Multisignal reagents for labeling analytes

• US 9,696,298 B2
• Filed: 07/24/2012
• Issued: 07/04/2017
• Est. Priority Date: 04/03/2003
• Status: Expired due to Term

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Szczepanik, Maciej, Coleman, Jack, Jin, Richard
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/556,425
• Publication Number:
  US 20130034866A1
• Time in Patent Office: 1,806 Days
• Field of Search: 435 4, 435 71, 435 72, 436517, 436532, 536 266
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C09B 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/82 involving vitamins or their receptors
• CPC Class Codes: G01N 33/94 involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors

Abstract

Provided is a composition comprising an analyte bound covalently or through a first binding pair to a polymer. In this composition, the analyte is less than about 2000 MW; the polymer further comprises more than one signal or first member of a second binding pair; and the analyte is not a member of the first binding pair or the second binding pair. Also provided is an assay for an analyte. The assay comprises: combining a sample suspected of containing the analyte with the above-described composition and a binding agent that binds to the analyte; and detecting the signal or the first member of the second binding pair that is bound to the binding agent. Additionally provided is a multisignal labeling reagent comprising a first polymer covalently bound to (a) a reactive group or a first member of a first binding pair, and (b) more than one digoxigenin molecule.

Claims

1. A competitive assay method for quantifying the amount of an analyte in a sample, comprising the steps of:

(a) combining

(i) a sample suspected of containing the analyte,

(ii) a detection composition,

comprising the same analyte or an analog thereof bound covalently to a polymer or bound covalently to a first member of a first binding pair that binds a second member of the first binding pair that is bound to a polymer,

said polymer comprising

more than one signal moiety, or

a first member of a second binding pair bound to a second member of the second binding pair,

wherein a signal moiety is covalently bound to the second member of the second binding pair, and

wherein the analyte or analog thereof is not a member of the first binding pair or the second binding pair, and

(iii) a binding agent capable of binding to analyte of the sample and to the covalently bound analyte or analyte analog of the detection composition; and

(b) detecting signal from signal moieties of any detection composition that is bound to the binding agent,

wherein the amount of signal detected is inversely proportional to the amount of the analyte in the sample, and

wherein the signal moiety is selected from the group consisting of a fluorescent dye, a colored dye, a radioactive molecule, a chemiluminescent molecule, and an enzyme.

2. The competitive assay method of claim 1, further comprising removing any of the detection composition that is not bound to the binding agent between step (a) and step (b).

3. The competitive assay method of claim 1, wherein the binding agent is an antibody or analyte-binding fragment thereof.

4. The competitive assay method claim 1, wherein the binding agent is bound to a solid phase.

5. The competitive assay method of claim 1, wherein the analyte is less than about 2000 MW.

6. The competitive assay method of claim 4, wherein the analyte is a mammalian cellular metabolite, a metabolite of a microorganism, a medication, an illicit drug, a vitamin, an environmental pollutant, a pesticide, or a toxin.

7. The competitive assay method of claim 1, wherein the analyte is cAMP, cGMP, 8-bromoadenosine 3′

,5′

-cyclic monophosphate, cholesterol, a hydroxycholesterol, vitamin D, 25-hydroxy vitamin D, vitamin B12, vitamin E, vitamin B1, vitamin B6, ascorbic acid, retinol, biotin, folate, legumin, salbutamol, melamine, sulfaquinoxaline, an inositol phosphate, a phosphatidyl inositol phosphate, prednisone, pregnenolone, dexamethasone, triamcinolone, fludrocortisone, dihydrotachysterol, oxandrolone, testosterone, dihydrotestosterone, nandrolone, norethindrone, medroxyprogesterone acetate, progesterone, a glucocorticoid, aldosterone, estrogen, oxytocin, androstanediol glucuronide, bilirubin, warfarin, an estrogen, methotrexate, tobramycin, acetaminophen, encainide, fluoxetine, gentamycin, an aminoglycoside, amikacin, coenzyme Q10, theophylline, phenytoin, cimetidine, disulfiram, trazodone, ethanol, halothane, phenylbutazone, azapropazone, ibuprofen, amiodarone, imipramine, miconazole, metronidazole, nifedipine, chloramphenicol, trimethoprim, a sulfonamide, rifampin, cisplatin, vinblastine, bleomycin, oxacillin, nitrofurantoin, phenobarbital, primidone, carbamazepine, metoclopramide, cholestyramine, colestipol, neomycin, sulfasalazine, digoxin, indomethacin, diltiazem, erythromycin, tetracycline, itraconazole, nicardipine, triamterene, spironolactone, chlorpromazine, a cyclosporin, nortriptyline, ethosuximide, imipramine, codeine, lorazepam, topiramate, disopyramide, levetiracetam, clobazam, oxcarbazepine, fructosamine, caffeine, methaqualone, meprobamate, fluphenazine, a barbiturate, a phenothiazine, serotonin, valproic acid, digitoxin, maprotiline, lidocaine, mexiletine, primidone, risperidone, OH-rispiridone, a porphyrin, haloperidol, flecainide, tocainide, acetazolamide, a sulfonamide, verapamil, a metanephrine, an oxidized nucleotide, a mycotoxin, tetrahydrocannabinol, a cannabinoid, cocaine, LSD, an amphetamine, a barbiturate, heroin, methadone, nicotine, cotinine, a benzodiazepine, bis(2-ethylhexyl) phthalate, bisphenol A, hydralazine, atrazine, an organochloride insecticide, an organophosphate insecticide, or a carbamate insecticide.

8. The competitive assay method of claim 1, wherein the analyte is a steroid hormone, a glucocorticoid, oxytocin, cAMP, cGMP, hydroxycholesterol, vitamin D or 25-hydroxy vitamin D.

9. The competitive assay method of claim 1, wherein the analyte is cAMP or vitamin D.

10. The competitive assay method of claim 1, wherein the signal moiety is an enzyme.

11. The competitive assay method of claim 10, wherein the enzyme is horseradish peroxidase, alkaline phosphatase or luciferase.

12. The competitive assay method of claim 1, comprising the steps of:

(a) combining a sample suspected of containing the analyte with a detection composition and a binding agent that binds to the analyte,

wherein the detection composition comprises the same analyte or an analog thereof bound covalently to a polymer comprising more than one signal moiety; and

(b) detecting signal from the more than one signal moiety covalently bound to the polymer of any detection composition that is bound to the binding agent,

wherein the amount of the signal detected is inversely proportional to the amount of analyte in the sample,

wherein the signal moiety is selected from the group consisting of a fluorescent dye, a colored dye, a radioactive molecule, a chemiluminescent molecule, and an enzyme.

13. The competitive assay method of claim 12, further comprising the step of:

removing any of the detection composition that is not bound to binding agent between step (a) and step (b).

14. The competitive assay method of claim 1, comprising the steps of:

(a) combining a sample suspected of containing the analyte with a detection composition,

wherein the detection composition comprises the same analyte or an analog thereof bound covalently to a first member of a first binding pair that binds a second member of the first binding pair that is bound to a polymer,

wherein said polymer comprises a first member of a second binding pair bound to a second member of the second binding pair, wherein a signal moiety is covalently bound to the second member of the second binding pair, and

wherein the analyte or analog thereof is not a member of the first binding pair or the second binding pair; and

(b) detecting signal from the signal moiety of any detection composition that is bound to the binding agent,

wherein the amount of the signal detected is inversely proportional to the amount of the analyte in the sample,

wherein the signal moiety is selected from the group consisting of a fluorescent dye, a colored dye, a radioactive molecule, a chemiluminescent molecule, and an enzyme.

15. The competitive assay method of claim 14, further comprising the step of:

removing any of the detection composition that is not bound to the binding agent between step (a) and step (b).