US5279936A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Method of separation employing magnetic particles and second medium
Assignee: Siemens Healthcare Diagnostics GmbH Germany
•
Filing: 1989-12-22
•
Granted: 1994-01-18
•
Expires: 2009-12-22
Abstract
Abstract Methods are disclosed for separating a component of interest from a mixture containing the component of interest and other components. The method comprises contacting a first liquid medium containing the component of interest and other components with a second liquid medium that is of different density than and/or of different viscosity than the first liquid medium. The contact is carried out in such a way that mixing of the media is minimized or avoided. The component of interest is bound to magnetic particles. The contacted first liquid medium and second liquid medium are subjected to a magnetic field gradient to allow the magnetic particles to migrate into the second liquid medium and separation of the component of interest from other components is realized. Also disclosed are assays employing the present method. Kits for carrying out the present method and assays are also disclosed.
Inventors
- 👤 John Vorpahl
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 22:02
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/569; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; G01N33/536; G01N33/537; G01N33/5375; G01N33/543; G01N33/54313; G01N33/54326; G01N2333/00; G01N2333/435; G01N2333/76; G01N2333/765; G01N2446/00; G01N2446/20; G01N2446/80; G01N2446/86; Y; Y10; Y10S; Y10S436/00; Y10S436/806; Y10S436/824
Claims Analysis
Total Claims: 87
Claim 1
Independent Claim
169 words
A method for separating a particulate biologic material (PBM), selected from the group consisting of microorganisms, cells, and organelles, from a mixture containing said PBM and other components, which method comprises: contacting a first liquid medium containing said PBM and said other components with a second liquid medium that is of different density and/or viscosity than said first liquid medium without mixing said media, said PBM being nonspecifically bound to magnetic particles (PBMMP) wherein said magnetic particles are selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances and subjecting said first liquid medium and said second liquid medium to a magnetic field gradient whereby said PBMMP migrates into said second liquid medium.
Claim 2
Independent Claim
169 words
A method for separating a particulate biologic material (PBM), selected from the group consisting of microorganisms, cells, and organelles, from a mixture containing said PBM and other components, which method comprises: contacting a first liquid medium containing said PBM and said other components with a second liquid medium that is of different density and/or viscosity than said first liquid medium without mixing said media, said PBM being nonspecifically bound to magnetic particles (PBMMP) wherein said magnetic particles are selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances and subjecting said first liquid medium and said second liquid medium to a magnetic field gradient whereby said PBMMP migrates into said second liquid medium.
Claim 3
Dependent Claim
16 words
The method of claim 1 wherein said second liquid medium is a solution of a polysaccharide.
Claim 4
Dependent Claim
10 words
The method of claim 2 wherein said polysaccharide is sucrose.
Claim 5
Dependent Claim
24 words
The method of claim 1 wherein the density of said second liquid medium is at least 1.1 times that of said first liquid medium.
Claim 6
Dependent Claim
23 words
The method of claim 1 wherein the viscosity of said second liquid medium is at least twice that of said first liquid medium.
Claim 7
Dependent Claim
23 words
The method of claim 1 wherein the viscosity of said first liquid medium is at least twice that of said second liquid medium.
Claim 8
Dependent Claim
13 words
The method of claim 1 wherein said magnetic particles are paramagnetic or superparamagnetic.
Claim 9
Dependent Claim
13 words
The method of claim 7 wherein said particles are 10-100 mn average diameter.
Claim 10
Dependent Claim
17 words
The method of claim 1 wherein said first liquid medium is layered above said second liquid medium.
Claim 11
Dependent Claim
33 words
The method of claim 1 wherein said PBM is a negatively charged non-magnetic particle and said PBM is bound to said magnetic particles, which are negatively charged, by means of a polycationic reagent.
Claim 12
Dependent Claim
27 words
The method of claim 1 wherein said PBM is a non-magnetic particle and said PBM is bound to said magnetic particles by means of a polycationic reagent.
Claim 13
Dependent Claim
11 words
The method of claim 11 wherein said polycationic reagent is polybrene.
Claim 14
Dependent Claim
14 words
The method of claim 1 which comprises separating said PBMMP from said second medium.
Claim 15
Dependent Claim
11 words
The method of claim 13 wherein said separated PBMMP is washed.
Claim 16
Dependent Claim
17 words
The method of claim 13 which comprises reversing the binding of said PBM to said magnetic particles.
Claim 17
Dependent Claim
10 words
The method of claim 1 which comprises detecting said PBM.
Claim 18
Dependent Claim
16 words
The method of claim 1 wherein said PBM has, or is caused to have, a label.
Claim 19
Dependent Claim
23 words
The method of claim 1 wherein said PBM is bound to said magnetic particles by means of ligand-receptor binding or nucleic acid hybridization.
Claim 20
Independent Claim
155 words
A method for separating cells from a mixture containing said cells and other components, which method comprises: layering an aqueous medium containing said cells and said other components with a second liquid medium that is of different density and/or viscosity than said aqueous medium, said cells being non-specifically bound to superparamagnetic or paramagnetic particles wherein said superparamagnetic particles and paramagnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances and, subjecting said aqueous medium and said second liquid medium to a magnetic field gradient sufficient to cause said coaggregate to migrate into said second medium.
Claim 21
Independent Claim
155 words
A method for separating cells from a mixture containing said cells and other components, which method comprises: layering an aqueous medium containing said cells and said other components with a second liquid medium that is of different density and/or viscosity than said aqueous medium, said cells being non-specifically bound to superparamagnetic or paramagnetic particles wherein said superparamagnetic particles and paramagnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances and, subjecting said aqueous medium and said second liquid medium to a magnetic field gradient sufficient to cause said coaggregate to migrate into said second medium.
Claim 22
Dependent Claim
15 words
The method of claim 19 which comprises separating said coaggregate from said second liquid medium.
Claim 23
Dependent Claim
16 words
The method of claim 20 which comprises reversing the coaggregation of said cells and said particles.
Claim 24
Dependent Claim
16 words
The method of claim 19 wherein said second liquid medium is a solution of a polysaccharide.
Claim 25
Dependent Claim
10 words
The method of claim 22 wherein said Polysaccharide is sucrose.
Claim 26
Dependent Claim
23 words
The method of claim 19 wherein the density of said second liquid medium is at least 1.1 times that of said aqueous medium.
Claim 27
Dependent Claim
22 words
The method of claim 19 wherein the viscosity of said second medium is at least two times that of said aqueous medium.
Claim 28
Dependent Claim
15 words
The method of claim 19 wherein said particles are 10 to 100 nm average diameter.
Claim 29
Dependent Claim
16 words
The method of claim 19 wherein said magnetic field gradient is at least about 200 0e.
Claim 30
Dependent Claim
16 words
The method of claim 19 wherein said aqueous medium is layered above said second liquid medium.
Claim 31
Dependent Claim
10 words
The method of claim 19 wherein said cells are erythrocytes.
Claim 32
Dependent Claim
19 words
The method of claim 19 wherein said cells are bound to said particles by means of a cationic reagent.
Claim 33
Dependent Claim
11 words
The method of claim 30 wherein said cationic reagent is polybrene.
Claim 34
Dependent Claim
11 words
The method of claim 19 wherein said separated coaggregate is washed.
Claim 35
Dependent Claim
10 words
The method of claim 19 which comprises detecting said cells.
Claim 36
Dependent Claim
16 words
The method of claim 19 wherein said cells have, or are caused to have, a label.
Claim 37
Independent Claim
169 words
A method for separating erythrocytes from immunoglobulins in a blood sample, which method comprises: combining said blood sample with negatively charged magnetic particles of 10 to 100 nm average diameter wherein said magnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances and a cationic polymer in a first aqueous medium to form a non-specifically bound erythrocyte-magnetic particle complex (EMPC), layering said aqueous medium with a second aqueous medium having at least a two-fold different viscosity than said first aqueous medium, subjecting said layered media to a magnetic field to cause said EMPC to migrate into said second aqueous medium; and separating said EMPC from said second aqueous medium.
Claim 38
Independent Claim
169 words
A method for separating erythrocytes from immunoglobulins in a blood sample, which method comprises: combining said blood sample with negatively charged magnetic particles of 10 to 100 nm average diameter wherein said magnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances and a cationic polymer in a first aqueous medium to form a non-specifically bound erythrocyte-magnetic particle complex (EMPC), layering said aqueous medium with a second aqueous medium having at least a two-fold different viscosity than said first aqueous medium, subjecting said layered media to a magnetic field to cause said EMPC to migrate into said second aqueous medium; and separating said EMPC from said second aqueous medium.
Claim 39
Dependent Claim
14 words
The method of claim 35 wherein said second aqueous medium is a sucrose solution.
Claim 40
Dependent Claim
17 words
The method of claim 35 wherein said first aqueous medium is layered above said second aqueous medium.
Claim 41
Dependent Claim
11 words
The method of claim 35 wherein said cationic polymer is polybrene.
Claim 42
Dependent Claim
11 words
The method of claim 35 wherein said separated EMPC is washed.
Claim 43
Dependent Claim
17 words
The method of claim 35 which comprises reversing any coaggregation of said cells and said magnetic particles.
Claim 44
Dependent Claim
10 words
The method of claim 35 which comprises detecting said cells.
Claim 45
Dependent Claim
16 words
The method of claim 35 wherein said cells have, or are caused to have, a label.
Claim 46
Dependent Claim
120 words
A kit comprising in packaged combination and separated in two or more containers: (a) negatively charged magnetic particles wherein said magnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances, (b) a high density or high viscosity liquid medium, (c) an aqueous medium of a density or viscosity less than said liquid medium of (b) and (d) a polyionic reagent.
Claim 47
Dependent Claim
120 words
A kit comprising in packaged combination and separated in two or more containers: (a) negatively charged magnetic particles wherein said magnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances, (b) a high density or high viscosity liquid medium, (c) an aqueous medium of a density or viscosity less than said liquid medium of (b) and (d) a polyionic reagent.
Claim 48
Dependent Claim
9 words
The kit of claim 43 comprising a polycationic reagent.
Claim 49
Dependent Claim
9 words
The kit of claim 44 comprising a releasing agent.
Claim 50
Dependent Claim
19 words
The kit of claim 43 wherein said magnetic particles have a member of a specific binding pair bound thereto.
Claim 51
Dependent Claim
8 words
The kit of claim 43 comprising non-magnetic particles.
Claim 52
Dependent Claim
19 words
The kit of claim 47 wherein said non-magnetic particles have a member of a specific binding pair bound thereto.
Claim 53
Dependent Claim
17 words
The kit of claim 43 comprising a member of a specific binding pair conjugated to a label.
Claim 54
Dependent Claim
14 words
The kit of claim 49 wherein said label is an enzyme or a chromogen.
Claim 55
Dependent Claim
127 words
A composition comprising: (a) a first liquid medium containing magnetic particles wherein said magnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances, to which are non-specifically bound a particulate biologic material (PBM), selected from the group consisting of microorganisms, cells, and organelles, and (b) a second liquid medium having a different density and/or viscosity than said first liquid medium and in contact but substantially unmixed therewith.
Claim 56
Dependent Claim
127 words
A composition comprising: (a) a first liquid medium containing magnetic particles wherein said magnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances, to which are non-specifically bound a particulate biologic material (PBM), selected from the group consisting of microorganisms, cells, and organelles, and (b) a second liquid medium having a different density and/or viscosity than said first liquid medium and in contact but substantially unmixed therewith.
Claim 57
Dependent Claim
19 words
The composition of claim 51 wherein said PBM is bound to said magnetic particles by means of charge-charge interactions.
Claim 58
Dependent Claim
14 words
The composition of claim 51 wherein said PBM is a cell or a microorganism.
Claim 59
Independent Claim
155 words
A method for conducting an assay for particulate biologic material (PBM), selected from the group consisting of microorganisms, cells, and organelles, said method comprising: contacting a first liquid medium containing said PBM with a second liquid medium that is of different density and/or viscosity than said first liquid medium without mixing said media, said PBM being non-specifically bound to magnetic particles wherein said magnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances, subjecting said media to a magnetic field gradient whereby said magnetic particles migrate into said second liquid medium, and detecting said PBM.
Claim 60
Independent Claim
155 words
A method for conducting an assay for particulate biologic material (PBM), selected from the group consisting of microorganisms, cells, and organelles, said method comprising: contacting a first liquid medium containing said PBM with a second liquid medium that is of different density and/or viscosity than said first liquid medium without mixing said media, said PBM being non-specifically bound to magnetic particles wherein said magnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances, subjecting said media to a magnetic field gradient whereby said magnetic particles migrate into said second liquid medium, and detecting said PBM.
Claim 61
Dependent Claim
17 words
The method of claim 54 wherein said PBM is detected by means of a signal producing system.
Claim 62
Dependent Claim
13 words
The method of claim 55 wherein said signal producing system includes a label.
Claim 63
Dependent Claim
18 words
The method of claim 56 wherein said label is selected from the group consisting of enzymes and chromogens.
Claim 64
Dependent Claim
19 words
The method of claim 56 wherein said label is conjugated to a member of a specific binding pair (sbp).
Claim 65
Dependent Claim
17 words
The method of claim 58 wherein said sbp member is reciprocal to an antigen of said PBM.
Claim 66
Dependent Claim
16 words
The method of claim 54 wherein said second liquid medium is a solution of a polysaccharide.
Claim 67
Dependent Claim
10 words
The method of claim 60 wherein said polysaccharide is sucrose.
Claim 68
Dependent Claim
24 words
The method of claim 54 wherein the density of said second liquid medium is at least 1:1 times that of said first liquid medium.
Claim 69
Dependent Claim
23 words
The method of claim 54 wherein the viscosity of said second liquid medium is at least twice that of said first liquid medium.
Claim 70
Dependent Claim
23 words
The method of claim 54 wherein the viscosity of said first liquid medium is at least twice that of said second liquid medium.
Claim 71
Dependent Claim
13 words
The method of claim 54 wherein said magnetic particles are Paramagnetic or supermagnetic.
Claim 72
Dependent Claim
13 words
The method of claim 65 wherein said particles are 10-100 nm average diameter.
Claim 73
Dependent Claim
17 words
The method of claim 54 wherein said first liquid medium is layered above said second liquid medium.
Claim 74
Dependent Claim
11 words
The method of claim 54 wherein said PBM is an erythrocyte.
Claim 75
Dependent Claim
20 words
The method of claim 54 wherein said PBM is bound to said magnetic particles by means of a polycationic reagent.
Claim 76
Dependent Claim
11 words
The method of claim 69 wherein said polycationic reagent is polybrene.
Claim 77
Dependent Claim
15 words
The method of claim 54 which comprises separating said magnetic particles from said second medium.
Claim 78
Dependent Claim
12 words
The method of claim 71 wherein said separated magnetic particles are washed.
Claim 79
Dependent Claim
17 words
The method of claim 71 which comprises reversing the binding of said PBM to said magnetic particles.
Claim 80
Dependent Claim
23 words
The method of claim 54 wherein said PBM is bound to said magnetic particles by means of ligand-receptor binding or nucleic acid hybridization.
Claim 81
Dependent Claim
166 words
In an assay for a particulate biologic material PBM, selected from the group consisting of microorganisms, cells, and organelles, wherein said assay comprises the step of separating said PBM from other components in a mixture, the improvement comprising: contacting a first liquid medium containing said PBM with a second liquid medium that is of different density and/or viscosity than said first liquid medium without mixing said media, said PBM being non-specifically bound to magnetic particles wherein said magnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances and subjecting said media to a magnetic field gradient whereby said magnetic particles migrate into said second liquid medium.
Claim 82
Dependent Claim
166 words
In an assay for a particulate biologic material PBM, selected from the group consisting of microorganisms, cells, and organelles, wherein said assay comprises the step of separating said PBM from other components in a mixture, the improvement comprising: contacting a first liquid medium containing said PBM with a second liquid medium that is of different density and/or viscosity than said first liquid medium without mixing said media, said PBM being non-specifically bound to magnetic particles wherein said magnetic particles are selected from the group consisting of uncoated pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements; and coated particles containing a core selected from the group consisting of pure metals, alloys, complex salts, oxides, borides, and sulfides of iron, cobalt, nickel and rare earth elements and a coating selected from the group consisting of proteins, carbohydrates and amphiphilic substances and subjecting said media to a magnetic field gradient whereby said magnetic particles migrate into said second liquid medium.
Claim 83
Dependent Claim
17 words
The method of claim 75 wherein said PBM is detected by means of a signal producing system.
Claim 84
Dependent Claim
13 words
The method of claim 76 wherein said signal producing system includes a label.
Claim 85
Dependent Claim
18 words
The method of claim 77 wherein said label is selected from the group consisting of enzymes and chromogens.
Claim 86
Dependent Claim
19 words
The method of claim 79 wherein said label is conjugated to a member of a specific binding pair (sbp).
Claim 87
Dependent Claim
17 words
The method of claim 79 wherein said sbp member is reciprocal to an antigen on said PBM.
References Cited (Backward Citations)
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