Method for separating cells using immunorosettes and magnetic particles
The present invention relates to methods for separating target cells from non-target cells using immunorosettes and magnetic particles. The method involves contacting a sample containing target cells and secondary targets such as erythrocytes with an antibody composition which allows immunorosettes of the target cells and the secondary targets to form. The sample is subsequently contacted with a second antibody composition which allows the binding of magnetic particles to the formed immunorosettes and free secondary targets. The immunorosettes and secondary targets labeled with magnetic particles are separated from non-target cells using a magnetic field. The antibody composition optionally contains bifunctional antibodies or tetrameric antibody complexes.
1. A selection method for separating target cells from non-target cells in a sample comprising target cells, secondary targets and non-target cells, the method comprising:
a) contacting the sample with a first antibody composition comprising both (a) at least one antibody that binds to the target cells and (b) at least one antibody that binds to the secondary targets, wherein the (a) at least one antibody that binds to the target cells is linked, either directly or indirectly, to (b) at least one antibody that binds to the secondary targets, and wherein the contacting is performed under conditions to allow immunorosettes of the target cells and the secondary targets to form;
b) contacting the sample with particles that are not cells and a second antibody composition comprising
(i) both (a) at least one antibody that binds to the secondary targets either singly or within the immunorosettes, and (b) at least one antibody that binds to a polymer coated on the particles wherein the (a) at least one antibody that binds to the secondary targets is linked, either directly or indirectly, to (b) the at least one antibody that binds to the polymer coated on the particles, or
(ii) both (a) at least one antibody that binds to the secondary targets either singly or within the immunorosettes, and (b) at least one antibody that also binds to the secondary targets either singly or within the immunorosettes, wheren the (a) at least one antibody that binds to the secondary targets is linked, either directly or indirectly, to (b) the st least one antibody that binds to the secondary targets to form an antibody complex, the antibody complex directly bound to the particles, or
(iii) at least one antibody that binds to the secondary targets either singly or within the immunorosettes, wherein the at least one antibody is directly bound to the particles,
under conditions to allow binding of the particles to the immunorosettes and/or the secondary targets to form immunorosette-particle complexes and/or secondary target-particle complexes, and
c) separating the immunorosette-particle complexes and/or the secondary target-particle complexes from the sample to separate the target cells from the non-target cells.
2. The method of claim 1 wherein the second antibody composition is bound to the particles prior to contacting with the sample.
3. The method of claim 1 wherein the second antibody composition is bound to the particle and added to the sample prior to contacting the sample with the first antibody composition.
4. The method of claim 1 , wherein the secondary targets are erythrocytes, granulocytes, or beads, optionally polystyrene beads.
5. The method of claim 1 , wherein the secondary targets are added to the sample.
6. The method of claim 1 , wherein the particles are non-magnetic particles.
7. The method of claim 1 , wherein the selection method is a positive selection method to select for a desired cell and the target cell are the desired cell or a negative selection method to remove a non-desired cell from the sample and the target cell are the non-desired cell.
8. The method of claim 1 , wherein the secondary targets have a density similar to the target cells and the particles have a density different from the target cells.
9. The method of claim 1 , wherein the immunorosette- particle complexes are separated from the sample in step c) by density separation or by sedimentation.
10. The method of claim 7 , wherein the method further comprises:
d) separating the target cells from the immunorosettes-particle complexes.
11. The method of claim 8 , wherein the target cells are separated from the immunorosette-magnetic particle complexes by physical, chemical, enzymatic or thermal dissociation.
12. The method of claim 4 , wherein the method further comprises:
d) lysis of the erythrocytes in the immunorosettes and
e) separation of the target cells from the lysed erythrocytes and the particles.
13. The method of claim 1 , wherein the sample is blood, whole blood, bone marrow, fetal liver, cord blood, a buffy coat suspension, a leukapheresis sample, a pleural and periotoneal effusion, a sample of thymocytes or a sample of splenocytes.
14. The method of claim 1 , wherein the target cells are stem cells, progenitor cells, monocytes, lymphocytes, or granulocytes.
15. The method of claim 14 , wherein the lymphocytes are T cells.