Microbubbles for affinity separation
The present invention relates to methods, compositions and kits for affinity isolation, affinity purification and affinity assay based on microbubbles coated with an affinity molecule. Particularly, the invention provides protein microbubbles coated with an affinity molecule. In addition, the invention provides glass microbubbles coated with an affinity molecule. Methods of using the microbubbles of the invention for isolating analytes and cells are specifically provided.
1. A composition for use in affinity isolation or affinity assay comprising heat-denatured, air-filled serum albumin microbubbles which are not stabilized by fixing by crosslinking the serum albumin or by treatment with Cr+++, wherein the air does not comprise a fluorocarbon gas, and microbubbles are suspended in a buffer containing polyvinyl alcohol (PVA), and further wherein the microbubbles are coated with a protein, polypeptide or peptide affinity molecule.
2. The composition of claim 1 , wherein the serum albumin microbubbles are formed by the introduction of air into a solution of serum albumin.
3. The composition of claim 2 , wherein the introduction comprises sonication.
4. The composition of claim 2 , wherein the introduction comprises heating the solution of serum albumin.
5. The composition of claim 1 , wherein the affinity molecule is selected from the group consisting of: a receptor, a protein, polypeptide or peptide ligand, and an antibody.
6. The composition of claim 1 , wherein the affinity molecule is biotin.
7. The composition of claim 1 , wherein the affinity molecule is avidin or streptavidin.
8. The composition of any of claims 5 - 7 , wherein the affinity molecule is directly coupled to the serum albumin.
9. The composition of claim 8 , wherein the affinity molecule is directly coupled to the protein using a heterobifunctional reagent.
10. The composition of claim 9 , wherein the heterobifunctional reagent is sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.
11. The composition of any of claims 5 - 7 , wherein the affinity molecule is indirectly coupled to the serum albumin.
12. The composition of claim 11 , wherein the affinity molecule is indirectly coupled to the serum albumin through the interaction of at least one other molecule.
13. The composition of claim 12 , wherein the serum albumin is directly coupled to streptavidin and the affinity molecule is biotinylated, wherein the streptavidin and biotin interact, thereby indirectly coupling the affinity molecule to the serum albumin.
14. The composition of claim 12 , wherein the affinity molecule is directly coupled to streptavidin and the serum albumin is biotinylated, wherein the streptavidin and biotin interact, thereby indirectly coupling the protein affinity molecule to the serum albumin.
15. The composition of claim 4 , wherein the introduction comprises heating the solution of serum albumin to a process temperature of 73° C.