EP0487289A2
Expired / Lapsed
EP
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Cyclosporin immunoassay
Assignee: Siemens Healthcare Diagnostics GmbH Germany
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Filing: 1991-11-19
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Granted: —
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Expires: 2011-11-19
Abstract
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.
Inventors
- 👤 Dariush Davalian
- 👤 Maureen H. Beresini
- 👤 Svetlana Alexander
- 👤 Mae W. Hu
- 👤 Edwin F. Ullman
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-29 06:08
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/5306; C; C07; C07K; C07K16/00; C07K16/44; C07K7/00; C07K7/64; C07K7/645; G01N33/531; G01N33/532; G01N33/535; G01N33/94; G01N33/9493; Y; Y02; Y02A; Y02A50/00; Y02A50/30; Y10; Y10S; Y10S435/00; Y10S435/962; Y10S436/00; Y10S436/815; Y10S436/825; Y10S436/829; Y10S530/00; Y10S530/807
Claims Analysis
Total Claims: 28
Claim 1
Independent Claim
59 words
A method for measuring the amount of cyclosporin in a sample suspected of containing cyclosporin which comprises the step of: combining in an aqueous medium: 1) said sample suspected of containing cyclosporin, 2) cyclosporin conjugated at one of its alanine nitrogen atoms to a label (cyclosporin label conjugate), and 3) antibodies capable of binding to said cyclosporin label conjugate.
Claim 2
Dependent Claim
76 words
A method according to Claim 1 for measuring the amount of cyclosporin in a sample suspected of containing cyclosporin which comprises the steps of: a) combining in an aqueous medium: 1) said sample suspected of containing cyclosporin, 2) cyclosporin conjugated at one of its alanine nitrogen atoms to glucose-6-phosphate dehydrogenase (cyclosporin G6PDH conjugate), 3) antibodies capable of binding to said cyclosporin G6PDH conjugate, and 4) substrates of glucose-6-phosphate dehydrogenase; and b) measuring the glucose-6-phosphate dehydrogenase activity.
Claim 3
Dependent Claim
27 words
A compound comprising a cyclosporin conjugated to an organic radical of at least 5 atoms at an alanine nitrogen atom of the cyclic backbone of said cyclosporin.
Claim 4
Dependent Claim
44 words
The compound of Claim 3 wherein said cyclosporin is bound to said organic radical by means of a bond, or a linking group of less than about 20 atoms other than hydrogen having a chain of not more than about 15 atoms in length.
Claim 5
Dependent Claim
25 words
The compound of Claim 3 or 4 wherein said organic radical comprises a label, an immunogenic carrier, or a residue of a small organic molecule.
Claim 6
Dependent Claim
21 words
Antibodies prepared in response to the compound of Claim 3, 4 or 5 wherein the organic radical is an immunogenic carrier.
Claim 7
Dependent Claim
12 words
The antibodies of Claim 6 which comprise antibodies capable of recognizing cyclosporin.
Claim 8
Dependent Claim
13 words
The antibodies of Claim 6 wherein said cyclosporin is cyclosporin A metabolite M1.
Claim 9
Dependent Claim
15 words
A compound that is a conjugate of the antibodies of Claim 6 and a label.
Claim 10
Dependent Claim
32 words
A compound according to claim of the formula: wherein one member of the group β1 and β2 is hydrogen and the other member is an organic radical of at least 5 atoms.
Claim 11
Dependent Claim
25 words
The compound of Claim 10 wherein said organic radical comprises a chain of about 3 to 15 atoms other than hydrogen bound to glucose-6-phosphate dehydrogenase.
Claim 12
Dependent Claim
18 words
Antibodies prepared in response to the compound of Claim 10 wherein the organic radical is an immunogenic carrier.
Claim 13
Dependent Claim
54 words
The compound of Claim 10, wherein either (i) β2 is hydrogen and β1 is selected from the group consisting of: wherein β3 is a poly(amino acid), or wherein (ii) one member of β1 and β2 is hydrogen and the other member is selected from the group consisting of: wherein β3 is a poly(amino acid).
Claim 14
Dependent Claim
168 words
A process for preparing a compound of Claim 10, which process comprises (i) wherein said organic radical comprises a two carbon chain terminated in a hydroxy group, the steps of: a) reacting a cyclosporin with a base which is sufficiently basic to deprotonate a secondary amide, b) reacting the deprotonated compound of a) with an epoxide, and c) removing protecting groups from said cyclosporin; (ii) wherein said organic radical comprises a carboxybenzyl group, the steps of: a) reacting a cyclosporin with a base which is sufficiently basic to deprotonate a secondary amide, b) reacting the deprotonated compound of a) with an α-halomethylbenzoic acid, lower alkyl ester, and c) removing protecting groups from said cyclosporin; or (iii) wherein said organic radical is an enzyme label, the step of: a) preparing an activated ester of the compound of Claim 10 wherein said organic radical comprises a chain of about 2 to 14 atoms other than hydrogen bound to a carboxyl group, and b) adding said activated ester to an enzyme.
Claim 15
Independent Claim
58 words
A method of inactivating interfering cross-reactive material in an assay for measuring the amount of cyclosporin in a sample suspected of containing cyclosporin which comprises combining an assay medium containing said sample with an antibody capable of binding to said interfering cross-reactive material wherein said antibody does not interfere with said assay for measuring the amount of cyclosporin.
Claim 16
Dependent Claim
16 words
The method of Claim 15 wherein said interfering cross-reactive material is a metabolite of said cyclosporin.
Claim 17
Dependent Claim
29 words
The method of Claim 15 or 16 wherein said antibody is an antibody which binds to cyclosporin metabolite M-1 and which is substantially incapable of binding to cyclosporin A.
Claim 18
Dependent Claim
77 words
A method according to Claim 15, for measuring the amount of cyclosporin in a sample suspected of containing cyclosporin comprising the steps of: a) combining in an aqueous medium: 1) said sample suspected of containing cyclosporin, 2) cyclosporin conjugated to label (cyclosporin conjugate), 3) a first antibody capable of binding to cyclosporin and to said cyclosporin conjugate, and 4) a second antibody capable of binding to metabolites of cyclosporin; and b) determining the activity of said label.
Claim 19
Dependent Claim
32 words
The method of Claim 18 wherein said second antibody is an antibody capable of binding to cyclosporin metabolite M1 but is substantially incapable of binding to cyclosporin A or said cyclosporin conjugate.
Claim 20
Dependent Claim
83 words
The method of Claim 18 or 19 wherein said second antibody is an antibody raised in response to a compound of the formula: wherein: Z is an immunogenic carrier; m is a number from 1 up to the molecular weight of Z divided by 5000; and A is a cyclosporin derivative of the formula: wherein: Y is a bond or a linking group of 1 to 60 atoms other than hydrogen having a chain of not more than about 30 atoms in length.
Claim 21
Dependent Claim
19 words
A composition comprised of an immune complex of a glucose-6-phosphate dehydrogenase-hapten conjugate (G6PDH conjugate) and an antibody to cyclosporin.
Claim 22
Dependent Claim
27 words
The composition of Claim 21 which includes a second antibody which binds to a cyclosporin metabolite but does not bind substantially to cyclosporin or said G6PDH conjugate.
Claim 23
Dependent Claim
110 words
A compound of the formula: wherein: Z is a hydroxyl, label, or group of molecular weight greater than 5000; m is a number from 1 up to the molecular weight of Z divided by 5000; and A is a cyclosporin derivative of the formula: wherein: Y is a bond or a linking group of 1 to 60 atoms other than hydrogen having a chain of not more than about 30 atoms in length; R₁, R₂, and R₃ are hydrogen, or hydroxyl with the proviso that at least one member of R₁, R₂ and R₃ is hydroxyl; R₄ is hydrogen or lower alkyl; and R₅ is hydroxyl and R₆ is hydrogen.
Claim 24
Dependent Claim
13 words
The compound of Claim 23 wherein R₅ is hydroxyl and R₆ is hydrogen.
Claim 25
Dependent Claim
20 words
The compound of Claim 23 or 24 wherein R₁ is hydroxyl, R₂ and R₃ are hydrogen and R₄ is methyl.
Claim 26
Dependent Claim
14 words
The compound of Claim 23, 24 or 25 wherein Y is of the formula:
Claim 27
Dependent Claim
42 words
Antibodies prepared in response to the compound of Claim 23, wherein Z is an immunogenic carrier, wherein said antibodies are capable of binding to the compound of Claim 23 wherein Z is a label and are substantially incapable of binding to cyclosporin.
Claim 28
Dependent Claim
69 words
A process of preparing the compound of Claim 23 comprising the steps of: a) oxidatively cleaving a cyclosporin metabolite at the olefinic side chain of amino acid residue number 1, b) reacting the product of a) with an aminooxyalkanoic acid, c) activating the product of b) for conjugation, d) forming the compound of Claim 23 by reacting the activated product of c) with a label or an immunogenic carrier.
References Cited (Backward Citations)
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