APTAMER BASED AFFINITY CAPTURE METHODS FOR THE SELECTIVE ENRICHMENT OF HUMAN IMMUNOGLOBULIN FC DOMAINS

Abstract

A method of capturing human immunoglobulin Fc domains in a biofluid sample is provided. The method includes providing an affinity capture device. The affinity capture device includes a surface having an aptamer that is at least 80% identical to SEQ ID NO 1 immobilized onto the surface of the affinity capture device. The biofluid sample is diluted with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) a magnesium cation at a concentration between about 10 µM to about 20 mM; and (C) a total monovalent cation concentration from 0 to no greater than 100 mM. The human immunoglobulin Fc domains in the biofluid sample are adsorbed to the aptamer with the binding buffer.

Description

Claims

1. A method of capturing human Fc domains found in a biofluid sample, the method comprising: providing an affinity capture device comprising a surface having an aptamer that binds to an Fc region;diluting the biofluid sample with a binding buffer comprising: (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES);(B) a magnesium cation at a concentration between about 10 µM to about 20 mM; and(C) a total monovalent cation concentration from 0 to no greater than 100 mM; andadsorbing the human Fc domains in the biofluid sample to the aptamer with the binding buffer.

2. The method of claim 1, wherein the aptamer is a 23-nucleotide aptamer.

3. The method of claim 1, wherein the aptamer is non-covalently immobilized onto the surface of the affinity capture device.

4. The method of claim 1, wherein the aptamer is covalently immobilized onto the surface of the affinity capture device.

5. The method of claim 1, wherein the binding buffer has a concentration of monovalent cations less than about 50 mM.

6. The method of claim 1, wherein the pH of the binding buffer is between about 5 and about 9.

7. The method of claim 1, wherein the biofluid sample is diluted by a factor of 2, 10, or 20.

8. The method of claim 1, wherein the biofluid sample is diluted to obtain a total monovalent cation concentation of the biofluid sample of no greater than 100 mM.

9. The method of claim 1, further comprising eluting the adsorbed human Fc domains from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM to about 1000 mM, wherein the ammonium is in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate.

10. The method of claim 9, wherein the eluent has a pH between about 6.5 to about 8.0.

11. The method of claim 1, further comprising washing the adsorbed human Fc domains with the binding buffer.

12. The method of claim 1, further comprising washing the adsorbed human Fc domains with a buffer comprising Ca+.

13. The method of claim 1, wherein the concentration of the magnesium cation is between about 50 µM to about 1 mM.

14. The method of claim 9, further comprising analyzing the eluted human Fc domains with a detector.

15. The method of claim 14, wherein the detector is a sandwiched enzyme linked immunosorbent assay or a mass spectrometer.

16. A method of capturing human Fc domains found in a biofluid sample, the method comprising: providing an affinity capture device comprising a surface having an anti-human Fc aptamer;diluting the biofluid sample with a binding buffer comprising: (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES);(B) a magnesium cation at a concentration between about 10 µM to about 20 mM; and(C) a total monovalent cation concentration from 0 to no greater than 100 mM; andadsorbing the human Fc domains in the biofluid sample to the aptamer with the binding buffer.

17. A method of capturing human Fc domains found in a biofluid sample, the method comprising: providing an affinity capture device comprising a surface having a metal-dependent aptamer that binds to an Fc region;diluting the biofluid sample with a binding buffer comprising: (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES);(B) a magnesium cation at a concentration between about 10 µM to about 20 mM; and(C) a total monovalent cation concentration from 0 to no greater than 100 mM; andadsorbing the human Fc domains in the biofluid sample to the aptamer with the binding buffer.