Homogeneous enzyme immunoassay for simultaneous detection of multiple analytes

• US 20030224373A1
• Filed: 06/04/2002
• Published: 12/04/2003
• Est. Priority Date: 06/04/2002
• Status: Active Grant

• Current Assignee: Lin-Zhi International Incorporated
• Original Assignee: Lin-Zhi International Incorporated
• Inventors: Chang, Chiu Chin, Chia, Tom, Lin, Marie
• Granted Patent: US 7,560,239 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/946 CNS-stimulants, e.g. cocaine, amphetamines
• CPC Class Codes: G01N 33/948 Sedatives, e.g. cannabinoids, barbiturates opiates G01N33/9486
• CPC Class Codes: G01N 33/9486 Analgesics, e.g. opiates, aspirine

Abstract

Methods and kits are provided for the simultaneous detection of multiple analytes, i.e. to the determination of whether one or more of a plurality of analytes is present in a sample, particularly the plurality of analytes are structurally unrelated or significantly different, particularly to whether one or more of such analytes is present in a sample in a concentration that is above a predetermined minimum or cutoff value, and particularly for such analytes with different cutoffs. The methods and kits are particularly useful for-screening for the presence of a plurality of drugs (licit and/or illicit), performance-enhancing substances, and other chemicals, and involve a competitive enzyme immunoassay employing one or more conjugates of G6PDH with a plurality of the analytes.

Claims

1. A method for detecting the presence of one or more non-serologically cross-reactive analyte types in a sample using a competitive homogeneous assay:

where the assay detects a plurality of different analyte types that are non-serologically cross-reactive and, where the assay involves analyte and receptor binding pairs such that the presence of one or move different analyte types is determine d by enzyme activity reflecting the concentration of analyte when present in excess of a predetermined concentration of the cutoff said method comprising the steps of;

(I) combining in an aqueous medium;

(a) G6PDH-analyte binding pair member conjugates covalently linked to a plurality of different analyte binding pair members of which at least two are non-serologically cross-reactive;

(b) receptors reactive to both analytes and the G6PDH-analyte binding pair member conjugates; and

,

(c) a sample to be tested for the presence of any of the plurality of analyte types and,

(II) detecting increased G6PDH activity in the aqueous medium due to competitive binding of the receptors with the analytes in the sample;

provided that;

(i) concentrations of G6PDH-analyte binding pair member conjugates and of the receptors are adjusted in the aqueous mixture so that the enzyme rate at the predetermined cutoff concentrations is approximately the same for the different analyte types whose presence is to be detected;

(ii) wherein the G6PDH is deactivated by from about 20% to about 85% resulting from the covalent linkage to the analyte binding pair member; and

(iii)wherein the deactivated G6PDH is inhibited by from about 20% to about 85% when bound to the receptors.

2. A method of claim 1 where at least two of the analyte types have a different cutoff concentration marking a predetermined concentration above which a positive signal is generated.

3. A method according to claim 1 in which the conjugates comprise individual G6PDH molecules covalently linked to a plurality of different types of analytes.

4. A method according to claim 1 in which the conjugates comprise individual G6PDH molecules covalently linked to a plurality of identical analyte types.

5. A method according to claim 1 in which at least two of the analytes are immunologically distinct from each other.

6. A method according to claim 1 in which the analytes are selected from group comprising:

licit and illicit drugs, sugars, amino acids, peptides, nucleic acids, nucleosides, nucleotides, vitamins, hormones, steroids, toxins, chemical and biological warfare agents, pesticides, and industrial chemicals, and analogs, derivatives and metabolites thereof.

7. A method according to claim 1 in which the analytes are selected from licit and illicit drugs and analogs, derivatives and metabolites thereof.

8. A method according to claim 1 in which at least two of the analytes are selected from opium, opioid analgesics, amphetamines, cocaine, methadone, alkaloids, catecholamines, methylendioxyamphetamines (MDMA, MDA, and MDEA, etc.), PCP, propoxyphene, methaqualone, barbiturates, benzodiazepines, tricyclic antidepressants, tranquilizers, tetrahydrocannabinol, LSD, ketamine, GHB, and other drugs of abuse, including amino acids, hormones, and steroids, and analogs, metabolites, and derivatives thereof.

9. A method according to claim 8 in which at least two of the analytes are selected from drugs of abuse having two different predetermined cutoff concentrations.

10. A method according to claim 1 in which at least two of the analytes are selected from opioid analgesics, amphetamines, cocaine, tetrahydrocannabinol, PCP, methylendioxyamphetamines, ketamine, GHB, LSD, methadone, methaqualone, and propoxyphene.

12. A method according to claim 1 in which at least two of the analytes are selected from alkaloids, peptides, nucleic acids, nucleosides, nucleotides, vitamins, hormones, food supplements, sugars, steroids, amino acids, and other performance-enhancing agents, and analogs, metabolites, and derivatives thereof.

13. A method according to claim 1 in which at least two of the analytes are selected from chemical and biological warfare agents, toxins, pesticides, herbicides, and industrial chemicals and pollutants.

14. A method according to claim 1 in which the G6PDH is deactivated by from about 20 to about 60%.

15. A method according to claim 1 in which the enzyme activity of the deactivated G6PDH-analyte conjugate is inhibited by from about 40 to about 80%

16. A method according to claim 1 in which the G6PDH is a recombinant G6PDH.

11. A method according to claim I in which at least two of the analytes are selected from barbiturates, tricyclic anti-depressants, tranquilizers, and benzodiazepines, and analogs, metabolites, and derivatives thereof.

17. A kit for testing the presence of analytes in a sample using a competitive homogeneous assay where the assay simultaneously detects the presence of a plurality of different analyte types that are non-serologically cross-reactive said kit comprising:

(a) a container containing a mixture of G6PDH-analyte binding pair member conjugates covalently linked to one or more different analytes types; and

,

(b) a container containing receptors reactive to both analytes and the G6PDH-analyte binding pair members;

where the concentrations of the said conjugates and receptors are adjusted in the containers to yield approximately the same enzyme rate for each analyte present at its predetermined cutoff concentration so that sample containing one or more of a plurality of the analytes in an excess of its predetermined cutoff concentration can be identified.

18. A kit of claim 17 for simultaneous detection of a plurality of analytes among which at least two having different predetermined cutoff concentrations with which any of the analytes when present in an excess of their predetermined cutoff concentrations can be detected.

19. A kit of claim 17 wherein the G6PDH is deactivated by from about 20% to about 85% resulting from the covalent linkage to the analyte binding pair member;

and wherein the deactivated G6PDH is inhibited by from about 20% to about 85% when bound to the receptors.

20. A kit according to claim 17 further comprising antibodies reactive to the plurality of analytes.

21. A kit according to claim 17 in which the conjugate comprises one or more conjugate(s) containing individual G6PDH covalently linked to a plurality of different analyte types.

22. A kit according to claim 17 in which the conjugate comprises a plurality of conjugates of individual G6PDH covalently linked to a single type of analyte.

23. A kit according to claim 17 in which the analytes are selected from the group consisting of:

licit and illicit drugs, sugars, amino acids, peptides, nucleic acids, nucleosides, nucleotides, vitamins, hormones, steroids, toxins, chemical and biological warfare agents, pesticides, and industrial chemicals, and analogs, derivatives, and metabolites thereof.

24. A kit according to claim 17 in which the analytes are selected from licit Hand illicit drugs and analogs, derivatives, and metabolites thereof.

25. A kit according to claim 17 in which the at least two of the analytes are selected from the group consisting of:

opium, opioid analgesics, amphetamines, cocaine, methadone, alkaloids, catecholamines, barbiturates, methylendioxyamphetamines, PCP, propoxyphene, methaqualone, barbiturates, benzodiazepines, tricyclid antidepressants, tranquilizers, tetrahydrocannabinol, LSD, ketamine, GHB, and other drugs of abuse, including amino acids, hormones, and steroids, and analogs, metabolites, and derivatives thereof.

26. A kit according to claim 17 in which the analytes are selected from the group consisting of drugs of abuse, and particularly those of significantly different structures, and/or of different cutoffs.

27. A kit according to claim 25 in which at least two of the analytes are selected from the group consisting of:

opioid analgesics, amphetamines, cocaine, tetrahydrocannabinol, PCP, methylendioxyamphetamines, ketamine, GHB, LSD, methadone, methaqualone, and propoxyphene.

28. A kit according to claim 25 in which at least two of the analytes are selected from barbiturates, tricyclic anti-depressants, tranquilizers, and benzodiazepine, and analogs, metabolites, and derivatives thereof.

29. A kit according to claim 17 in which at least two of the analytes are selected from alkaloids, vitamins, hormones, food supplements, sugars, peptides, nucleic acids, nucleosides, nucleotides, steroids, amino acids and other performance-enhancing agents, and analogs, metabolites, and derivatives thereof.

30. A method according to claim 17 in which at least two of the analytes are selected from chemical and biological warfare agents, toxins, pesticides, herbicides, and industrial chemicals and pollutants.