Cyclosporin immunoassay

• US 6,054,303 A
• Filed: 03/10/1995
• Issued: 04/25/2000
• Est. Priority Date: 11/20/1990
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Products GmbH (Siemens AG)
• Original Assignee: Dade Behring Marburg GmbH (Siemens AG)
• Inventors: Davalian, Dariush, Ullman, Edwin F., Beresini, Maureen H., Hu, Mae Wan-Leng, Alexander, Svetlana
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US08/401,827
• Time in Patent Office: 1,873 Days
• Field of Search: 435/188, 530/807, 530/403, 436/546, 436/527, 436/531, 436/829, 436/815
• US Class Current: 435/188
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: C07K 7/645 Cyclosporins; Related peptides
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/9493 Immunosupressants
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation
• CPC Class Codes: Y10S 530/807 Hapten conjugated with peptide or protein

Abstract

A method of measuring the amount of cyclosporin in a sample suspected of containing cyclosporin is disclosed. A method of inactivating interfering cross-reactive material in an assay for measuring the amount of cyclosporin in a sample suspected of containing cyclosporin is also disclosed. Compositions wherein cyclosporin is conjugated to an immunogenic carrier or a label, optionally through a linking group, at an alanine nitrogen atom of the cyclic backbone of cyclosporin are also disclosed. Compositions wherein atiocyclosporin is conjugated, optionally through a linking group, to an immunogenic carrier or a label are also disclosed. Where cyclosporin is conjugated to an immunogenic carrier, the conjugates may be used as immunogens for the preparation of antibodies which are capable of recognizing cyclosporin. Where atiocyclosporin is conjugated to an immunogenic carrier, the conjugates may be used as immunogens for the preparation of antibodies which are capable of recognizing interfering cross-reactive material but substantially incapable of recognizing cyclosporin or cyclosporin-label conjugates. Where cyclosporin is conjugated to a label, the conjugates may be used as part of a signal producing system in cyclosporin assays. Both the antibodies and label conjugates are useful in the disclosed assay methods.

Claims

1. A cyclosporin compound conjugated to an organic radical at an alanine nitrogen atom of said cyclosporin, wherein said compound is useful in an immunological determination, and further wherein,

said organic radical is an immunogenic carrier comprising a poly(amino acid) having a molecular weight greater than 2000;

a ligand;

a residue of a small organic compound, wherein said small organic compound has a molecular weight less than about 2000;

or a label;

said organic radical is conjugated to the cyclosporin through a linking group selected from the group consisting of;

(A) an alkyleneoxide chain of the formula --(CH₂)_n O--, wherein an alanine nitrogen atom of cyclosporin is conjugated to a carbon atom of the alkyleneoxide, n is an integer of 1 to 6, the alkyleneoxide oxygen atom is bound to a first end of from 1 to 3 carboxamide groups linked together by alkylene chains, and the organic radical is bound to a second end of the from 1 to 3 carboxamide groups;

or

(B) a carbonyloxybenzyl group of the formula;

##STR18## wherein an alanine nitrogen atom of cyclosporin is conjugated to the benzylic carbon atom, an oxygen atom of the carbonyloxy group is bound to a first end of from 1 to 3 carboxamide groups linked together by alkylene chains, and the organic radical is bound to a second end of the from 1 to 3 carboxamide groups;

such that if the linking group is the carboxamide extended alkyleneoxide chain, then the alanine nitrogen is part of cyclosporin amino acid residue no. 7, and if the linking group is the carboxamide extended carbonyloxybenzyl group, then the alanine nitrogen is part of cyclosporin amino acid residue no. 7, 8, or nos. 7 and 8.

2. The cyclosporin compound as claimed in claim 1, which is conjugated at the alanine nitrogen atom of amino acid residues No. 7 and 8 through a linking group of the formula:

##STR19## to keyhole limpet hemocyanin.

3. The cyclosporin compound as claimed in claim 1, which is conjugated at the alanine nitrogen atom of amino acid residue No. 7 through a linking group of the formula:

##STR20## to glucose-6-phosphate dehydrogenase.

4. The compound of claim 3 wherein said cyclosporin is selected from the group consisting of cyclosporin A, cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin E, cyclosporin F, cyclosporin G, cyclosporin H, and cyclosporin I.

5. The compound of claim 3 wherein said cyclosporin is cyclosporin A.

6. The compound of claim 1 wherein said organic radical comprises a label.

7. The compound of claim 6 wherein said label is selected from the group consisting of enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, fluorophores, dyes, particles, solid supports, liposomes, chemiluminescers, luminescers, radioactive isotopes, catalysts, ligands, haptens, and sensitizers.

8. The compound of claim 6 wherein said label is an enzyme.

9. The compound of claim 8 wherein said enzyme is a dehydrogenase.

10. The compound of claim 9 wherein said dehydrogenase is glucose-6-phosphate dehydrogenase.

11. The compound of claim 1 wherein said organic radical comprises an immunogenic carrier.

12. The compound of claim 1 wherein said organic radical comprises a residue of a small organic molecule.

13. The compound of claim 1 wherein said organic radical comprises a ligand.

14. The compound of claim 13 wherein said ligand is biotin or a hapten.

15. The compound as claimed in claim 1, wherein said cyclosporin is cyclosporin A.

16. The compound as claimed in claim 15, wherein n is 2, 3, or 4.

17. The compound as claimed in claim 16, wherein the alkyleneoxide chain is extended by 1 or 2 carboxamide groups.

18. The compound as claimed in claim 16, wherein the carbonyloxybenzyl group is extended by 1 or 2 carboxamide groups.

19. The compound as claimed in claim 17 or claim 18, wherein n is 2.

20. The compound as claimed in claim 18, wherein the carbonyloxy group is para to benzylic carbon.

21. The compound as claimed in claim 20, wherein n is 2.

22. The compound as claimed in claim 16, wherein the carbonyloxy group is ortho or para to the benzylic carbon.

23. The compound as claimed in claim 22, wherein n is 2.

24. The compound as claimed in claim 22, wherein the organic radical is an immunogenic carrier selected from the group consisting of keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) and the linking group is the carbonyloxybenzyl group extended by 1 or 2 carboxamide groups linked together by alkylene chains.

25. The compound as claimed in claim 16, wherein the label is an enzyme and the linking group is a alkyleneoxide group extended by 1 or 2 carboxamide groups.

26. The compound as claimed in claim 25, wherein the enzyme is glucose-6-phosphate dehydrogenase.

35. The compound of claim 24 wherein said ligand is biotin or a hapten.

27. A compound comprising a cyclosporin conjugated via a linking group to an organic radical at an alanine nitrogen atom of said cyclosporin, said compound being useful in an immunological determination;

wherein said linking group is selected from the group consisting of;

##STR21## wherein the organic radical is an immunogenic carrier, a ligand, or a label.

28. The compound of claim 27 wherein said organic radical comprises a label.

29. The compound of claim 28 wherein said label is selected from the group consisting of enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, fluorophores, dyes, particles, solid supports, liposomes, chemiluminescers, luminescers, radioactive isotopes, catalysts, ligands, haptens, and sensitizers.

30. The compound of claim 29 wherein said label is an enzyme.

31. The compound of claim 30 wherein said enzyme is glucose-6-phosphate dehydrogenase.

32. The compound of claim 27 wherein said organic radical is an immunogenic carrier.

33. The compound of claim 32 wherein said immunogenic carrier is selected from the group consisting of antigenic poly(amino acids), lipopolysaccharides and particles.

34. The compound of claim 27 wherein said organic radical comprises a ligand.

36. A compound of the formula:

##STR22## wherein either B₁ or B₂ is hydrogen and the other is a linking group selected from the group consisting of;

##STR23## wherein the linking group connects an alanine nitrogen atom of amino acid residue No. 7 or No. 8 to an immunogenic poly(amino acid) having a molecular weight of at least 2000.

37. A cyclosporin A compound conjugated to an organic radical at an alanine nitrogen atom of said cyclosporin, wherein said compound is useful in an immunological determination, and further wherein,

said organic radical is an immunogenic carrier or an enzyme conjugated to the cyclosporin A through a linking group selected from the group consisting of:

(A) an alkyleneoxide of the formula --(CH₂)₂ O--, wherein the alanine nitrogen atom of cyclosporin is conjugated to a carbon atom of the alkyleneoxide, the alkyleneoxide oxygen atom is bound to a first end of 1 carboxamide group linked together by alkylene chains, and the organic radical is bound to a second end of the carboxamide group;

or

(B) a carbonyloxybenzyl group of the formula;

##STR24## wherein the alanine nitrogen atom of cyclosporin is conjugated to the benzylic carbon atom, the carbonyloxy group is para to the benzylic carbon, an oxygen atom of the carbonyloxy group is bound to a first end of 2 carboxamide groups linked together by alkylene chains, and the organic radical is bound to a second end of the 2 carboxamide groups;

such that if the linking group is the carboxamide extended, alkyleneoxide chain, then the alanine nitrogen is part of cyclosporin amino acid residue no. 7, and if the linking group is the carboxamide extended carbonyloxybenzyl group, then the alanine nitrogen is part of cyclosporin amino acid residue no. 7 or 8;

wherein the immunogenic carrier is selected from the group consisting of keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA);

or

wherein the enzyme is glucose-6-phosphate dehydrogenase.

38. A compound comprising a cyclosporin conjugated via a linking group to an organic radical at an alanine nitrogen atom of said cyclosporin, said compound being useful in an immunological determination;

wherein said linking group is selected from the group consisting of;

##STR25## wherein the organic radical is an immunogenic carrier, a ligand, or a label.