Cell separation method using a release system for cell-antibody-substrate conjugates containing a polyethylene glycol spacer unit

1 . A releasable conjugate, comprising:

a biotinylated ligand having a biotin moiety,

a ligand moiety (Ligand 1 ) and a biotin-binding molecule (bbm) bound to the biotin moiety of the biotinylated ligand wherein the biotin moiety and the ligand moiety of the biotinylated ligand are separated by a spacer group consisting of polyethylene glycol according to the general formula I

wherein n is between 1 and 500 inclusive, and wherein X and Y are substituted alkyl groups having between 1 and 20 carbon atoms inclusive with amine, amide, and/or thioether residues, wherein the ligand is a first antibody and the biotin-binding molecule (bbm) is a second antibody which comprises a detection means selected from the group consisting of a chromophor unit, a fluorescence unit, a radioactive unit, and solid support.

2 . The releasable conjugate of claim 1 , wherein the biotinylated ligand is an antibody or a fragment of an antibody directed against antigen expressed intracellularly or extracellularly.

3 . The releasable conjugate of claim 1 , wherein the releasable conjugate has a formula selected from the group IIa, IIb, and IIc

4 . The releasable conjugate of claim 1 , wherein the biotinylated ligand comprises at least one of a chromophor unit, a fluorescence unit, and a radioactive unit as a detection means.

5 . A method for cleaving a releasable conjugate, comprising:

providing the biotinylated ligand according to claim 1 ; and

adding a release agent in a sufficient concentration to displace the biotin-binding molecule (bbm) from the biotin moiety of the biotinylated ligand.

6 . The method of claim 5 , wherein the release agent is at least one of biotin and streptavidin.

7 . The method of claim 5 , further comprising:

providing an additional auxiliary release agent selected from the group of VII, VIII, and IX:

wherein R 1-3, 5-13 is chosen from the group consisting of hydrogen, substituted and unsubstituted alkyl residues having between 1 and 20 carbon atoms, inclusive, and R 4 is chosen from the group consisting of substituted and unsubstituted alkyl residues having between 1 and 20 carbon atoms, inclusive.

8 . A method for the separation of target cells from a cell sample comprising the steps:

incubating the cell sample with a releasable conjugate according to claim 1 , wherein the ligand is a first antibody for which target cells express an antigen;

incubating the cell sample with a second antibody coupled to a solid support;

separating the cells labeled with the solid support from the cell sample by applying an magnetic field; and

adding a release agent in a sufficient concentration to displace the biotin-binding molecule (bbm) from the biotin moiety of the biotinylated ligand.

9 . The method of claim 8 , further comprising:

separating the biotin-binding molecule (bbm) from the target cells.

10 . The method of claim 9 , wherein separating the biotin-binding molecule from the target cells comprises applying a magnetic field to the target cells.

11 . The method of claim 10 , wherein separating the biotin-binding molecule from the target cells further comprises separating the biotin-binding molecule from the target cells in at least one column containing ferromagnetic material.

12 . The method of claim 8 , wherein adding the release agent further comprises:

adding an auxiliary release agent.

13 . The method of claim 9 , further comprising detecting at least one of the biotinylated ligand and the biotin-binding molecule (bbm) subsequent to separating the target cells.

14 . The method of claim 13 , wherein detecting the at least one of the biotinylated ligand and the biotin-binding molecule (bbm) comprises using at least one of a chromophor unit, a fluorescence unit, and a radioactive unit as a detection means.

15 . The method of claim 12 , wherein the release agent is selected from the group consisting of VII, VIII, and IX:

wherein R 1-3, 5-13 is chosen from the group consisting of hydrogen, substituted and unsubstituted alkyl residues having between 1 and 20 carbon atoms, inclusive, and R 4 is chosen from the group consisting of substituted and unsubstituted alkyl residues having between 1 and 20 carbon atoms, inclusive.

16 . The method of claim 8 , wherein the biotinylated ligand is an antibody or a fragment of an antibody directed against antigen expressed intracellularly or extracellularly.

17 . The method of claim 8 , wherein the release agent is at least one of biotin and streptavidin.

18 . The method of claim 8 , wherein the releasable conjugate has a formula selected from the group of IIa, IIb, and IIc: