Gel-shell beads with adsorbed or bound biomolecules

• US 20050112655A1
• Filed: 10/26/2004
• Published: 05/26/2005
• Est. Priority Date: 10/28/2003
• Status: Active Grant

• Current Assignee: BioArray Solutions Limited (Werfen SA)
• Original Assignee: BioArray Solutions Limited (Werfen SA)
• Inventors: Banerjee, Sukanta, Tan, Enqing
• Granted Patent: US 7,582,488 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386

Abstract

Disclosed are gel-coated beads (including Hydrogel™-coated beads), which are capable of adsorbing, or absorbing, proteins and other biomolecules onto or into the gel coating. The gel-coated beads with absorbed or adsorbed biomolecules are suitable for use in an assays, purification or other purposes. The beads have a core made from any of a number of materials, including latex, coated with the gel shell. The biomolecules can be retained within the gel, following adsorption, by covalent attachment, or, by selection of conditions of ambient pH and/or ionic strength such that they are retained without further reaction. Therefore, adsorbed proteins would retain the ability to bind to their respective ligands.

Claims

1. A method of detecting particular binding molecules, comprising:

providing beads having a core and a shell, where the shell includes an ionic gel and wherein a biomolecule is immobilized in the ionic gel;

contacting the beads with said binding molecules; and

determining whether binding takes place based on detection of signal from labeling associated with the binding molecules.

2. The method of claim 1 wherein the biomolecule is a protein or an oligonucleotide.

3. The method of claim 2 wherein the biomolecule is a protein and the binding molecules are antibodies, antigen or enzymes.

4. The method of claim 2 wherein the biomolecule is an oligonucleotide and the binding molecule is an oligonucleotide.

5. The method of claim 3 wherein the labeling is with a labeled secondary antibody, or another labeled molecule, which binds to the antibodies, antigen or enzymes.

6. The method of claim 1 wherein the ionic gel is Hydrogel™

.

7. The method of claim 2 wherein the protein is immobilized by covalent bonding between functional groups in the protein and the ionic gel.

8. The method of claim 7 wherein the covalent bonding is by forming an amide bond between carboxyl groups in the Hydrogel™

and the accessible free amine groups present in the side chains of the amino acids on the protein molecule by using an EDAC-mediated coupling reaction.

9. The method of claim 7 wherein the covalent bonding is by the carboxyl group being first attached to a homo or hetero-bifunctional ligand and then the functional group of the ligand utilized for covalent attachment of the protein

10. The method of claim 2 wherein the protein is immobilized under conditions where pH is about 3 and the ionic concentration is less than about 100 mM.

11. The method of claim 10 wherein the ion monitored in determining ionic concentration is sodium.

12. The method of claim 1 wherein the ionic concentration is greater than about 1M.

13. The method of claim 12 wherein the ion monitored in determining ionic concentration is sodium.

14. A method of detecting and purifying protein-binding molecules, comprising:

providing a plurality of beads having a core and a shell inside of a transparent column, where the shell includes an ionic gel and wherein protein is immobilized in the ionic gel;

contacting the beads with said protein-binding molecules;

determining, without removing beads from the column, whether binding takes place between the protein-binding molecules and the protein based on detection of signal from labels associated with the protein-binding molecules; and

removing the beads and removing the protein-binding molecules from the beads.

15. The method of claim 14 wherein the protein-binding molecules are proteins including antibodies, antibody fragments, antigen or enzymes.

16. The method of claim 15 wherein the ionic gel is Hydrogel™

.

17. The method of claim 15 wherein the protein is immobilized by covalent bonding between functional groups in the protein and the ionic gel.

18. The method of claim 16 wherein the covalent bonding is by forming an amide bond between carboxyl groups in the Hydrogel™

and the accessible free amine groups present in the side chains of the amino acids on the protein molecule by using an EDAC-mediated coupling reaction.

19. The method of claim 17 wherein the covalent bonding is by the carboxyl group being first attached to a homo or hetero-bifunctional ligand and then the functional group of the ligand utilized for covalent attachment of the protein

20. The method of claim 17 wherein the protein is immobilized under conditions where pH is about 3 and the ionic concentration is less than about 100 mM.

21. The method of claim 20 wherein the ion monitored in determining ionic concentration is sodium.

22. The method of claim 21 wherein the ionic concentration is greater than about 1M.

23. The method of claim 22 wherein the ion monitored in determining ionic concentration is sodium.

25. The method of claim 2415 wherein the molecules are proteins including antibodies, antibody fragments, antigen or enzymes.

24. A method of monitoring reactions of molecules, comprising:

providing a plurality of beads having a core and a shell, where the shell includes an amphiphilic gel, having hydrophilic and hydrophobic domains, and wherein hydrophilic biomolecules are immobilized in hydrophilic domains of the amphiphilic gel and hydrophobic biomolecules are immobilized in hydrophobic domains of the amphiphilic gel;

contacting the beads with molecules which bind to hydrophilic biomolecules and with molecules which bind to hydrophobic biomolecules; and

determining whether binding takes place between the molecules and the biomolecules based on detection of signal from labels associated with the molecules.

26. A method of permitting certain reactions between biomolecules in a gel and certain molecules to take place and preventing reactions among other molecules, comprising:

providing a plurality of beads having a core and a shell, where the shell includes a gel which contains biomolecules, and molecules of greater than a certain size or bearing a designated charge cannot enter the gel;

contacting the beads with a sample of molecules such that some molecules in the sample enter the gel and react with the biomolecules but wherein other molecules are excluded.

27. The method of claim 26 further including determining whether a reaction takes place between the molecules and the biomolecules based on detection of signal from labels associated with the molecules.

28. The method of claim 26 wherein the molecules are proteins including antibodies, antibody fragments, antigen or enzymes.