Method of heterogenous purification using a bidentate conjugate

• US 5,534,620 A
• Filed: 05/25/1995
• Issued: 07/09/1996
• Est. Priority Date: 09/30/1987
• Status: Expired due to Term

• Current Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Original Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Inventors: Kearns, Elizabeth K., Oh, Chan S.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/450,652
• Time in Patent Office: 411 Days
• Field of Search: 530/413, 530/412, 530/344, 530/345, 436/501, 436/536, 436/543, 435/174, 435/177, 435/181, 435/6
• US Class Current: 530/413
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/539 involving precipitating reagent , e.g. ammonium sulfate
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/94 involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors

Abstract

A novel heterogenous purification method is disclosed. The method removes a macromolecule from a liquid by allowing the macromolecule to undergo a specific binding affinity reaction with a bidentate conjugate. The method is carried out by immobilizing the bidentate conjugate on a solid phase, contacting the bidentate conjugate with the liquid comprising the macromolecule, and separating the immobilized bidentate conjugate from contact with the liquid, thereby removing the macromolecule from the liquid to obtain either purified macromolecule or a purified liquid.

Claims

1. A heterogenous purification method with a solid phase and a liquid phase, the method comprising the sequential steps of:

(a) providing a bidentate conjugate comprising a first bidentate member attached to a second bidentate member through a spacer moiety wherein the spacer moiety comprises between about 16 atoms and about 22 atoms and the spacer moiety has a length between about 21 Å and

about 29 Å

, said first and second bidentate members being different small molecule ligands, each of the ligands being capable of specifically binding to their respective and different first and second macromolecular specific binding partners;

(b) immobilizing said bidentate conjugate on a solid phase;

(c) contacting the immobilized bidentate conjugate with a liquid phase comprising a first macromolecular specific binding partner capable of adhering in a specific binding affinity reaction to the bidentate conjugate;

(d) separating the immobilized bidentate conjugate with the adhered first macromolecular specific binding partner from contact with the liquid phase;

(e) eluting from said bidentate conjugate a first macromolecular specific binding partner with a substantially undiminished biological activity as compared to the biological activity of the first macromolecular specific binding partner prior to adherence to the bidentate conjugate, thereby obtaining a purified first macromolecular binding partner; and

(f) regenerating the solid phase to thereby render it reusable for the purification of additional first macromolecular binding partner.

2. The method of claim 1, wherein the bidentate conjugate is immobilized by attaching the second bidentate member of the bidentate conjugate to the solid phase.

3. The method of claim 2, further comprising the step of attaching the second macromolecular specific binding partner to the solid prior to the immobilizing step.

4. The method of claim 3, wherein the bidentate conjugate is immobilized on the solid by contacting the second bidentate member of the bidentate conjugate with the second macromolecular specific binding partner attached to the solid.

5. The method of claim 1, wherein the eluting step is carried out by a method selected from the group consisting of contacting the immobilized bidentate conjugate with an acidic medium, by mass-displacement, and by changing the pH, or the ionic strength or the temperature of an eluting buffer used to elute the first macromolecular specific binding partner.

6. The method of claim 1, wherein the first macromolecular specific binding partner is an antibody to the first bidentate member.

7. The method of claim 1, wherein the first bidentate member has affinity binding characteristics which are identical or analogous to an analyte of interest.

8. The method of claim 7 wherein the analyte of interest is selected from the group consisting of haptens, hormones, polypeptides, oligonucleotides, and vitamins.

9. The method of claim 8 wherein the analyte of interest is a hapten with a molecular weight between about 100 and 1500 Daltons.

10. The method of claim 9 wherein the hapten is selected from the group consisting of theophylline;

digoxin;

disopyramide;

lidocaine;

N-acetylprocainamide;

procainamide;

propanolol;

quinidine;

amykamycin;

chloramphenicol;

gentamicin;

kanamycin;

netilmycin;

tobramycin;

tricyclic antidepressants;

ethosuximide;

phenobarbital;

phenytoin;

primidone;

valproic acid;

acetaminophen;

acetylsalicylic acid;

methotrexate;

morphine;

codeine; and

heroin;

dinitrophenol;

1-substituted-4-hydroxy-2-nitrobenzene; and

4-substituted-2-nitro-trialkylanilinium.

11. The method of claim 1 wherein the second bidentate member is biotin.

12. The method of claim 1 wherein the first bidentate member is N-acetylprocainamide.

13. The method of claim 1 wherein the spacer moiety is at least about 22 Å

in length.

14. The method of claim 1 wherein both the first and second macromolecular specific binding partners are multivalent.

15. A heterogenous purification method, comprising the sequential steps of:

(a) selecting a bidentate conjugate comprising a first bidentate member attached to a second bidentate member through a spacer moiety, wherein the first and second bidentate members are different small molecule ligands and the spacer moiety comprises between about 16 atoms and about 22 atoms and the spacer moiety has a length between about 21 Å and

about 29 Å

, and each of the first and second bidentate members is capable of specifically binding to its respective and different first and second macromolecular specific binding partners;

(b) attaching the second macromolecular specific binding partner to a solid phase;

(c) immobilizing a bidentate conjugate on the solid phase by attaching the second bidentate member of the bidentate conjugate to the second macromolecular specific binding partner attached to the solid phase;

(d) contacting the immobilized bidentate conjugate with a liquid comprising the first macromolecule specific binding partner; and

,

(e) separating the immobilized bidentate conjugate from contact with the liquid, thereby obtaining a purified first macromolecular specific binding partner.

16. An affinity chromatography method for the isolation of a specific antibody for a ligand from a solution containing the antibody, comprising the steps of:

(a) attaching a bidentate conjugate to a solid, wherein the bidentate conjugate comprises a ligand and a spacer moiety, the spacer moiety comprising between about 16 atoms and about 22 atoms and the spacer moiety has a length between about 21 Å and

about 29 Å

;

(b) contacting the bidentate conjugate with a solution comprising a specific antibody for the ligand;

(c) separating the bidentate conjugate with the ligand bound antibody from contact with the solution; and

(e) regenerating the solid to thereby render it reusable for the isolation of additional specific antibody.

17. The method of claim 16, further comprising the step of removing the antibody from contact with the ligand of the bidentate conjugate.