Restricted access media and methods for making restricted access media
The present invention is directed to restricted access media (RAM), methods for preparing restricted access media, and kits for preparing restricted access media that contain protected ligand binding agents or protected enzymes. Certain RAM provided contain a plurality of protected regions of the support that contain ligand binding agents that are protected by blocking agents. Certain RAM provided contain a plurality of protected regions of the support that contain unbound ligand binding agents or enzymes that are retained in the protected regions by a capping agent. Methods of making the RAM of the invention and associated kits are also provided.
1. A restricted access media comprising a support wherein a plurality of protected regions of said support contain one or more covalently linked ligand binding agent molecule(s) and wherein a plurality of unprotected regions of said support contain one or more covalently linked blocking agent molecule(s) wherein the blocking agent molecules exhibit reduced binding or no binding to the ligand that is recognized by the ligand binding agent.
2. The restricted access media of claim 1 , wherein said support comprises an inorganic material, a biological material, an organic material, an organic polymer, a composite support, or a modified support.
3. The restricted access media of claim 1 , wherein said ligand binding agent molecule comprises a protein, a glycoprotein, a DNA, a RNA, a nucleoprotein, or a carbohydrate containing agent.
4. The restricted access media of claim 3 , wherein the protein comprises an antibody or ligand binding fragment thereof.
5. The restricted access media of claim 1 , wherein said blocking agent molecule comprises a sub-fragment of said ligand binding agent molecule wherein ligand binding activity is reduced or eliminated.
6. A method for preparing a restricted access media comprising:
a. incubating an activated support with a solution comprising a plurality of ligand binding agent molecules or ligand binding agent molecules that are conjugated to linking agents to form a ligand bound support wherein said ligand binding agent molecules are covalently linked to protected and unprotected regions of said support either directly or by said linking agent;
b. treating said ligand bound support with a size restricted cleaving agent that cleaves said ligand binding agent molecules that are covalently bound to unprotected regions of said ligand bound support or that cleaves the ligand binding agent molecules that are conjugated to linking agent molecules that are covalently bound to unprotected regions of said ligand bound support, wherein cleavage of the ligand binding agent molecules or cleavage of the ligand binding agent-linking agent molecules that are covalently bound to the unprotected regions of the support by the cleaving agent forms covalently linked blocking agent molecules in the unprotected regions; and
c. removing said cleaving agent and unbound fragments of cleaved ligand binding agent molecules or linking agent—ligand binding agent conjugate molecules, thereby preparing a restricted access media comprising a support wherein a plurality of protected regions of said support contain one or more covalently linked ligand binding agent molecule(s) and wherein a plurality of unprotected regions of said support contain one or more covalently linked blocking agent molecule(s).
7. The method of claim 6 , wherein the support comprises a pore size in the range of about 50 Å to about 500 Å.
8. The method of claim 6 , wherein said ligand binding agent molecule comprises a protein, a glycoprotein, a DNA, a RNA, a nucleoprotein, or a carbohydrate containing agent or wherein said linking agent comprises a protein, a glycoprotein, a DNA, a RNA, a nucleoprotein, or a carbohydrate containing agent.
9. The method of claim 8 , wherein said protein comprises an antibody.
10. The method of claim 6 , wherein said ligand binding agent molecule comprises a protein and said size restricted cleaving agent comprises a protease.
11. The method of claim 6 , wherein said activated support is activated by an aldehyde or a hydrazide.
12. A kit comprising a restricted access media comprising: i) a support wherein a plurality of protected regions of said support contain one or more covalently linked ligand binding agent molecules and wherein a plurality of unprotected regions of said support contain one or more covalently linked blocking agent molecule(s) wherein the blocking agent molecules exhibit reduced binding or no binding to the ligand that is recognized by the ligand binding agent and ii) at least one container.
13. The kit of claim 12 , wherein said support comprises an inorganic material, a biological material, an organic material, an organic polymer, a composite support, or a modified support.
14. The kit of claim 12 , wherein said ligand binding agent molecule comprises a protein, a glycoprotein, a DNA, a RNA, a nucleoprotein, or a carbohydrate containing agent.
15. The kit of claim 14 , wherein the protein comprises an antibody or ligand binding fragment thereof.
16. The kit of claim 12 , wherein said blocking agent molecule comprises a sub-fragment of said ligand binding agent molecule wherein ligand binding activity is reduced or eliminated.
17. The kit of claim 12 , wherein said covalently linked ligand binding agent is covalently linked to said support with a linking agent and the blocking agent is a sub-fragment of the linking agent.