US4510244A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Cell labeling with antigen-coupled microspheres
Assignee: Leland Stanford Junior University
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Filing: 1982-09-22
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Granted: 1985-04-09
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Expires: 2002-09-22
Abstract
Abstract Method for isolating specific antibody hybridomas from a hybridoma cell mixture employing antigen-conjugated labeled microspheres and a label activated cell sorter. By selecting for labeled cells which produce light scatter and low red autofluorescence, viable single cells can be isolated and cloned which produce the desired antibodies.
Inventors
- 👤 David R. Parks
- 👤 Virginia M. Bryan
- 👤 Leonard A. Herzenberg
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 10:28
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/10; C12N5/12; C12N5/16; C12N5/163; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/569; G01N33/56966; G01N33/58; G01N33/585; C12N2510/00; C12N2510/02; Y; Y10; Y10S; Y10S436/00; Y10S436/824
Claims Analysis
Total Claims: 7
Claim 1
Independent Claim
124 words
A method for selectively isolating a hybridoma cell producing antibodies to a specific determinant site which comprises: combining a heterogeneous mixture of hybridoma cells producing a variety of antibodies including an antibody for said determinant site with said determinant site bound fluorescent labeled microspheres, wherein said label permits mechanical selection of cells labeled with said microspheres as compared to cells which are unlabeled; centrifuging said mixture of hybridoma cells and microspheres to provide a monolayer of sedimented cells, followed by separating bound microspheres from unbound microspheres; selectively segregating by means of said microsphere label from cells having fewer microspheres than said predetermined number individual cells labeled with said microspheres at a predetermined minimum number of microspheres per cell; and growing said selectively segregated cell.
Claim 2
Independent Claim
124 words
A method for selectively isolating a hybridoma cell producing antibodies to a specific determinant site which comprises: combining a heterogeneous mixture of hybridoma cells producing a variety of antibodies including an antibody for said determinant site with said determinant site bound fluorescent labeled microspheres, wherein said label permits mechanical selection of cells labeled with said microspheres as compared to cells which are unlabeled; centrifuging said mixture of hybridoma cells and microspheres to provide a monolayer of sedimented cells, followed by separating bound microspheres from unbound microspheres; selectively segregating by means of said microsphere label from cells having fewer microspheres than said predetermined number individual cells labeled with said microspheres at a predetermined minimum number of microspheres per cell; and growing said selectively segregated cell.
Claim 3
Dependent Claim
15 words
A method according to claim 1, wherein said segregation employs a fluorescence activated cell sorter.
Claim 4
Independent Claim
34 words
A method according to any of claims 1 to 2, wherein said cells are further selectively segregated by a level of light scattering and endogeneous red fluorescence to selectively segregate live from dead cells.
Claim 5
Dependent Claim
32 words
A method according to claim 1, wherein said microspheres are organic polymer microspheres of from about 0.1 to 2 micrometer diameter having fluorescent chromophore functionalities having excitation at greater than 450 nm.
Claim 6
Independent Claim
123 words
A method for producing specific monoclonal antibodies which comprises: combining a heterogeneous mixture of hybridoma cells capable of producing a variety of antibodies including said specific monoclonal antibodies with a mixture of fluorescent microspheres, capable of excitation above about 450 nm and to which the homologous determinant site for said monoclonal antibodies is covalently bonded, wherein said microspheres are coated with sufficient protein to inhibit aggregation; centrifuging the mixture of microspheres and hybridoma cells to produce a cell layer; incubating said cell layer; separating unbound microspheres from said cell layer; selectively segregating cells having a minimum of a predetermined number of microspheres bound to said cells from cells having fewer bound microspheres; and growing said selectively segregated cells to produce said monoclonal antibody.
Claim 7
Independent Claim
123 words
A method for producing specific monoclonal antibodies which comprises: combining a heterogeneous mixture of hybridoma cells capable of producing a variety of antibodies including said specific monoclonal antibodies with a mixture of fluorescent microspheres, capable of excitation above about 450 nm and to which the homologous determinant site for said monoclonal antibodies is covalently bonded, wherein said microspheres are coated with sufficient protein to inhibit aggregation; centrifuging the mixture of microspheres and hybridoma cells to produce a cell layer; incubating said cell layer; separating unbound microspheres from said cell layer; selectively segregating cells having a minimum of a predetermined number of microspheres bound to said cells from cells having fewer bound microspheres; and growing said selectively segregated cells to produce said monoclonal antibody.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-04-08 | 1988-10-08 | 1989-04-09 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-04-08 | 1992-10-08 | 1993-04-09 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-04-08 | 1996-10-08 | 1997-04-09 | $7700.00 | Window Closed / Lapsed |