US4879220A
Expired / Lapsed
US
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Crosslinking receptor-specific probes for electron microscopy
Assignee: University of Oregon
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Filing: 1986-11-18
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Granted: 1989-11-07
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Expires: 2006-11-18
Abstract
Abstract A method and probes are disclosed for the localization of functional receptors by electron microscopy (EM). Electron dense probes are directed to receptors by means of specific ligand-receptor interactions and subsequently anchored covalently to target cellular material. A particular probe is a complex between colloidal gold and dextran derivatized with diaminohexane and containing photo-activatable nitroarylazido groups. Ligands with a specific affinity for certain receptors can be covalently attached to the complex to provide receptor-specific markers. For example, ouabain can be covalently attached to the dextran matrix to produce a probe specific for functional units of Na,K-ATPase. Covalent attachment of the bound probes at or near their respective receptors greatly enhances retention of probe during washing and standard procedures of fixation and dehydration in preparation for EM.
Inventors
- 👤 Randall J. Mrsny
- 👤 G. Bruce Birrell
- 👤 Johannes J. Volwerk
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 00:14
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/58; G01N33/585; G01N33/53; G01N33/573
Claims Analysis
Total Claims: 42
Claim 1
Independent Claim
80 words
A method for labeling cellular material with colloidal sized particles, the method comprising: providing a probe which includes a colloidal sized particle, a crosslinking agent and a ligand bound to the particles; contacting the probe with a cellular material having a receptor for the ligand so that the ligand joins with a receptor of the cellular material; and inducing the crosslinking agent to react with the cellular material to provide a covalent bond between the probe and the cellular material.
Claim 2
Independent Claim
80 words
A method for labeling cellular material with colloidal sized particles, the method comprising: providing a probe which includes a colloidal sized particle, a crosslinking agent and a ligand bound to the particles; contacting the probe with a cellular material having a receptor for the ligand so that the ligand joins with a receptor of the cellular material; and inducing the crosslinking agent to react with the cellular material to provide a covalent bond between the probe and the cellular material.
Claim 3
Dependent Claim
33 words
The method of claim 1 wherein the ligand and crosslinking agent are attached to the particle by a polymer having functional groups suitable for attachment of both the ligand and the crosslinking agent.
Claim 4
Dependent Claim
21 words
The method of claim 2 wherein the polymer is a water soluble polysaccharide or a derivative thereof having pendant functional groups.
Claim 5
Dependent Claim
15 words
The method of claim 1 wherein the ligand is attached to the particle by dextran.
Claim 6
Dependent Claim
26 words
The method of claim 1 wherein the crosslinking agent has a functional group which can be induced to react with the cellular material by photo irradiation.
Claim 7
Dependent Claim
13 words
The method of claim 1 wherein the crosslinking agent is 6(4'-azido-2'-nitrophenylamino) hexanoyl (SANPAH).
Claim 8
Dependent Claim
10 words
The method of claim 1 wherein the ligand is ouabain.
Claim 9
Dependent Claim
43 words
The method of claim 1 wherein the ligand is a polymer that: has a terminal glucose residue; is water soluble; has a sufficient number of functional groups to allow for covalent attachment of crosslinking moieties; and is capable of binding to the particle.
Claim 10
Dependent Claim
38 words
The method of claim 1 wherein: the ligand is attached to the particle by a dextran derivative; the crosslinking agent is 6(4'-azido-2'-nitrophenylamino) hexanoyl (SANPAH), which is bound to the dextran derivative; and the colloidal sized particle is gold.
Claim 11
Dependent Claim
14 words
A specimen of cellular material prepared for study by the method of claim 1.
Claim 12
Dependent Claim
15 words
The method of claim 1 wherein the colloidal sized particle is an electron dense particle.
Claim 13
Dependent Claim
12 words
The method of claim 11 wherein the particle is a heavy metal.
Claim 14
Dependent Claim
10 words
The method of claim 12 wherein the particle is gold.
Claim 15
Dependent Claim
53 words
A probe for labeling cellular material with a colloidal sized particle, the probe comprising: a colloidal sized particle; a ligand capable of joining with a receptor on cellular material; and a cross linking agent capable of reacting with the cellular material to provide a covalent bond between the probe and the cellular material.
Claim 16
Dependent Claim
53 words
A probe for labeling cellular material with a colloidal sized particle, the probe comprising: a colloidal sized particle; a ligand capable of joining with a receptor on cellular material; and a cross linking agent capable of reacting with the cellular material to provide a covalent bond between the probe and the cellular material.
Claim 17
Dependent Claim
15 words
The probe of claim 14 wherein the ligand is attached to the particle by dextran.
Claim 18
Dependent Claim
26 words
The probe of claim 14 wherein the crosslinking agent has a functional group which can be induced to react with the cellular material by photo irradiation.
Claim 19
Dependent Claim
13 words
The probe of claim 14 wherein the crosslinking agent is 6(4'-azido-2'-nitrophenylamino) hexanoyl (SANPAH).
Claim 20
Dependent Claim
10 words
The probe of claim 14 wherein the ligand is ouabain.
Claim 21
Dependent Claim
43 words
The probe of claim 14 wherein the ligand is a polymer that: has a terminal glucose residue; is water soluble; has a sufficient number of functional groups to allow for covalent attachment of crosslinking moieties; and is capable of binding to the particle.
Claim 22
Dependent Claim
27 words
The probe of claim 14 wherein: the crosslinking agent is 6(4'-azido-2'-nitrophenylamino) hexanoyl (SANPAH), which is bound to the dextran derivative; and the colloidal sized particle is gold.
Claim 23
Dependent Claim
32 words
The probe of claim 14 wherein the ligand and crosslinking agent are attached to the particle by a polymer having functional groups suitable for attachment of the ligand and the crosslinking agent.
Claim 24
Dependent Claim
21 words
The method of claim 21 wherein the polymer is a water soluble polysaccharide or a derivative thereof having pendant functional groups.
Claim 25
Dependent Claim
15 words
The probe of claim 14 wherein the colloidal sized particle is an electron dense particle.
Claim 26
Dependent Claim
12 words
The probe of claim 23 wherein the particle is a heavy metal.
Claim 27
Dependent Claim
10 words
The probe of claim 24 wherein the particle is gold.
Claim 28
Dependent Claim
79 words
A method for isolating molecules or complexes of molecules having receptors, the method comprising: labeling target molecules or a complex of molecules by contacting the molecule or the complex with the probe according to claim 14 and inducing the crosslinking agent to react with the molecule or complex to provide a covalent bond between the probe and the molecule or the complex; and separating the labeled molecule or the complex by action on a physical parameter of the probe.
Claim 29
Independent Claim
48 words
A method for making a probe for labeling cellular material with colloidal-sized particles, the method comprising binding a ligand and crosslinking agent to a colloidal-sized particle, the crosslinking agent being capable of reacting with cellular material to provide a covalent bond between the probe and the cellular material.
Claim 30
Independent Claim
48 words
A method for making a probe for labeling cellular material with colloidal-sized particles, the method comprising binding a ligand and crosslinking agent to a colloidal-sized particle, the crosslinking agent being capable of reacting with cellular material to provide a covalent bond between the probe and the cellular material.
Claim 31
Dependent Claim
19 words
The method of claim 27 wherein the ligand and the crosslinking agent are attached to the particle by dextran.
Claim 32
Dependent Claim
29 words
The method of claim 27 wherein the crosslinking agent has a functional group which can be induced to react with the cellular material by the application of photo irradiation.
Claim 33
Dependent Claim
13 words
The method of claim 27 wherein the crosslinking agent is 6(4'-azido-2'-nitrophenylamino) hexanoyl (SANPAH).
Claim 34
Dependent Claim
10 words
The method of claim 27 wherein the ligand is ouabain.
Claim 35
Dependent Claim
43 words
The method of claim 27 wherein the ligand is a polymer that: has a terminal glucose residue; is water soluble; has a sufficient number of functional groups to allow for covalent attachment of crosslinking moieties; and is capable of binding to the particle.
Claim 36
Dependent Claim
42 words
The method of claim 27 wherein the ligand is selected from the group consisting of biogenic amine neurotransmitters, neurologically active amino acids, neuropeptides, hormones, growth factors, ion channel blockers, enzyme substrates or inhibitors, drugs and tumor promoters, oligonucleotides and complementary DNA fragments.
Claim 37
Dependent Claim
35 words
The method of claim 27 wherein: the ligand and the crosslinking agent are attached to the particle by a dextran derivative; the crosslinking agent is 6(4'-azido-2'-nitrophenylamino) hexanoyl (SANPAH); and the colloidal sized particle is gold.
Claim 38
Dependent Claim
33 words
The method of claim 27 wherein the ligand and the crosslinking agent are attached to the particle by a polymer having functional groups suitable for attachment of the ligand and the crosslinking agent.
Claim 39
Dependent Claim
21 words
The method of claim 35 wherein the polymer is a water soluble polysaccharide or a derivative thereof having pendant functional groups.
Claim 40
Dependent Claim
15 words
The method of claim 27 wherein the colloidal sized particle is an electron dense particle.
Claim 41
Dependent Claim
12 words
The method of claim 37 wherein the particle is a heavy metal.
Claim 42
Dependent Claim
10 words
The method of claim 38 wherein the particle is gold.
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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) | 1992-11-06 | 1993-05-08 | 1993-11-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-11-06 | 1997-05-08 | 1997-11-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-11-06 | 2001-05-08 | 2001-11-07 | $7700.00 | Window Closed / Lapsed |