Immobilized cyclophilin and methods of using such

• US 5,047,512 A
• Filed: 06/23/1987
• Issued: 09/10/1991
• Est. Priority Date: 05/03/1985
• Status: Expired due to Fees

• Current Assignee: David W. Speicher, Harding, Matthew W., Robert E. Handschumacher
• Original Assignee: David W. Speicher, Harding, Matthew W., Robert E. Handschumacher
• Inventors: Harding, Matthew W., Speicher, David W., Handschumacher, Robert E.
• Primary Examiner(s): Draper, Garnette D.
• Application Number:
  US07/065,395
• Time in Patent Office: 1,540 Days
• Field of Search: 530/402, 530/408, 530/409, 530/411, 530/412, 530/413, 530/350, 530/810, 530/812-815, 436/501, 436/161, 436/503, 436/518, 436/528-532, 436/824
• US Class Current: 530/402
• CPC Class Codes: A61K 35/26 Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: Y10S 436/824 Immunological separation techniques
• CPC Class Codes: Y10S 530/81 Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
• CPC Class Codes: Y10S 530/812 Peptides or proteins is immobilized on, or in, an organic carrier
• CPC Class Codes: Y10S 530/813 Carrier is a saccharide
• CPC Class Codes: Y10S 530/814 Cellulose or derivatives thereof
• CPC Class Codes: Y10S 530/815 Carrier is a synthetic polymer

Abstract

Homogeneous cyclophilin, a soluble binding protein, having a specific binding activity of above 50 ug cyclosporin A per mg protein and a molecular weight of about 17,600 daltons, reversibly binds immunosuppressants or antibodies thereto such as cyclosporin or anti-cyclophilin. It is isolated from the cytosol of several different mammalian tissues and can be used in various diagnostic and purifications procedures.

Claims

1. Cyclophilin, a homogeneous cytosolic binding protein having a specific binding activity above 50 ug cyclosporin A per mg of protein bound and a molecular weight of about 17,600 daltons, immobilized on a support.

2. The immobilized cyclophilin of claim 1 in which said cyclophilin has a specific binding activity above 65 ug cyclosporin A per mg of protein bound and said support comprises agarose having a carboxy terminated 12 carbon atom spacer arm.

3. In a method of determining the presence or quantity of a ligand in a sample by effecting a specific binding assay during which the sample is combined with a specific binding partner of the ligand under binding conditions, the improvement which comprises the specific binding partner being cyclophilin, a homogeneous cytosolic binding protein having a specific binding activity above 50 ug cyclosporin A per mg of protein bound and a molecular weight of about 17,600 daltons, and said ligand being a cyclophilin receptor.

4. The method of claim 3, wherein said cyclophilin is immobilized.

5. The method of claim 4, in which said cyclophilin has a specific binding activity above 65 ug cyclosporin A per mg of protein bound immobilized on agarose having a carboxy terminated 12 carbon atom spacer arm.

6. The method of claim 5, wherein said sample is sera of a patient suspected of having systemic lupus erythamatosis.

7. The method of claim 6, further comprising the steps of separating said sera and said cyclophilin and thereafter contacting said cyclophilin with labeled systemic lupus erythamatosis antibodies.

8. The method of claim 3 used to purify the endogenous ligand for cyclophilin from a sample containing said ligand.

9. The method of claim 3 which is used to purify cyclosporin A.

10. The method of claim 5, wherein said immobilized cyclophilin has been equilibrated with tritiated cyclosporin A, whereby the ligands capable of displacing the tritated cyclosporin A are detected.

11. The method of claim 4, wherein said immobilized cyclophilin has been equilibrated with tritiated cyclosporin A, whereby the ligands capable of displacing the tritated cyclosporin A are detected.

12. The method of claim 3, wherein said ligand is an immunosuppressant or anti-cyclophilin antibodies.

13. The method of claim 12, wherein the immunosuppressant is cyclosporin A or an analog thereof.

14. In a method of isolating an receptor from its environment by contact with immunoadsorbent, the improvement which comprises employing as immunoadsorbent, cyclophilin, a homogeneous cytosolic binding protein having a specific binding activity above 50 ug cyclosporin A per mg of protein bound and a molecular weight of about 17,600 daltons and wherein said receptor is adsorbable to said cyclophilin.

15. The method of claim 20 wherein said cyclophilin is immobilized.