-bearing magnetic capture agent.
Claim 14
Dependent Claim
45 words
A method as claimed in claim 1, wherein each of said first capture agent and said at least one other capture agent is in the form of antibody-bound magnetic particles comprising a magnetic metal compound as said solid support and an antibody as said receptor.
Claim 15
Dependent Claim
23 words
A method as claimed in claim 1, wherein the receptor of at least one capture agent is chemically attached to its solid support.
Claim 16
Dependent Claim
26 words
A method as claimed in claim 1, wherein the receptor of at least one capture agent is attached to its solid support via a disulfide bond.
Claim 17
Dependent Claim
26 words
A method as claimed in claim 1, wherein the receptor of at least one capture agent is attached to its solid support via a polynucleotide linkage.
Claim 18
Dependent Claim
20 words
A method as claimed in claim 16, wherein said polynucleotide linkage comprises a recognition sequence for a site-specific cleaving agent.
Claim 19
Dependent Claim
33 words
A method as claimed in claim 14, wherein said capture agent is dissociated from its target subset by chemically disrupting the attachment between the receptor of said capture agent and its solid support.
Claim 20
Dependent Claim
22 words
A method as claimed in claim 1, wherein the receptor of at least one capture agent is adsorbed to its solid support.
Claim 21
Dependent Claim
32 words
A method as claimed in claim 1, wherein said capture agent is dissociated from its target subset by disrupting the attachment between the receptor of said capture agent and its solid support.
Claim 22
Dependent Claim
16 words
A method as claimed in claim 20, wherein said chemical disruption is effected by pH change.
Claim 23
Dependent Claim
15 words
A method as claimed in claim 20, wherein said disruption is effected by temperature change.
Claim 24
Dependent Claim
18 words
A method as claimed in claim 20, wherein said disruption is effected under the influence of electromagnetic radiation.
Claim 25
Dependent Claim
18 words
A method as claimed in claim 20, wherein said disruption is effected under the influence of electric charge.
Claim 26
Dependent Claim
15 words
A method as claimed in claim 20, wherein said disruption is effected by enzyme activity.
Claim 27
Dependent Claim
34 words
A method as claimed in claim 1, wherein the receptor of at least one capture agent is bound directly to its target subset, said receptor being directly or indirectly attached to its solid support.
Claim 28
Dependent Claim
38 words
A method as claimed in claim 26, wherein said capture agent is dissociated from its target subset by disrupting the attachment between the receptor of said capture agent and the determinant of the biological entities comprising said subset.
Claim 29
Dependent Claim
66 words
A method as claimed in claim 1, wherein the receptor of at least one capture agent is bound sequentially to its target subset by a specific-binding substance which specifically binds to the characteristic determinant of the target subset of said capture agent, the receptor of said capture agent binding specifically to said specific binding substance, said receptor being directly or indirectly attached to its solid support.
Claim 30
Dependent Claim
33 words
A method as claimed in claim 28, wherein said capture agent is dissociated from its target subset by disruption of the attachment between the receptor component and solid support of said capture agent.
Claim 31
Dependent Claim
28 words
A method as claimed in claim 1, wherein said mixed population of biological entities is pre-treated to increase the relative content of said selected subset in said population.
Claim 32
Dependent Claim
13 words
A method as claimed in claim 2, wherein the recovered cells are cultured.
Claim 33
Dependent Claim
13 words
A method as claimed in claim 4, wherein the recovered cells are cultured.
Claim 34
Dependent Claim
67 words
A method as claimed in claim 1, wherein said isolated biological entities are subject to analysis by an analytical procedure selected from the group consisting of cell counting, flow cytometry, hematological analysis, polymerase chain reaction (PCR), ligase chain reaction (LCR), image analysis, gene product amplification, message amplification, colorimetric analysis, isotope counting, enzyme-linked immunosorbent assay (ELISA), in situ hybridization, fluorescence in situ hybridization, fluorescent microscopy and light microscopy.
Claim 35
Dependent Claim
37 words
A method as claimed in claim 1, wherein said test sample is a biological fluid selected from the group consisting of blood, bone marrow, sputum, urine, or other bodily fluid, tissue homogenate, disaggregated tissue and cultured cells.
Claim 36
Independent Claim
354 words
A method for isolating a selected subset of cells from among a plurality of cell subsets in mixed cell population in a blood sample, said plurality of subsets comprising a first subset having a first characteristic antigen and at least one other subset having at least a second characteristic antigen, one of said first subset and said at least one other subset being said selected subset, said method comprising the steps of: (a) providing a plurality of capture agents, each capture agent comprising an antibody attached to a solid support, said plurality of capture agents including a first capture agent comprising an antibody which specifically binds, either directly or indirectly, to said first characteristic antigen and at least a one other capture agent comprising an antibody which specifically binds, either directly or indirectly, to said second characteristic antigen, the antibody of each of said first and at least one other capture agent being dissociably attached to its solid support and the attachment of antibody to solid support in said first capture agent being different from the attachment of antibody to solid support in said at least one other capture agent, the subset to which each said capture agent binds being its target subset; (b) contacting said blood sample with said plurality of capture agents, whereby the antibody of said first capture agent specifically binds to said first subset and the antibody of said at least one other capture agent specifically binds to said at least one other subset; (c) collecting on a collection surface the bound subsets from said test sample and separating from said bound subsets any subset of said population that is not bound to a capture agent; (d) selectively releasing one dissociably linked target subset, among said bound subsets, from said collection surface by dissociating said one dissociably linked subset from all or part of its solid support; (e) isolating said selectively released target subset; and (f) dissociating the receptor of the other of said first or at least one other capture agent from its solid support; and (g) isolating the subset that had been bound to said other capture agent.
Claim 37
Independent Claim
354 words
A method for isolating a selected subset of cells from among a plurality of cell subsets in mixed cell population in a blood sample, said plurality of subsets comprising a first subset having a first characteristic antigen and at least one other subset having at least a second characteristic antigen, one of said first subset and said at least one other subset being said selected subset, said method comprising the steps of: (a) providing a plurality of capture agents, each capture agent comprising an antibody attached to a solid support, said plurality of capture agents including a first capture agent comprising an antibody which specifically binds, either directly or indirectly, to said first characteristic antigen and at least a one other capture agent comprising an antibody which specifically binds, either directly or indirectly, to said second characteristic antigen, the antibody of each of said first and at least one other capture agent being dissociably attached to its solid support and the attachment of antibody to solid support in said first capture agent being different from the attachment of antibody to solid support in said at least one other capture agent, the subset to which each said capture agent binds being its target subset; (b) contacting said blood sample with said plurality of capture agents, whereby the antibody of said first capture agent specifically binds to said first subset and the antibody of said at least one other capture agent specifically binds to said at least one other subset; (c) collecting on a collection surface the bound subsets from said test sample and separating from said bound subsets any subset of said population that is not bound to a capture agent; (d) selectively releasing one dissociably linked target subset, among said bound subsets, from said collection surface by dissociating said one dissociably linked subset from all or part of its solid support; (e) isolating said selectively released target subset; and (f) dissociating the receptor of the other of said first or at least one other capture agent from its solid support; and (g) isolating the subset that had been bound to said other capture agent.
Claim 38
Dependent Claim
18 words
A method as claimed in claim 35, wherein at least one of said isolated cell subsets is recovered.
Claim 39
Dependent Claim
18 words
A method as claimed in claim 36, wherein at least one of said recovered cell subsets is cultured.
Claim 40
Dependent Claim
26 words
A method as claimed in claim 35, wherein said first and second capture agents each comprise a monoclonal antibody attached to a particulate, magnetic solid support.
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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) | 2000-07-07 | 2001-01-06 | 2001-07-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-07-07 | 2005-01-06 | 2005-07-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-07-07 | 2009-01-06 | 2009-07-08 | $7700.00 | Window Closed / Lapsed |