Multisignal reagents for labeling analytes
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. In this assay, the amount of the signal or the first member of the second binding pair bound to the binding agent is inversely proportional to the analyte in the sample. 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.
1. A competitive binding assay composition comprising:
(a) a sample suspected of containing an analyte;
(b) a detection reagent comprising
the analyte or an analog thereof bound covalently to a first nucleic acid strand, and
a second nucleic acid strand complementary with and hybridized to the first nucleic acid strand,
wherein the second nucleic acid strand comprises more than one first member of a binding pair of which the analyte is not a member, and
wherein the binding pair is a sugar-lectin, an antigen-antibody, a ligand-ligand receptor, a hormone-hormone receptor, an enzyme-substrate, a biotin-avidin, or a biotin-streptavidin binding pair; and
(c) a binding agent capable of binding to the analyte or the analog thereof of the detection reagent and to analyte contained in the sample,
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-risperidone] paliperidone, a porphyrin, haloperidol, flecainide, tocainide, acetazolamide, a sulfonamide, verapamil, a metanephrine, an oxidized nucleotide, a mycotoxin, tetrahydrocannabinol, a cannabinoid, cocaine, LSD, an amphetamine, heroin, methadone, nicotine, cotinine, a benzodiazepine, bis(2-ethylhexyl) phthalate, bisphenol A, hydralazine, atrazine, an organochloride insecticide, an organophosphate insecticide, or a carbamate insecticide.
2. The composition of claim 1 , wherein the analyte is oxytocin, cAMP, cGMP, hydroxycholesterol, vitamin D, or 25-hydroxy vitamin D.
3. The composition of claim 2 , wherein the analyte is cAMP or vitamin D.
4. The composition of claim 1 , wherein the binding agent is an antibody or an analyte-binding fragment thereof.
5. The composition of claim 1 , wherein the binding agent is bound to a solid phase.
6. The composition of claim 5 , wherein the binding agent is an antibody or an analyte-binding fragment thereof.
7. The composition of claim 1 , further comprising second members of the binding pair bound to the first members of the binding pair.
8. The composition of claim 7 , wherein the second members of the binding pair further comprise a covalently bound signaling molecule.
9. The composition of claim 8 , wherein the signaling molecule is a fluorescent dye or a chemiluminescent molecule.
10. The composition of claim 1 , wherein the detection reagent (b) consists essentially of:
the analyte or an analog thereof bound covalently to the first nucleic acid strand, and
the second nucleic acid strand complementary with and hybridized to the first nucleic acid strand,
wherein the second nucleic acid strand comprises more than one first member of a binding pair of which the analyte is not a member, and
wherein the binding pair is a sugar-lectin, an antigen-antibody, a ligand-ligand receptor, a hormone-hormone receptor, an enzyme-substrate, a biotin-avidin, or a biotin-streptavidin binding pair.
11. The composition of claim 10 , wherein the analyte is oxytocin, cAMP, cGMP, hydroxycholesterol, vitamin D, or 25-hydroxy vitamin D.
12. The composition of claim 11 , wherein the analyte is cAMP or vitamin D.
13. The composition of claim 10 , wherein the binding agent is an antibody or an analyte-binding fragment thereof.
14. The composition of claim 10 , wherein the binding agent is bound to a solid phase.
15. The composition of claim 14 , wherein the binding agent is an antibody or an analyte-binding fragment thereof.