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 method for generating coated, gas-filled albumin microbubbles for use in affinity isolation or affinity assay comprising:
(a) heating a solution of albumin;
(b) sonicating the heated solution from step (a) to introduce gas into the solution wherein the solution does not contain any andehyde or Cr+++, thereby generating albumin microbubbles that have not been stabilized by crosslinking the albumin with an aldehyde or by treatment with Cr+++;
(c) suspending the albumin microbubbles from step (b) in a buffered solution comprising polyvinyl alcohol; and
(d) coating the microbubbles from step (c) with an affinity molecule.
2. A method for generating affinity-modified gas-filled protein microbubbles for use in affinity isolation or affinity assay comprising:
(a) providing a protein in a solution, wherein the protein is coupled to an affinity molecule;
(b) heating the solution of protein from step (a);
(c) sonicating the heated solution from step (b) to introduce gas into the solution wherein the solution does not contain any andehyde or Cr+++, thereby generating affinity-modified protein microbubbles, wherein the protein microbubbles are not stabilized by fixing by crosslinking the protein with an aldehyde or by treatment with Cr+++; and
(d) suspending the affinity-modified protein microbubbles from step (c) in a buffered solution comprising polyvinyl alcohol.
3. A method for generating coated gas-filled microbubbles for use in affinity isolation or affinity assay comprising:
(a) heating a solution of protein;
(b) sonicating the heated solution from step (a) to introduce gas into the solution to generate protein microbubbles, wherein the protein microbubbles are not stabilized by crosslinking the protein with an aldehyde or by treatment with Cr+++;
(c) suspending the protein microbubbles from step (b) in a buffered solution comprising polyvinyl alcohol; and
(d) coating the microbubbles from step (c) with an affinity molecule.
4. The method of any one of claim 1 , 2 or 3 , wherein the affinity molecule is selected from the group consisting of a receptor, a ligand, a nucleic acid and an antibody.
5. The method of claim 3 , wherein the affinity molecule is biotin.
6. The method of claim 3 , wherein the affinity molecule is avidin or streptavidin.
7. The method of any one of claim 1 , 2 or 3 , wherein the coating comprises covalently coupling the affinity molecule to an amine group on the microbubble.
8. The method of claim 3 , wherein the coating comprises covalently coupling the affinity molecule to an epoxy group on the microbubble.
9. The method of any one of claim 1 , 2 or 3 , wherein the coating comprises coupling the affinity molecule to the microbubble through a heterobifunctional reagent.
10. The method of claim 9 wherein the method further comprises treating microbubbles with the heterobifunctional reagent sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (s-SMCC)) to form s-SMCC microbubbles.
11. The method of claim 10 wherein the method further comprises treating the affinity molecule with n-succinimidyl S-acetylthioacetate (SATA), and the protective acetyl group is removed just prior to reaction with s-SMCC microbubbles.