Cell labeling with antigen-coupled microspheres

• US 4,510,244 A
• Filed: 09/22/1982
• Issued: 04/09/1985
• Est. Priority Date: 04/17/1980
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Herzenberg, Leonard A., Parks, David R., Bryan, Virginia M.
• Primary Examiner(s): Kepplinger, Esther M.
• Application Number:
  US06/421,668
• Time in Patent Office: 930 Days
• Field of Search: 435/7, 435/68, 435/172, 435/241, 435/240, 435/172.2, 436/548, 436/526, 436/527, 436/531, 436/533, 436/534, 436/544, 436/545, 436/546, 436/824, 436/800, 436/804, 436/805, 436/806, 436/519
• US Class Current: 435/325
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/163 one of the fusion partners being a B or a T lymphocyte
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: Y10S 436/824 Immunological separation techniques

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.

Claims

1. 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.

2. A method according to claim 1, wherein said segregation employs a fluorescence activated cell sorter.

3. 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.

4. 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.

5. 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.