DE1066523T1

DE1066523T1 - CONTROL DETERMINATION METHODS FOR DRUGS CONSTRUCTED FROM SMALL MOLECULES

Info

Publication number: DE1066523T1
Application number: DE1066523T
Authority: DE
Inventors: E. Shindelman
Original assignee: Microgenics Corp
Current assignee: Microgenics Corp
Priority date: 1998-03-27
Filing date: 1998-03-27
Publication date: 2001-09-20
Also published as: EP1066523A2; WO1998026644A2; WO1998026644A9; WO1998026644A3

Claims

PATENT CLAIMS What is claimed as an invention:

1. A set of reagents for determining an analyte in a sample in a confirmatory assay, comprising:

(a) a detection antibody for the analyte; and
b) a neutralizing antibody for the analyte;

wherein binding of the neutralization antibody to the analyte in the confirmatory assay inhibits binding of the analyte to the detection antibody.

2. A set of reagents according to claim 1, further comprising a competitive binding compound,

wherein the detection antibody binds the competitive binding compound in a manner that is specifically inhibitable by the analyte.

3. Reagent for adapting an immunoassay for the determination of an analyte in a sample to enable differentiation of the analyte from an interfering substance in a confirmatory assay,

comprising a neutralizing antibody for the analyte, accompanied by written information for performing the confirmatory assay;
wherein the immunoassay comprises the step of preparing a reaction mixture comprising the sample, a detection antibody for the analyte and a competitive binding compound, and

4. Assay method for determining an analyte in a sample, comprising the following steps:

(a) performing a direct test to determine the amount of analyte and/or interfering substance in the sample, the direct test comprising the steps of manufacturing

preparing a reaction mixture comprising the sample, a detection antibody for the analyte and a competitive binding compound, but no neutralization antibody, and then measuring the amount of detection antibody bound to the competitive binding compound in the reaction mixture;

b) performing a confirmatory test to determine the amount of interfering substance in the sample, the confirmatory test comprising the steps of preparing a reaction mixture comprising the sample, a detection antibody for the analyte and a competitive binding compound, and wherein either a neutralization antibody is introduced into the reaction mixture or the

sample is pretreated with a neutralization antibody and then measuring the amount of detection antibody bound to the competitive binding compound in the reaction mixture; and

c) determining either (i) the presence of the analyte in the sample if the amount measured in step b) differs significantly from that measured in step a)

amount; or: (ii) the absence of detectable analyte in the sample if the amount measured in step b) does not differ significantly from the amount measured in step a).

5. Assay procedure to confirm the presence of an analyte in a sample which results in a positive test result for the analyte in a direct test,
wherein the direct assay comprises the steps of preparing a reaction mixture comprising the sample, a detection antibody for the analyte and a competitive binding compound, but not the neutralization antibody, and then measuring the amount of detection antibody bound to the competitive binding compound in the reaction mixture;

wherein the method of confirmation comprises performing a confirmation test comprising the following steps:
a) preparing a reaction mixture comprising the sample, a detection antibody for the analyte, a competitive binding compound,

b) either a neutralization antibody is introduced into the reaction mixture or the sample is pretreated with a neutralization antibody ;

c) measuring the amount of detection antibody bound to the competitive binding compound in the reaction mixture prepared in step a); and

(d) determining either: (i) the presence of analyte in the sample if the amount measured in step (c) differs significantly from the amount determined in the direct assay procedure; or: (ii) the absence of detectable analyte in the sample if the amount measured in step (c) does not differ significantly from the amount measured in the direct assay procedure under

separates.

6. Use of an antibody specific for a small molecule analyte in the manufacture of a reagent, set of reagents or kit for performing a confirmatory assay, wherein binding of the antibody to the analyte in a sample tested in the confirmatory assay reduces the amount of analyte measured.

7. Reagent(s) according to at least one of claims 1 to 3,

wherein the α-αιδαιδα has been raised against a first conjugate in which a hapten is bound to a carrier;

wherein the neutralization αηηδ^φβΓ was raised against a second conjugate in which a hapten is bound to a support through a position of the hapten which is different from that of the hapten in the first conjugate; and wherein binding of the αηηδ-αηδ^φβΓ to the analyte inhibits binding of the analyte to the detection antibody.

8. Reagent(s) according to claim 2 or claim 3, wherein the competitive binding compound preferentially binds the ΔNαδιελεηδα-Δαε^γφβΓ as compared to the neutralizing anti- Δαε^.

9. Reagent(s) according to claim 8 having one or more of the following properties:

• · ■· ■

i) the binding between the competitive binding compound and the detection antibody exhibits an affinity which is at least about 100 times higher than that of the binding between the hapten derivative and the neutralization antibody ;

(ii) the binding between the competitive binding compound and the detection

Antibody exhibits an affinity of at least about 10 ⁹ M"¹; or

iii) the binding between the competitive binding compound and the neutralizing antibody exhibits an affinity of not more than about 10 ⁶ M" ¹ .

10. The assay method of claim 4 or claim 5, wherein the neutralization antibody is included in the reaction mixture of the confirmatory test and the competitive binding compound preferentially binds the detection antibody compared to the neutralization antibody.

11. Assay method according to claim 10 having one or more of the following properties:

(ii) the binding between the competitive binding compound and the detection antibody exhibits an affinity of at least about 10 ⁹ M"¹; or

iii) the binding between the competitive binding compound and the neutralizing antibody exhibits an affinity of not more than about 10 ⁶ M" ¹ .
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12. Use according to claim 6, wherein the competitive binding compound preferentially binds the detection antibody compared to the neutralization antibody.

13. Reagent(s) according to at least one of claims 1 to 3,

wherein the neutralization antibody preferentially binds the analyte compared to an interfering substance: and

wherein the neutralizing antibody is aliquoted such that the amount in each aliquot is sufficient to prevent the analyte, but not all of the interfering substance in the sample, from binding the detection antibody in the confirmatory assay; or the reagent(s) is accompanied by an indication of that amount.
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14. Reagent(s) according to claim 13, wherein the neutralization antibody is insolubilized.

15. Reagent(s) according to claim 14, wherein the detection antibody and the neutralization antibody are the same antibody in soluble or insolubilized form, respectively.

16. An assay method according to claim 4 or claim 5, wherein the amount of neutralization antibody incorporated into the reaction mixture of the confirmatory test or used to pretreat the sample is sufficient to prevent the analyte, but not all of the interfering substance in the sample, from binding the detection antibody.

17. The assay method of claim 16, wherein the neutralization antibody is insolubilized.

18. The assay method of claim 16 or claim 17, wherein the sample is pretreated with the neutralizing antibody prior to preparation of the reaction mixture in the confirmatory test and the amount of neutralizing antibody is sufficient to remove the analyte but not all of the interfering substance from the sample.

19. Use according to claim 6, wherein the antibody is aliquoted such that the amount in each aliquot is sufficient to prevent the analyte, but not all of the interfering substance in the sample, from binding the detection antibody in the confirmatory assay; or the reagent, set of reagents or kit is accompanied by an indication of the amount.

20. Use according to claim 19, wherein the antibody is insolubilized.

21. At least one of the reagents claimed above comprising a competitive binding compound which is a hapten derivative selected from the group consisting of:

(i) a fluorescent hapten or a fluorescent quenching hapten;

(ii) a radiolabelled hapten;
(iii) an insolubilised hapten;
(iv) a hapten bound to an enzyme; and
(v) a hapten bound to an enzyme donor.
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22. At least one of the assay methods claimed above having at least one of the following properties:

(i) wherein each competitive binding compound is a fluorescent hapten or a fluorescence quenching hapten and measuring the bound detection antibody comprises measuring fluorescence emission or fluorescence polarization;

(ii) wherein each competitive binding compound is a hapten conjugated to an enzyme, wherein each reaction mixture further comprises a substrate for the respective enzyme, and measuring the bound detection antibody comprises measuring the enzyme-catalyzed conversion of the respective substrate to a product; or

(iii) wherein each competitive binding compound is a hapten conjugated to an enzyme donor, wherein each reaction mixture further comprises both an enzyme acceptor which complements the respective enzyme donor to form an active enzyme when the detection antibody is not bound to the heptane in the conjugate, and a substrate for the respective active enzymes, and

Measuring the bound detection antibody involves measuring the enzyme-catalyzed conversion of the respective substrate to a product.

23. At least one of the assay methods claimed above, which is a homogeneous assay method.

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24. At least one of the assay methods claimed above having one or more of the following properties:

(i) wherein the analyte has a molecular weight of not more than about 1000;
(ii) wherein the analyte is a substance of abuse or a metabolite thereof;
(iii) wherein the sample is a urine sample;

(iv) where the sample is a plasma or serum sample.

25. At least one of the reagent(s), assay methods or uses claimed above, wherein the assay is an assay for one or more of amphetamine or amphetamine metabolites.

26. At least one of the reagent(s), assay methods or uses claimed above, wherein the assay discriminates between one or more of amphetamine or amphetamine metabolites and an interfering substance selected from the group consisting of ephedrine, pseudoephedrine, phenylpropanolamine, phentermine and fenfluramine.

27. At least one of the reagent(s), assay methods or uses claimed above, wherein one of the detection and neutralization antibodies is raised against a conjugate in which an amphetamine derivative is bound to a support through a position in the phenol ring and the other is raised against a conjugate in which the amphetamine is bound to a support through the alkylamino group.

28. At least one of the reagent(s), assay methods or uses claimed above, wherein the detection antibody is raised against a conjugate in which

an amphetamine derivative is linked to a support through a position in the phenol ring, and the assay comprises binding the detection antibody to a conjugate in which an amphetamine derivative is linked to a support through a position in the phenol ring.
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29. At least one of the reagent(s), assay methods or uses claimed above, wherein the assay is an assay for LSD or an LSD metabolite.

30. At least one of the reagent(s), assay methods or uses claimed above, wherein the assay is either of the following:

(i) an assay that distinguishes LSD from an LSD metabolite; or
(ii) an assay in which LSD or an LSD metabolite is isolated from an interfering substance,

selected from the group consisting of methamphetamine, fenfluramine, ambroxol and chlorpromazine.

31. At least one of the reagent(s), assay methods or uses claimed above, wherein one of the detection and neutralization antibodies is raised against a conjugate in which an LSD derivative is linked to a support through the indole ring and the other is raised against a conjugate in which an LSD derivative is linked to a support through the alkylamide group.

32. At least one of the reagent(s), assay methods or uses claimed above, wherein the detection antibody is raised against a conjugate in which an LSD derivative is bound to a support through a position in the indole ring, and the assay comprises binding the detection antibody to a conjugate in which an LSD derivative is bound to a support through a position in the indole ring.