JP2002539450A - ELISA kit for metabolic phenotyping - Google Patents

Abstract

  (57) [Summary] The present invention relates to metabolic phenotypes, including but not limited to CYP1A2, N-acetyltransferase-1 (NAT-1), CYP2P6, CYP2E1 and CYP3A4, which can be used on a routine basis in clinical laboratories. It relates to an enzyme-linked immunosorbent assay (ELISA) for rapid determination. The ELISA kit allows the physician to predict a) personalized treatment of drugs such as theophylline, tamoxifen and clozapine, and b) susceptibility to carcinogen-induced diseases such as colorectal cancer. To reduce the number of patients undergoing clinical trials, select patients with the appropriate phenotype that are most likely to respond.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] BACKGROUND OF THE INVENTION (a) Technical Field The present invention following enzymes, CYP 1A2, N-acetyltransferase -1
(NAT-1), CYP 2D6, CYP 2E1 and CYP 3A4, including but not limited to an enzyme-linked immunosorbent assay (ELISA) for rapid determination of metabolic phenotype. The use of ELISA kits is used on a routine basis in clinical laboratories, and provides physicians with a) personalized treatment of multiple drugs that are metabolized by these enzymes, b) for carcinogen-induced diseases, including many cancers. Includes, but is not limited to, predicting susceptibility, and reducing the number of patients undergoing clinical trials by selecting patients with the most likely responsive phenotype Absent. (B) Prior Art For most drugs (or non-biological substances) administered to humans, their fate is metabolized in the liver to less toxic and lipophilic forms, which are subsequently excreted in the urine That is. Their metabolism involves two systems that work in series: a cytochrome P450 system, which catalyzes the oxidation reaction and contains at least 20 enzymes that are localized in the microsomal fraction, and at least 5 enzymes. Conjugation systems including. The rate of drug metabolism varies between individuals and races due to the presence of enzyme polymorphisms within each system. Two or three phenotypes can be distinguished: poor metabolites (PM), advanced metabolites (EM) and very advanced metabolites (UEM). Phenotypic knowledge is clinically useful for the following reasons: a) Phenotype is associated with toxicity in chemical, plant, disease and cancer. b) Allow physicians to prescribe personalized drug regimens. c) Provides rationale in the design of therapeutic agents.

[0002] Metabolites of drugs or probe drugs in urine samples by using high performance liquid chromatography (HPLC) or capillary electrophoresis (CE) methods, whose phenotype is not readily available in clinical laboratories Is determined by measuring the molar ratio.

The drug enzymes NAT-1, CYP1A2, CYP2D6, CYP2E1 and CYP3 metabolized by the patented metabolic enzymes
A4 is involved in the metabolism of many drugs. Table 1 lists a wide range of drug therapies that are metabolized and the enzymes involved. These include asthma (theophylline), malaria (dapsone), breast cancer (tamoxifen), cardiovascular disease (procainimide), organ transplantation (cyclosporine), analgesics (acetaminophen, codeine), general anesthetics (lidocaine) and mild stability Includes drugs used for various diseases, including common medications such as drugs (barium). The wide range of drug therapies that screening can apply to these enzymes has shown that rapid phenotypic screening has the potential and impact to determine the outcome and safety of patient treatment. Table 1 Drugs Metabolized by Non-Biological Enzymes Phenotyped by CMPD

[0004]

[Table 1]

[0005] Metabolic enzymes and related metabolic enzymes with altered cancer susceptibility are involved in the metabolism of many carcinogenic compounds. Thus, alterations in the activity of these enzymes alter the biological activity of many carcinogens. Table 2 lists the non-living substances metabolized by the enzyme. Table 2 Enzymes and carcinogens they metabolize

[0006]

[Table 2]

[0007] Metabolic enzymes associated with cancer The factors affecting the phenotype of cancer are multifactorial, and it is difficult to relate only one factor to cancer. However, current studies have linked different metabolic phenotypes to an increased risk of certain cancers.

[0008] Table 3 lists metabolic enzymes phenotyped by these enzymes and cancers whose altered phenotype is associated with increased susceptibility. Table 3 Non-biomaterial metabolic enzymes associated with cancer development

[0009]

[Table 3]

NAT1 The NAT1 gene has long been classified as monomorphic. However, it has now been suggested that NAT1 is as polymorphic as other N-acetyltransferases (NAT2). NAT1 has two phenotypes, slow and fast metabolites (eg, each NAT1 ^* 4 vs. NAT1 ^* 10 genotype). NA
Measurement of T1 activity is of clinical interest for the following reasons. Polymorphism NAT1 is polymorphic and can distinguish two metabolic phenotypes: fast and slow metabolites. NAT1 metabolizes several drugs and food components, including p-aminobenzoic acid, p-aminosalicylic acid, and dapsone.

[0011] In addition, NAT1 is an environmental procarcinogen, especially diaminobenzidine, N-hydroxy-4-aminobiphenol, heterocyclic aromatic amines (MeIQx and Ph
IP). In one study, there was an increased risk of colorectal cancer in individuals carrying the NAT1 ^* 10 allele and thus undergoing rapid N-acetylation (OR = 1.9; CI = 1.2-3. Although 2) is shown, another study has shown an increased risk of bladder cancer (metabolizing benzidine). Racial Differences NAT1 activity varies widely with a given population. Slow and fast NAT
One phenotype is distinguished. The NAT1 ^* 10 genotype associated with a fast metabolic phenotype was monitored in three different ethnic groups, Indians, Malaysians, and Chinese. The frequency of the NAT1 ^* 10 allele was 17%, 39% and 30%, respectively. In contrast, the NAT1 ^* 4 genotype associated with the slow metabolite had a frequency of 50%, 30% and 35%, respectively, in the same population. Thus, in drug metabolism studies, each race must be studied separately for evidence of polymorphism, and its antinodes should not be extrapolated from one race to another That makes sense. Dapson The classic example of phenotyping in drug administration is that of dapsone. Dapson has been used to treat malaria and is being investigated for the treatment of Pneumocystis carinii pneumonia in AIDS patients. Side effects include rash, anemia, methemoglobinemia, granulocytopenia and liver failure. Dapson is cleared from the body by the NAT1 metabolic system. A correlation has been shown between slow acetylation and increased side effects of dapsone (46% vs. each for slow and fast acetylated forms).
17%). For these reasons, the utility of a reliable phenotyping test is obvious. Personalized Therapy It is well known that personalized treatment is possible for many drugs (theophylline, digoxin, aminoglycosidase, dapsone, etc.). However, the individualization of treatments requires that the methods used for drug phenotyping be expensive, time consuming, and require high-performance liquid chromatography (experts that are not readily available in clinical laboratories) ( Development has been slow because it involves HPLC) or capillary electrophoresis (CE).

[0012] In order to predict the response and side effect profiles to a wide range of potentially toxic drugs, methods for determining the NAT1 phenotype of an individual using non-toxic drugs would be highly sought. .

Provide an enzyme-linked immunosorbent assay (ELISA) kit for NAT1 phenotyping that could be achieved by technicians with minimal training and on a routine basis that does not require complex equipment. It will be strongly required.

There would be a strong need to provide an enzyme-linked immunosorbent assay (ELISA) kit that would allow physicians to personalize the treatment of drugs such as dapsone. CYP1A2 CYP1A2 makes up 15% of all CYP450 enzymes in human liver. CYP
Measurement of 1A2 activity is of clinical interest for the following reasons: Polymorphism CYP1A2 may be polymorphic but has not been firmly established. Three metabolic phenotypes can be distinguished: fast, moderate and slow metabolites. CYP1
A2 metabolizes several drug and food ingredients including acetaminophen, antipyrine, 17β-estradiol, caffeine, cloipramine, clozapine, flutamide (antiandrogen), imipramine, paracetamol, phenacetin, tacrine and theophylline.

In addition, CYP1A2 activates environmental pre-carcinogens, especially heterocyclic and aromatic amines. In one study, individuals who undergo rapid N-acetylation and have high CYP1A2 activity have an increased risk of colorectal cancer (16).
35% relative to the% control, OR = 2.79 (P = 0.002)). Induction and Inhibition CYP1A2 is induced by many drugs and environmental factors such as omeprazole, iansoprazole, polyaromatic hydrocarbons and tobacco smoke. CYP1A
No. 2 is inhibited by oral contraceptives, ketoconazole, α-naphthoflavone, fluvoxamine (a seronin uptake inhibitor), and furaphyrin. Racial Differences The activity of CYP1A2 varies greatly (60 to 70 fold) in a given population. The slow, moderate and fast CYP1A2 phenotypes are distinguished. The proportions of these three CYP1A2 phenotypes vary between races and countries: moderate percentages in the United States, African Americans, China, Japan, Italy and Australia are 50, 70, 60, and> 95, respectively. , 60 and 20. It is important to note that in drug metabolism studies, each race must be studied separately for evidence of polymorphism, and that its antinodes should not be extrapolated from one race to another. Reasonable. Theophylline A classic example of the need for phenotyping in drug administration is theophylline. Theophylline is used to treat rashes. However, theophylline toxicity remains a common clinical problem, including life-threatening cardiovascular and neurological toxicities. Theophylline is cleared from the body by the CYP1A2 metabolic system.
Inhibition of CYP1A2 by quinolone antibiotics or serotonin reuptake inhibitors will result in theophylline toxicity. For these reasons, the utility of a reliable phenotyping test is obvious. Personalized treatment It is well known that personalized treatment is possible for many drugs (theophylline, digoxin, aminoglycosidase, etc.). However, the individualization of treatments requires that the methods used for drug phenotyping be expensive, time consuming, and require high-performance liquid chromatography (experts that are not readily available in clinical laboratories) ( Development has been slow because it involves HPLC) or capillary electrophoresis (CE).

[0016] In order to predict response and side effect profiles to a wide range of potentially toxic drugs, there will be a strong need for methods for determining the CYP1A2 phenotype of an individual using non-toxic drugs. .

Provide an enzyme-linked immunosorbent assay (ELISA) kit for CYP1A2 phenotyping that could be achieved by technicians with minimal training and on a routine basis that does not require complex equipment. Will be strongly required.

There would be a strong need to provide an enzyme-linked immunosorbent assay (ELISA) kit that would allow physicians to personalize the treatment of drugs such as theophylline, tamoxifen or clozapine.

CYP2D6 CYP2D6 makes up 1-3% of all CYP450 enzymes in human liver. CY
Measurement of P2D6 activity is of clinical interest for the following reasons: Polymorphism CYP2D6 is the first P450 enzyme to show polymorphic expression in humans. Three metabolic phenotypes are distinguishable: poor (PM), advanced (EM) and very advanced (UEM) phenotypes. CYP2D6 metabolizes a wide range of drugs and food ingredients, including: psychotropic drugs: amifuramine, amitriptyline, clomipramine, clozapine, desipramine, haloperidol, imipramine, maprotiline, methoxyphenamine, minaprine, nortriptyline, paroxetine, perphenazine, Remoxiprid, thioridazine, tomoxetine, trifloperidol, zuclopentixol Cardiovascular drugs: bufuralol, debrisoquine, encainide, flecainide, guanoxane, indolamine, metoprolol, mexiletine, n-propylazimarin, propaphenone, propranolol, sparteine , Timolol, verapamil Other drugs: chloropropamide, codeine, dextromethorphan, methampheta Down,
Perhexylene, phenformin.

[0020] In addition, CYP2D6 is involved in the metabolism of many carcinogens, but has not yet been reported as a major metabolite. In one study, individuals with fast CYP2D6 metabolites and slow N-acetylated forms have an increased risk of hepatocellular carcinoma (
OR = 2.6; 95% CI = 1.6-4). Induction and Inhibition CYP2D6 is inhibited in vitro by quinidine and by appetite suppressants such as viral protease inhibitors and D- and L-fenfluramide. Racial Differences The activity of CYP2D6 varies greatly with a given population. The poor (PM), advanced (EM) and very advanced (UEM) phenotypes of CYP2D6 are distinguished. The CYP2D6 gene is inherited as an autosomal recessive trait, and 90 and 10% of White and North Americans are advanced (EM) and poor (P
M) Differentiate into metabolite phenotype. In another study, P in different racial groups
A percentage of M has been observed, with white North Americans and Europeans 5-10%.
1.8% of black Americans, 1.2% of Thai natives, 1% of Chinese, and 2.1% of Malay natives had a PM, but the Japanese population had no PM phenotype. Did not exist.

In drug metabolism studies, each race must be studied separately for evidence of polymorphism, and its antinodes should not be extrapolated from one race to another The absence is reasonable. Dextromethorphan / Antidepressant An example of the need for phenotyping in drug administration is dextromethorphan. Dextromethorphan is a non-opioid antitussive with psychotropic effects. However, the dextromethorphan dose range of 0 to 6 mg / kg is based on individual patient tolerance. Dextromethorphan is activated via the CYP2D6 metabolic system. Dextromethorphan exerts qualitatively and quantitatively different objective and subjective influences on poor vs. poor. Advanced metabolites (average movement +
86 +/- 6% for PM vs. 95 +/- 0.5% for SE, EM; p <
0.05).

Another important agent for the CYP2D6 phenotyping is a tricyclic antidepressant. C
There is a risk of side effects in both the PM and UEM phenotypes of YP2D6. PM individuals given standard doses of these drugs produce toxic plasma concentrations and have unpleasant side effects, including xerostomia, hypotension, sedation, tremors, and in some cases life-threatening cardiotoxicity. May lead. Conversely, administration of these drugs to UEM individuals will fail treatment because the plasma concentration of the active drug is much lower at standard doses. For these reasons, the utility of a reliable phenotyping test is obvious. Personalized treatment It is well known that personalized treatment is possible for many drugs (theophylline, digoxin, aminoglycosidase, dextromethorphan and others). However, the individualization of treatments requires that the methods used for drug phenotyping be expensive, time consuming, and require high-performance liquid chromatography (experts that are not readily available in clinical laboratories) ( Development has been slow because it involves HPLC) or capillary electrophoresis (CE).

[0023] In order to predict the response and side effect profile to a wide range of potentially toxic drugs, methods for determining the CYP2D6 phenotype of an individual using non-toxic drugs will be highly sought. .

Provide an enzyme-linked immunosorbent assay (ELISA) kit for CYP2D6 phenotyping that could be achieved by technicians with minimal training and on a routine basis that does not require complex equipment It will be strongly required.

There would be a strong need to provide an enzyme-linked immunosorbent assay (ELISA) kit that would allow physicians to personalize the treatment of drugs such as dextromethorphan, clozapine or verapamil. .

CYP2E1 CYP2E1 makes up about 5% of all CYP450 enzymes in human liver. CYP
Measuring 2E1 activity is of clinical interest for the following reasons: Polymorphism Although there is some evidence of a CYP2E1 gene polymorphism in the human population, the molecular mechanism must be further characterized. Studies have shown the presence of two alleles, designated c1 and c2. Initial studies indicate a possible association of the c2 allele with higher CYP2E1 expression.

CYP2E1 is ethanol, acetone, acetaminophen, nitrosamine,
Metabolizes several drugs and food components, including nitrosodimethylamine, p-nitrophenol.

In addition, CYP2E1 activates environmental carcinogens, especially nitrosodimethylamine, nitrosopyrrolidone, benzene, carbon tetrachloride, 3-hydroxypyridine (tobacco smoke product). One study has shown that individuals with high CYP2E1 (c2) activity are at increased risk for gastric cancer (OR = 23.6-25.7). Induction and Inhibition CYP2E1 is induced by many drugs and environmental factors such as tobacco smoke, and by starvation, and in uncontrolled diabetes. CYP2E1 is inhibited by chloromethiazole, trans-1,2-dichloroethylene, and by the isoflavonoids genestein and equol. Ethnic differences The proportion of the CYP2E1 phenotype varies between races and countries: the frequency of rare c2 alleles is about 4% in Caucasians and 20% in Japanese, another study of polymorphisms Describe a rare C allele with a frequency of about 10% in Caucasians and 25% in Japanese. In one study, it has been shown that Japanese men have significantly lower levels of CYP2E1 activity compared to Caucasian men. Another study has shown that a mixed Nicaraguan population of Caucasian (Spanish) and Asian (Central American Indians) has an intermediate level of CYP1A2 allelic mutation when compared to the parental population. Thus, in drug metabolism studies, each race must be studied separately for evidence of polymorphism, and its antinodes should not be extrapolated from one race to another That makes sense. Acetaminophen An example where phenotyping is required for drug administration is in the case of acetaminophen.
Acetaminophen is a widely used analgesic. However, acetaminophen shows hepatotoxicity with low frequency. Hepatotoxicity is due to conversion via CYP2E1 to a reactive metabolite (N-acetyl-p-benzoquinone imine) that can bind to nucleophiles. For these reasons, the utility of a reliable phenotyping test is obvious. Personalized treatment It is well known that personalized treatment is possible for many drugs (theophylline, digoxin, aminoglycosidase, acetaminophen, etc.). However, the individualization of treatments requires that the methods used for drug phenotyping be expensive, time consuming, and require high-performance liquid chromatography (experts that are not readily available in clinical laboratories) ( Development has been slow because it involves HPLC) or capillary electrophoresis (CE).

There will be a strong need for a method for determining the CYP2E1 phenotype of an individual using non-toxic drugs to predict response and side effect profiles to a wide range of potentially toxic drugs. .

Provide an enzyme-linked immunosorbent assay (ELISA) kit for CYP2E1 phenotyping that could be achieved by technicians with minimal training and on a routine basis that does not require complex equipment It will be strongly required.

[0031] It will enable doctors to personalize the treatment of drugs such as acetaminophen,
It would be highly desirable to provide an enzyme-linked immunosorbent assay (ELISA) kit.

CYP3A4 The CYP3A family makes up about 25% of all CYP450 enzymes in human liver. Measurement of CYP3A4 activity is of clinical interest for the following reasons: Polymorphism Although a large degree of inter-individual variation of CYP3A4 isozyme expression in human liver has been demonstrated (> 20-fold), this polymorphism expression The genetic basis of has not been identified to date. CYP3A4 is benzodiazepine, erythromycin, dextromethorphan, dihydropyridine, cyclosporine, lidocaine, midazotam,
It metabolizes several drugs and food components, including nifedipine, terfenadine, and cyclosporin A.

In addition, CYP3A4 is an environmental precarcinogen, especially N′-nitrosonornicotine (
NNN), 4-methylnitrosamino-1- (3-pyridyl-1-butanone) (
NNK), 5-methylchrysene, 4,4'-methylene-bis (2-chloroaniline) (tobacco smoke product). Induction and Inhibition CYP3A4 is induced by many drugs such as dexamethasone, phenobarbital, primidone and the antibiotic rifampicin. Conversely, CYP3A4 contains erythromycin, grapefruit juice, indinavir, ketoconazole,
Inhibited by miconazole, quinine and saquinavir. An example where phenotyping is necessary for drug administration is the case of cyclosporine in the treatment of organ transplant patients. Cyclosporine helps prevent new organs from being rejected.
It is an immunosuppressant administered after transplantation. The plasma concentration of this drug is critical, with high levels leading to nephrotoxicity and low levels leading to organ rejection. Cyclosporin is CYP3
It is metabolized via the A4 system. Several studies have pointed out the importance of monitoring CYP3A4 activity in maintaining effective and safe levels of cyclosporin.
For these reasons, the utility of a reliable phenotyping test is obvious. Personalized treatment It is well known that personalized treatment is possible for many drugs (theophylline, digoxin, aminoglycosidase, cyclosporine and others). However, the individualization of treatments requires that the methods used for drug phenotyping be expensive, time-consuming, and require high-performance liquid chromatography (experts) that are not readily available in clinical laboratories ( HPLC) or capillary electrophoresis (C
E) is included, so development has not progressed easily.

[0034] In order to predict response and side effect profiles to a wide range of potentially toxic drugs, methods for determining the CYP3A4 phenotype of an individual using non-toxic drugs will be highly sought. .

Provide an enzyme-linked immunosorbent assay (ELISA) kit for CYP3A4 phenotyping that could be achieved by technicians with minimal training and on a routine basis that does not require complex equipment. It will be strongly required.

There would be a strong need to provide an enzyme-linked immunosorbent assay (ELISA) kit that would allow physicians to personalize the treatment of drugs such as cyclosporin.

[0037] SUMMARY One object of the present invention the invention is to provide a daily basis can be used in an enzyme-linked immunosorbent assay for the rapid determination of the metabolic enzyme phenotype (ELISA) kits in the clinical laboratory It is.

Another object of the invention is to enable physicians to: a) personalize treatment with drugs that are metabolized by these enzymes b) predict susceptibility to carcinogen-induced diseases such as various cancers To provide an ELISA kit.

Another object of the present invention is to provide a method for determining an individual's metabolic enzyme phenotype using non-toxic drugs to predict response and side effect profiles to a wide range of potentially toxic drugs. To provide.

The ELISA phenotyping kit will use a non-toxic probe agent to determine the spectrum of the individual's metabolic enzyme phenotype. Table 4 lists the probe drugs that should be used for each of the proposed enzymes. Table 4 Enzyme and probe drugs

[0041]

[Table 4]

These drugs were taken by the individual to be phenotyped and their urine was collected 4 hours after ingestion. Urine will be analyzed by the ELISA technique developed according to the present invention. Urine samples were monitored for the following probe drug derivatives (FIGS. 1-7):
), The molar ratio will be calculated to reveal the phenotype of the individual.

In Examples I and II, a detailed description of probe drug derivatives for CYP1A2 and ELISA development is provided. The materials and methods described for the development of the CYP1A2 ELISA kit, and the overall general method are described in NA
Metabolic phenotype E for T1, CYP2D6, CYP2E1 and CYP3A4
Applicable to and will be applicable to the development of LISA kits.

[0044] DETAILED DESCRIPTION different probes agents inventions can be used to determine the CYP1A2 phenotype (caffeine, theophylline). According to the present invention, suitable probe agents include, but are not limited to, caffeine, theophylline or acetaminophen.

[0045] Of these, caffeine is a preferred probe. Caffeine is widely consumed and relatively safe. In previous studies, the phenotypes were 1,7-dimethylxanthine (1,7 DMX) + 1,7-dimethyluric acid (1,7 DMU) and 1,3
, 7-trimethylxanthine (1,3,7TMX, caffeine). In these studies, subjects are orally administered a caffeine-containing substance and the urine concentration of the target metabolite is determined by HPLC (Kilban
e, A. J. et al. (1990) Clin. Pharmacol. The
r 47: 470-474; Tang, B .; -K. et al. (1991) C
lin. Pharmacol. Ther 49: 648-657) or CE (
Meachers et al. (1998) Biomarkers 3:20.
5-218).

Inhibition of CYP1A2 by quinolone antibiotics or serotonin uptake inhibitors
This causes theophylline toxicity. For these reasons, the utility of a reliable phenotyping test is obvious.

The enzyme-linked immunosorbent assay (ELISA) has been successfully applied to the determination of small amounts of drugs and other antigenic compounds in plasma and urine samples and is simple to perform. We have previously developed an ELISA for N-acetyltransferase (NAT2) phenotyping using caffeine as a probe drug (Wo
ng, P.S. , Leyland-Jones, B .; , And Wainer, I .;
W. (1995) J. Mol. Pharm. Biomed. Anal. 13: 1079-
1086). We subsequently tested and demonstrated the effectiveness of the ELISA for NAT2 phenotyping (Leyland-Jones et al. (1999) Ame.
r. Assoc. Cancer Res. 40: Abstract 356).
ELISA for NAT2 phenotyping is easier to perform than HPLC and CE.

According to the present invention, caffeine and two caffeine metabolites (1,7-dimethylxanthine (1,7DMX)) in an individual urine sample collected after caffeine consumption
, 1,7-dimethyluric acid (1,7 DMU)) is used to determine the molar ratio of the currently developed antibodies. The CYP1A2 phenotype of the individual is determined from this ratio.
Using these antibodies, an antigen-enzyme-linked immunosorbent assay (ELISA) will be performed to determine this ratio. The antibodies of the present invention will be polyclonal or monoclonal raised against caffeine and two different caffeine metabolites, which will allow the determination of the molar ratio of caffeine and their metabolites.

In accordance with the present invention, the molar ratio of caffeine metabolites is determined as follows for individual CYP1
Used to determine the A2 phenotype: (1,7-dimethylxanthine (1,7DMX) + 1,7-dimethyluric acid (1,
7DMU)) / caffeine distinguishes slow, medium and fast CYP1A2 metabolites at a molar ratio of 4 and 12 (Butler et al. (1992) Pharmacogenetics).
s 2: 116-117).

Materials and Methods Materials N-acetyl-p-aminophenol (acetaminophen), dioxane,
98-100% glass redistilled formic acid and isobutyl chloroformate are available from A & C Ame
rican Chemicals Ltd. (Ville St-Lauren
t, Que. Purchased from Canada; horseradish peroxidase is B
oehringer Mannheim (Montreal, Que., Can)
ada); ELISA plates (96-well Easy Wa)
sh ^™ modified flat bottom, high binding; Corning glassware, Corning, NY,
USA) and Falcon 96-well microtest tissue culture plate no. 3
072 (Beckton Dickinson Labware, Frankl
in, NJ, USA) is Fisher (Montreal, Quebec, C
alkaline phosphatase conjugated to goat anti-rabbit IgG, keyhole limpet hemocyanin (KLH) was purchased from Pierce
Chemical Co. (Rock ford, IL, USA); acetic anhydride, acetonitrile for HPLC, benzylurea, bovine serum albumin (catalog number A-3803), N-bromosuccinimide, caffeine metabolite, 1-ethyl-3 - (3-dimethylaminopropyl) carbodiimide hydrochloride solution (EDAC), 4-bromo-ethyl butyrate, ethyl 6-bromo-hexanoic acid, methyl cyanoacetate, deuterated chloroform (CDCl _3), deuterated dimethyl sulfoxide (d ₆ ), heavy water (D ₂ O), 1,4-diaminobutane, diethanolamine, dimethyl formamide, dimethyl sulfate, di -tert- butyl, ethyl chloroformate, Freund's adjuvant (complete and incomplete), glutaraldehyde (50% v / v), 1-methylxanthine, p-nitrophenol phosphate disodium salt, palladium (10 dry weight% activated carbon), o-phenylenediamine hydrochloride, polyethylene sorbitan monolaurate (Tween 20), pig skin gelatin, protein A-Sepharose 4B, Sephadex ^™ G25 fine,
Sodium hydride, sodium methoxide, theophylline, tributylamine,
Tween ^™ 20 is available from Sigma-Aldrich (St-Louis, Miss.
uri, USA); silica gel particle size 0.040-0.0
63mm (230-400 mesh) ASTM Emerck Darmsta
dt, Germany, VWR (Montreal, Que., Canada).
) Purchased from. The dioxane was dehydrated and refluxed with calcium hydride for 4 hours before use. Other reagents were ACS grade.

Synthetic Method The synthetic route for the production of caffeine, 1,7-dimethylxanthine, 1,7-dimethyluric acid derivative is shown in FIGS. 8 and 9.

Synthesis of 7-ethoxycarboxypentyl-1,3-dimethylxanthine (II) Compound II was prepared according to Dally et. al. (Dally, JW, Mueller
, C.I. , Shamin, M .; (1991) Pharmacology, 42: 3.
09-321). Dissolve 320 mg theophylline (I) (1.78 mmol) in 7 mL dry dimethylformamide,
290 mg of potassium carbonate (2.1 mmol) are added to the reaction mixture. 358μ
L of ethyl 6-bromohexanoate (2.02 mmol) is added slowly and the suspension is heated at 60 ° C. for 14 hours. The suspension is filtered to remove potassium carbonate.
After washing the potassium carbonate with a small amount of dimethylformamide, the solvent is evaporated under reduced pressure using a rotary evaporator and a high vacuum pump. The residue is dissolved in chloroform, and the solution is dried with magnesium sulfate (MgSO ₄ ). The solvent is evaporated under reduced pressure using a rotary evaporator. 480 mg of product (slightly yellow liquid, 1.49 mmol) are obtained, corresponding to a yield of 83.7%.

Synthesis of 7-carboxypentyl-1,3-dimethylxanthine (III) Compound III is synthesized as follows. Dissolve 225 mg of compound II (0.7 mmol) in 7 mL of dimethylformamide. 4 mL of 10% NaOH
Add the solution and reflux the solution for 30 minutes (100-125 ° C). The solvent is evaporated under reduced pressure using a rotary evaporator and a high vacuum pump. The residue is dissolved in 7 mL of water and the solution is acidified to pH 4 with a 6N HCl solution. When the solution is cooled to 4 ° C., the product crystallizes as needles. The crystals were placed in a 15 mL semi-solid glass funnel (10
-15 ASTM) and dry. 175 mg of product (0.595
Mmol), corresponding to an 85% yield.

Synthesis of 7-ethoxycarboxypentyl-1-methylxanthine (V) Compound V is synthesized as follows. 116 mg of 1-methylxanthine (I
V) (0.7 mmol) is dissolved in 4 mL of dimethylformamide. 129m
g potassium carbonate (0.93 mmol) are added and the resulting solution is stirred. 125
A solution of μL of ethyl 6-bromohexanoate (0.7 mmol) in 0.4 mL of dimethylformamide is slowly added in three portions. The reaction mixture is heated at 50 ° C for 1.5 hours and at 65 ° C for 1 hour. After cooling, the suspension is filtered and the filtrate is evaporated under reduced pressure using a rotary evaporator and a high vacuum pump. The product is a silica gel column (4) using an ethyl acetate-hexane solution (9: 1, v / v) as an eluent.
Purify by flash chromatography (0 × 1 cm).

Synthesis of 7-carboxypentyl-1-methylxanthine (VI) Compound VI is synthesized as follows. Dissolve 31 mg of compound V (0.1 mmol) in 1 mL of dimethylformamide and add 660 μL of 10% NaOH. The resulting solution is refluxed (100-120 ° C) for 30 minutes. After cooling to room temperature, the solvent is evaporated under reduced pressure using a rotary evaporator and a high vacuum pump.
The residue is dissolved in water and the solution is acidified to pH 4 with a 6N HCl solution. After cooling, the solution gives white needles, which are filtered and dried. 23 mg of product (0
. 082 mmol), corresponding to a yield of 82%.

Synthesis of 6-Amino-1-benzyluracil (IX) Compound IX was prepared as described below in Hutzenlaub and Pfeidere.
r (Hutzenlaub, W., and Pfeiderer, W. (197
9) Liebigs Ann. Chem. 1847-1854). 8.46 g of sodium methoxide (160 mmol)
Is dissolved in 71 mL of methanol. The solution was stirred and 7.55 g of benzyl urea (50 mmol) and 4.71 mL of methyl cyanoacetate (53.4 mmol)
Add. The suspension is refluxed at 68-70 ° C for 5.5 hours and cooled to room temperature. After filtration, the methanol is evaporated under reduced pressure using a rotary evaporator. The product is precipitated by dissolving the residue in warm distilled water and acidifying with glacial acetic acid to pH 3-4. After 2 hours (or overnight) at room temperature, the suspension is filtered under reduced pressure through a sintered glass funnel. The product is washed with water and dried. The yield is 62-65%.

Synthesis of 6-Amino-1-benzyl-5-bromouracil (X) Compound X was prepared as follows by Hutzenlaub and Pfeiderer
(Hutzenlaub, W., and Pfeiderer, W. (1979)
) Liebigs Ann. Chem. 1847-1854). 3.2 g of 6-amino-1-benzyluracil (15.8
(Mmol) in 100 mL of 60 mL of acetic acid and 3 mL of acetic anhydride. 2
. 85 g of N-bromosuccinimide (16 mmol) are added in portions over 30 minutes. The reaction mixture is stirred for 1 hour and cooled to room temperature. The precipitate is filtered, washed with a little cold ethanol and dried. 3.36 g of white crystals were obtained (12
Mmol), corresponding to a yield of 76%.

Synthesis of 6-amino-1-benzyl-5-[(N-4′-aminobutyl) -amino] uracil (XI) Compound XI is synthesized as follows. 3 g of compound X (10.71 mmol) was added to 30 mL of 50% 1,4-diaminobutane (bp 158-160 ° C .; do).
. 877) Dissolve in aqueous solution (v / v) and stir the solution at room temperature overnight. The solution is evaporated under reduced pressure using a rotary evaporator and a high vacuum pump. The obtained oil was dissolved in a minimum amount of ethyl acetate-methanol solution (4: 1; v / v), and a semi-molten glass funnel (150 mL) using an ethyl acetate-methanol solution as an eluent was used.
Purify by dry flash chromatography on silica gel packed in. In each successive fraction, 60% ethyl acetate / 40 with increasing solvent polarity
% Methanol to 45% ethyl acetate / 55% methanol (v / v). The product is isolated as a pale yellow oil. The amount of purified product obtained is 1.6 g (6.1 mmol), corresponding to a yield of 57%.

6-amino-1-benzyl-5- [N-4′-tert-butoxycarbonyl-
Synthesis of Amino] uracil (XII) Compound XII is synthesized as follows. Dissolve 1.63 g of compound XI (5.9 mmol) in 5.4 mL of 1 N NaOH. 270 mg of sodium bicarbonate (3.2 mmol) and 2.7 mL of water are added. 5.4 mL of di-t
tert-butyl dicarbonate in isopropanol (1.88 g (8.61 g)
(Mmol) in 5.4 mL isopropanol) is slowly added to the solution of compound XI. After stirring at room temperature for 3 hours, 13.4 mL of water was added, and unreacted di-ter was added.
Extract t-butyl dicarbonate twice with 20 mL of petroleum ether. The pH of the reaction mixture was adjusted to 7 by adding a 10% citric acid solution, and the solution was
Extract twice with ethyl acetate. The organic layer is dried over sodium sulfate (Na ₂ SO ₄ ) and concentrated under reduced pressure using a rotary evaporator. The product precipitates upon addition of a small amount of light petroleum ether to the concentrated solution. 0.99 g of off-white crystalline compound XII (2.62 mmol) are obtained, corresponding to a yield of 44%.

Synthesis of 6-amino-1-benzyl-5-[(N-4′-tert-butoxycarbonylaminobutyl-N-ethoxycarbonyl) -amino] uracil (XIII) Compound XIII was synthesized as follows. You. 806 mg of compound XII (2.
(14 mmol) are suspended in 7.5 mL of water and stirred vigorously. Add 0.5 mL of ethyl chloroformate (5.22 mmol). 3.75 mL of 1N NaOH
The solution is added dropwise and the resulting solution is stirred at room temperature for 2.5 hours. The white solid product is filtered, washed thoroughly with water and dried. 741 mg of product are obtained (1.77).
Mmol), corresponding to a yield of 82.7%.

6-amino-1-benzyl-5-[(N-4′-tert-butoxycarbonylaminobutyl-N-ethoxycarbonyl) -amino] -3-methyluracil (X
Synthesis of IV) Compound XIV is synthesized as follows. 712 mg of compound XIII (1.
(77 mmol) in 5.8 mL of water. 2.3 mL of 1N NaOH solution are added and the resulting solution is heated at 40 ° C. with vigorous stirring. 0.23 mL of dimethyl sulfate (2.43 mmol) is added slowly and the solution is stirred at 40 ° C. for 1.5 hours. The precipitate formed during the reaction is filtered, washed with water and dried. The product is purified from the precipitate by flash chromatography on a silica gel column (40 × 1 cm) using a dichloromethane solution containing 4% methanol as eluent. The product recrystallizes from ethyl acetate. 498 mg of compound XIV (1.15
Mmol), corresponding to a yield of 65%.

6-amino-5-[(N-4′-tert-butoxycarbonylaminobutyl-
Synthesis of N-ethoxycarbonyl) -amino] -3-methyluracil (XV) Compound XV is synthesized as follows. 440 mg of compound XIV (1.02
Mmol) in 12 mL of methanol and 252 mg of ammonium formate (
4 mmol). 240 mg of palladium on carbon (10%
). The catalytic hydrogenation is carried out at room temperature for 3 hours. The catalyst is filtered off and the filtrate is evaporated under reduced pressure using a rotary evaporator and a high vacuum pump. 341
mg product (0.99 mmol) is obtained, corresponding to a yield of 97%.

Synthesis of 7- (4′-aminobutyl) -1-methyluric acid (XVI) Compound XVI is synthesized as follows. 300 mg of compound XV (0.87
5 mmol) is dissolved in 4.5 mL of dry dimethylformamide and mixed with 144 mg of sodium hydride (6 mmol). The mixture is left at room temperature for 20 minutes and
Stir at 10-115 ° C for 30 minutes. The color gradually changes to dark yellow. After cooling, 6
. 5 mL of water are added and the solution is acidified to pH 0 with 6N HCl solution. The solvent is evaporated under reduced pressure using a rotary evaporator and a high vacuum pump, and the crude product is dissolved in an ethyl acetate-methanol solution (1: 4, v / v). The inorganic salts are filtered off and the yellow filtrate is purified by flash chromatography on a silica gel column (40 × 1 cm) using an ethyl acetate-methanol (3: 7, v / v) solution as eluent. After titration of the residue with isopropanol, the product was obtained as a pale yellow solid. 98.9 mg of product were obtained (0.391 mmol), 45
% Yield.

NMR Spectroscopy ¹ H NMR spectra of the compounds were obtained using a 500 MHz spectrophotometer (Varian XL 500 MHz, Varian Analytical).
Instruments, San Fernando, CA, USA).

Conjugation of hapten to bovine serum albumin and keyhole limpet hemocyanin Caffeine-BSA, 1,7-dimethylxanthine-BSA conjugate was Rojo
(Rojo et al. (1986) J Immunol. 137: 904).
-910). In a 25 mL Erlenmeyer flask, 15 mg of BSA was dissolved in 6 mL of a caffeine derivative (or 1,7-dimethylxanthine derivative) solution (1.25 μmol / mL aqueous solution), followed by 1.43 mL of EDAC solution ( (10 mg / mL aqueous solution). The solution is stirred overnight at room temperature and dialyzed against water (500 ml twice daily) for 48 hours at room temperature. The conjugate is stored in 0.5 mL portions at -20 ° C. Dimethyluric acid conjugate was prepared according to the method of Pescar et al. (Peskar (1972) Eur. J. Bioch).
em. 26: 191-195). 7.5 mg of 1,7-dimethyluric acid (0.03 mmol) is placed in a 5 mL round bottom flask and 1 mL of 0.1 M
Na ₂ PO ₄ -NaH ₂ PO ₄ buffer, dissolved in pH 7.0. 500 μl of 0.
021 M glutaraldehyde solution (42.5 μL of 50% per 10 mL of water
Glutaraldehyde (v / v)) is added to the stirring solution. After stirring for 2 hours, 100μ
L of 1 M lysine in 0.1 M Na ₂ PO ₄ -NaH ₂ PO ₄ buffer (pH 7.0) is added. The solution was stirred for 1 hour and 250 mL of 150 mM NaCl, 5 mM
Dialysis is performed for 48 hours against a Na ₂ PO ₄ -NaH ₂ PO ₄ buffer solution, pH 7.0, while exchanging the buffer solution 2-3 times per day. 1,7-dimethylxanthine-BSA
The conjugate is stored in 0.5 mL portions at -20 ° C. Caffeine-KLH and 1,7
-Dimethylxanthine-KLH conjugate is prepared as follows. 20mg KL
The lyophilized H powder is dissolved in 2 mL of 0.9 M NaCl solution and dialyzed by exchanging the solution twice with respect to 100 mL for 10 hours. In a 25 mL Erlenmeyer flask, 0.8 mL of a caffeine derivative or a 1,7-dimethylxanthine derivative (2.5 μmol / mL 0.9 M) is added to 1.1 mL of a KLH solution (about 10 mg / mL).
NaCl solution). 2 mL of EDAC solution (10 mg / mL 0.9 M
NaCl solution) and 1.8 mL of a 0.9 M NaCl solution are added successively to the derivative solution. The solution is stirred overnight (20 hours) at room temperature. The solution is 250 mL of 0
. The solution is dialyzed against 9M NaCl for 48 hours, exchanging the solution 2-3 times per day. Caffeine-KLH and 1,7-dimethylxanthine-KLH solutions are 0.5
Store at −20 ° C. in mL. The 1,7-dimethyluric acid-KLH conjugate was Pesk
The method of ar et al. (Peskar (1972) Eur. J. Biochem. 26:
191-195). 20 mg of KLH lyophilized powder was dissolved in 2 mL of 0.9 M NaCl solution, and 100 mL was added for 10 hours.
Change the solution twice and dialyze. 7.3 mg of 1,7-dimethyluric acid (about 0.03 mmol) is placed in a 5 mL round bottom flask and dissolved with 1 mL of KLH solution. 50
0 μL of a 0.021 M glutaraldehyde solution (42.5 per 10 mL of water)
μL of 50% glutaraldehyde (v / v)) is added dropwise to the stirring solution. After stirring for 2 hours, 100 μL of 1 M lysine in 0.1 M Na ₂ PO ₄ -NaH ₂ PO ₄ buffer (
pH 7.0) solution is added. The solution was stirred for 1 hour and 250 mL of 0.9 M Na
_{Cl, 5mM Na 2 PO 4 -NaH} 2 PO 4 buffer, pH 7.0, to be dialyzed for 48 hours with the change of day 2-3 buffer. 1,7-dimethyluric acid-
Store the KLH conjugate in 0.5 mL aliquots at -20 ° C.

Protein Determination by the Method of Lowry et al. (Lowry, OH et al. (1951) J. Biol. Chem.
Solution A: Dissolve ₂ g of Na ₂ CO ₃ in 50 mL of water and bring to a volume of 100 mL with 10 mL of 10% SDS and 10 mL of 1N NaOH, water. Freshly prepared Solution B: 1% NaK tartrate Solution C: 1% CuSO ₄ . 5H ₂ 0 solution D: 1N phenol (freshly prepared): 3mL folinic - Shiokarutofu E Nord reagent (2.0 N) and 3mL water solution E: 98 mL of the solution A, 1 mL of solution B, solution C of 1 mL, freshly Preparation BSA: 1 mg / mL. 0.10 g bovine serum albumin (fraction volume) / 100 mL assay standard curve Tube # (13 × 100 mm) solution 12 34 45 67 BSA (μl) 0 10 15 20 30 40 50 water (μl) 200 190 185 180 170 160 160 150 Solution E (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Vortex and leave at room temperature for 10 minutes. Solution D (μl) 200 200 200 200 200 200 200 200 Vortex and leave at room temperature for 1 hour. The absorbance is read at 750 nm using water as a blank. Unknown sample solution D. F ^a (in triplicate) Tube # (13 × 100 mm) Unknown sample (μl) xxx water (μl) y y y x + y = 200 μl Solution F (mL) 2.0 2.0 2.0 Vortex for 10 minutes at room temperature Neglect. Solution D (μl) 200 200 200 200 200 200 200 200 Vortex and leave at room temperature for 1 hour. The absorbance is read at 750 nm using water as a blank. Using a standard curve, the D.E. F. Calculate protein concentration taking into account (dilution ratio). a: D. F. (Dilution ratio): The absorbance of the unknown sample at 750 nm must be in the range of the absorbance of the standard curve.

The caffeine, 1,7-DMX or 1,7-incorporated per mg of KLH
Method for Determining the Number of MMUs in DMU This method gives a rough estimate. Useful because it is possible to determine whether the coupling has proceeded as expected. A) Solution-10% sodium dodecyl sulfate (SDS) solution-1% SDS solution-0.5 or 1 mg / mL caffeine-KLH (or 1,7-DMX-K
LH or 1,7-DMU-KLH) in 1% SDS solution (1 mL) -0.5 or 1 mg / mL in KLH in 1% SDS solution B) Method -Caffeine-KLH conjugate (or 1,7-DMX) -KLH or 1,7-D
The absorbance of MU-KLH is measured at the wavelength of maximum absorption of caffeine (or 1,7-DMX or 1,7-DMU) using a 1% SDS solution as a blank. , 7-DMX or 1,7-DM
Measured at the maximum absorption wavelength of U)-Equation:

[0068]

(Equation 1)

According to where K is the number of moles of caffeine per mg of KLH; ελ _max (caffeine) is the molar extinction coefficient of caffeine at the maximum absorption wavelength.
Caffeine (or 1,7-DMX or 1,7-
Calculate the moles of (DMU).

Hapten Coupling to Horseradish Peroxidase Caffeine and 1,7-dimethylxanthine derivatives and 1,7-dimethyluric acid derivatives (after succinylation with succinic anhydride) were converted to horseradish peroxidase (HRP) by the following method. Was conjugated to A 5 mL round bottom flask is charged with 0.12 mmol of derivative. 500 μL of dioxane, freshly dried with calcium chloride, was added by pipette. The suspension is stirred and cooled to 10 ° C. in a water bath using crushed ice. 31 μL of isobutyl chloroformate (0.24 mmol) (recently opened or purchased) and 114 μL of tributylamine (0.47 mmol) were added by pipette. Stir for 30 minutes at 10 ° C. While stirring, 1
Dissolve 3 mg of horseradish peroxidase (HRP) in 2 mL of water and cool the solution to 4 ° C. on crushed ice. After stirring for 30 minutes, 100 μL of 1N NaOH
Add the solution (freshly prepared) to the HRP solution at 4 ° C. and pour the alkaline HRP solution into a 5 mL flask all at once. The suspension is stirred at 10-12 ° C for 4 hours. 0.
The free derivative is separated from the HRP conjugate by filtration on a Sephadex G-25 ^™ fine column (1.6 × 30 cm), equilibrated and eluted with 1 M sodium phosphate buffer, pH 7.0. Collect 1.0-1.2 mL fractions manually or with a fraction collector. During the elution, two bands will be observed: H
Light yellow band behind RP and HRP conjugates. The HRP conjugate band eluted between fractions 11-16. The fractions containing the HRP conjugate are pooled in a 15 mL tissue culture vessel with a screw cap. Dilute part (usually 50μ
(L + 650 μL buffer) Determine HRP conjugate concentration at 403 nm. [HRP conjugate] (mg / mL) = A ₄₀₃ × 0.4 × D. F. The UV absorption spectrum (UV) between 320 and 220 nm is recorded. The presence of additional absorption peaks at 280 nm, 280 nm and 290 nm by caffeine-HRP, 1,7-DMX-HRP and 1,7-DMU-HRP conjugates
It is an indicator that the coupling has proceeded as expected. After the above measurement, 5 μL of 4% thiomersal solution is added per mL of caffeine-HRP, 1,7-DMX-HRP or 1,7-DMU-HRP conjugate solution. The conjugate is stored at 4 ° C.

Antibody Production Six mature female New Zealand white rabbits (Charles Riv)
er Canada, St-Constant, Que. , Canada) was used for antibody production. The protocol used in this study has been approved by the McGill University Animal Protection Commission according to Canadian Animal Protection Council guidelines. An isotonic salt solution (0.6 mL) containing 240 μg of KLH-conjugated antigen was added to 0.1 mL.
Emulsified with 6 mL complete Freund's adjuvant. 0.5 mL of the emulsion (100 μg of antigen) was injected intramuscularly or subcutaneously per rabbit. Rabbits were then boosted with 50 μg of antigen emulsified in incomplete Freund's adjuvant at 3 week intervals. 10-14 days after the boost, blood was collected without anticoagulant into a vacuum tube by venipuncture of the ear and kept at 4 ° C. After coagulation, centrifuge at 4 ° C.
. Sodium azide was added to the antiserum to a final concentration of 001% (1 mL
(1 μL of 1% sodium azide solution per antiserum). The antiserum was stored at −20 ° C. in 0.5 mL portions.

Antiserum titer: The wells of the microtiter plate were filled with 10 μg mL ⁻¹ of bovine serum albumin-
Caffeine (or 1,7-dimethylxanthine, 1,7-dimethyluric acid) conjugate (100 mM sodium carbonate buffer, pH 9.6) was coated overnight at 150C (150 μL / well). They are Nunc Immuno Wash 1
2 Using autoclavable TPBS (0.05% Tween 20)
Was washed 3 times with a phosphate buffer solution containing Unoccupied sites were blocked by a 2 hour incubation at room temperature with 150 μL / well TPBS containing 0.05% porcine gelatin. Wells were washed three times with TPBS and 150 μL of antiserum diluted in TPBS was added. After 2 hours at room temperature, the wells were washed three times with TPBS and 100 μL of goat anti-rabbit IgGs-alkaline phosphatase conjugate diluted in PBS containing 1% BSA was added. After 1 hour at room temperature, the wells were washed 3 times with TPBS.
Wash three times with water. Next, 150 μL of a diethanolamine buffer (10 mM, pH 9.8) solution containing MgCl ₂ (0.5 mM) and p-nitrophenol phosphate (3.85 mM) was added to the wells. After 30 minutes at room temperature, the absorbance at 405 nm was read on a microplate reader. Antibody titer is defined as the dilution required to change one unit (1 au) absorbance.

Isolation of IgG Antibodies Rabbit IgG antibodies to the KLH conjugate were purified by affinity chromatography on a protein A-Sepharose 4B column as follows.
A 0.9 × 15 cm Pharmacia chromatography column was packed with the Rotein A-Sepharose 4B suspension to a volume of 1 mL. Column is 0.15M N
0.01 M Na ₂ HPO contains aCl ₄ -NaH ₂ PO ₄ buffer (pH 8.
0) Wash thoroughly with (PBS), followed by 3-4 mL of 0.1 M trisodium citrate buffer (pH 3.0). The column was then thoroughly washed with PBS.
1 mL of rabbit antiserum was diluted with 1 mL of PBS, and the resulting solution was slowly added to the column. The column was washed with 15 mL of PBS and eluted with 0.1 M trisodium citrate buffer (pH 3.0). 0.8 mL of three fractions of 2.2 mL
Calibrated 15m containing 1M Tris-HCl buffer (pH 8.5)
Collected in L tubes. The purified rabbit IgG antibody was stored at 4 ° C. in the presence of 0.01% sodium azide.

Competitive antigen ELISA Additive-free buffer and water were passed through a 0.45 μM Millipore filter.
It was maintained for one week, except that the substrate buffer was freshly prepared. BSA, antibodies,
Tween^TM20 and horseradish peroxidase are added immediately before use in buffer and
Added to water. The urine sample is usually a cup of coffee (about 100 mg per cup)
Drink instant or roasted coffee containing caffeine
Collected after time and stored at -20 ° C in 1.5 mL microtubes 1 mL at a time
did. For ELISA, 3 × 10 5 in wells of a microtiter plate ^-6 Will give caffeine, 1,7-DMX and 1,7-DMU concentrations lower than M
As described above, a urine sample was prepared using an isotonic sodium phosphate buffer, pH 7.5 (310 mosM),
Dilute with. The wells of the ELISA plate are Nunc Immuno Wash
 Washed with 12 washer. 6.6 μg ml^-11 comprising the isolated IgG antibody of
A 6 mL solution was prepared in 100 mM sodium carbonate buffer (pH 9.6),
150 μL of the solution was pipetted into an 8-channel pipette (Brinkmann Transf
erpette^TM-8 50-200 μL) and 2 from Brinkmann.
Each well of a microtiter plate using a 00 μL Flex chip
Added. After coating the wells with antibody for 20 hours at 4 ° C.,
. Isotonic sodium phosphate buffer (IPB) containing 05% Tween TM20
Wash 3 times with T), invert plate and allow liquid to absorb on paper towel
To drain water appropriately. Adjust 30 mL of IPBT solution containing 1% BSA
150 μL of this solution was pipetted into an 8-channel pipette (Brinkmann T).
transferpette^TM-8 50-200 μL) and 200 μL yellow
-Chip (Sarsteadt for P200 Gilson Pipetman)
Each well was added using a yellow tip). After 3 hours at room temperature, the wells were
Washed three times with PBT solution and drained. Caffeine, 1,7-DMX and 1,7
-400 μL of sample for DMU determination was
And 1.5 mL micros using a P200 Gilson Pipetman.
Added to tubes. Sarstedt Yellow Chip and P200 Gils
Using on Pipetman, 200 μL of each sample was shown in FIG.
Falcon 96 well microtest tissue culture plate
Add to the double. 8-channel pipette (Brinkmann Transf
erpette^TM-850-200 μL) and use an 8-channel pipette.
Change to a chip (200μL Flex chip from Brinkmann)
In each row, 150 μL of sample was coated in a 96-well ELISA mask coated with antibody.
Transfer to the corresponding well of the microtitre plate. After sample addition, use microtiter
Cover the plate and leave at room temperature for 2 hours. While the plate is standing,
Substrate buffer without hydrogen hydride and o-phenylenediamine hydrochloride (25 mM
Enoic acid and 50 mM sodium phosphate dibasic buffer (pH 5.0) are prepared.
Microtiter plates were washed three times with IPBT solution and 0.05% Tween^TM And drain three times. 50 μL of hydrogen peroxide and 40 mg of o-phenylene
Add diamine dihydrochloride to the substrate buffer. 150 microliters (150 μL
) Is transferred to an 8-channel pipette (Brinkmann Transfer).
rpette^TM-8 50-200 μL) and 200 μL Flex chip (
Add each well using a Brinkmann). Microtiter press
Cover the plate and shake for 25-30 minutes at room temperature.
Brinkmann Transferpette^TM-8 50-200 μL)
And 50 μL using a 200 μL Flex chip (Brinkmann)
Stop by adding / well 2.5M HCl. Gently shake for 3 minutes
After shaking, the absorbance at 405 nm was read on a microplate reader.

Standard Solution of Caffeine, 1,7-DMX and 1,7-Dimethyluric Acid Solution for ELISA Using a 100 mL volumetric flask, 310 mosM sodium phosphate buffer,
Caffeine, 1,7-DM at a concentration of 6.00 × 10 ⁻⁴ M in pH 7.5 (IPB)
Prepare 100 mL of X and 1,7-DMU stock solution. Stir the solution to ensure complete dissolution.

The stock solution is stored at −20 ° C. in 1 mL portions. On the day of the ELISA, melt one and warm to room temperature. Preparation to prepare the following standard solution of the compound # [Compound] Composition 1 6.00x10 ^-4 M stock solution ^{2 2.00x10 -4 M 200μLS1 + 400μLIPB 3} 1.12x10 -4 M 200μLS1 + 868μLIPB 4 6.00x10 -5 M 100μLS1 + 900μLIPB ^{5 3.56x10 -5 M 60μLS1 + 951μLIPB 6} 2.00x10 -5 M 100μLS2 + 900μLIPB 7 1.12x10 -5 M 100μLS3 + 900μLIPB 8 6.00x10 -6 M 100μLS4 + 900μLIPB 9 3.56x10 -6 M 100μLS5 + 900μLIPB 10 2.00x10 -6 M 100μLS6 + 900μLIPB 11 1 ^{.12x10 -6 M 100μLS7 + 900μLIPB 12 6.00x10} -7 M 100μLS8 + 9 ^{0μLIPB 13 3.56x10 -7 M 100μLS9 + 900μLIPB} 14 2.00x10 -7 M 100μLS10 + 900μLIPB 15 1.12x10 -7 M 100μLS11 + 900μLIPB 16 6.00x10 -8 M 100μLS12 + 900μLIPB 17 3.56x10 -8 M 100μLS13 + 900μLIPB 18 2.00x10 -8 M 100μLS14 + 900μLIPB 19 2.00 × ^{10 −9} M 100 μLS15 + 900 μL IPB 20 2.00 × ^{10 −10} M 100 μLS15 + 900 μL IPB 21 2.00 × ^{10 −11} M 100 μLS15 + 900 μL IPB 22 2.00 × 10 ⁻¹² M 100 μLS15 + 900 μL IPB 23 2.00 × 10 ¹³ μL

Antibody Specificity To ensure the accuracy of the CYP1A2 phenotyping ELISA measurement, the antibody must have specificity for its individual caffeine metabolites and will not recognize any other derivatives Little or no recognition. To confirm its selectivity, an ELISA will be performed with standard solutions of the compounds listed in Table 5. Ideal antibody specificity results are also postulated in Table 5. Table 5 Caffeine-Abs, 1,7 for caffeine metabolites and structural analogs
-Reactivity of DMX-Ab and 1,7-DMU-Ab

[0078]

[Table 5]

A, number 0 means no inhibition or higher than 40% at the highest concentration tested in the ELISA (5 × 10 ⁻³ M); competitive antigen E
The concentrations of caffeine, 1,7-dimethylxanthine and 1,7-dimethyluric acid required for 50% inhibition in LISA will be determined; b, 1,3-dimethylxanthine, theophylline; c, 1,7-dimethylxanthine, paraxanthine; d, 3,7-dimethylxanthine, theobromine; e, AAU, 5-acetamido-6-aminouracil; f, AAMU, 5-acetamido-6-amino-3-methyluracil G, AADMU, 5-acetamido-6-amino-1,
3-dimethylxanthine. Results As determined by ELISA, strong sedimentation lines after double immunodiffusion on agar plates of derivatives conjugated to antisera and rabbit serum albumin, and low cross-reactivity with other caffeine derivatives, 30, Positive production of antibodies to caffeine, 1,7-DMX and 1,7-DMU with antibody titers of 000-100,000 could be observed. These results constitute constructive conditions for the development of a competitive antigen ELISA, according to the methods as described in the section above on materials and methods.

According to one embodiment of the present invention, a competitive antigen ELISA will be developed for CYP1A2 phenotyping using caffeine as a probe agent. Contrary to current methods used for phenotyping, this assay is sensitive, rapid, and can be easily performed on a routine basis by even the least trained and untrained technicians in clinical laboratories . Example II Caffeine in urine by ELISA kit, 1,7-dimethylxanthine (1,
Determination of 7-DMX) and 1,7-dimethyluric acid (1,7-DMU) Table 6 Contents of ELISA kit and storage conditions

[0081]

[Table 6]

Dilution of urine samples for [caffeine], [1,7-DMX] and [1X] determination by ELISA Required for determination of caffeine, 1,7-DMX and 1,7-DMU Dilution of urine samples was determined by sensitivity of competitive antigen ELISA and caffeine, 1,7-
It is a function of DMX and 1,7-DMU concentration. It is suggested that AAMU and 1X dilute the urine sample by a factor such that it is about 3 × 10 ⁻⁶ M in the wells of the microtiter plate.

[0083]

[Table 7]

A: Stir the microtube containing the urine sample before pipetting. Store the diluted urine sample at -20 ° C in a box for microtubes. Buffer B: Dissolve the contents of one vial B / 100 mL.

[Caffeine], [1,7-DMX] and [Caffeine] in diluted urine samples by ELISA
1,7-DMU]. Warning The substrate is carcinogenic. When handling buffer E (substrate buffer), surgical gloves should be worn. Each sample is determined in duplicate. Excellent pipetting techniques are required. If this technique is mastered, the double absorption value should be less than 5%. Buffers C, D and E are prepared fresh. Buffer E-H ₂ O ₂ is prepared just before pipetting into the microtiter plate wells. Sample preparation: Prepare and print Table 8 on a computer. This table shows the contents of each of the 96-well microtiter plates. Enter the name (or number) of the urine sample in the corresponding well location in Table 8. Choose the dilution ratio (DF) for each urine sample and fill in the corresponding positions in Table 8. Record the dilution of each urine sample with buffer B in the corresponding position in Table 8: F. (100 μL of 10 × diluted urine sample + 900 μL buffer B) and 100/900. For prepared samples of different dilutions, see "Dilution of urine samples by ELISA" above. 1
Prepare urine samples of different dilutions in 1.5 mL microtubes using a styrofoam support for 00 microtubes. Make Table 9 on a computer and print it. Using a styrofoam support (100 microtubes), prepare the following 48 microtubes in the order shown in Table 9. Table 8 Location of blank, control and urine samples in microtiter plates

[0086]

[Table 8]

Table 9 Contents of different microtubes

[0088]

[Table 9]

Solution Buffer C: Dissolve the contents of one vial C / 50 mL. Add 25 mL Tween ^™ 20. Buffer C: Dissolve the contents of one vial D / 25 mL.

Add 25 mL Tween ^™ 20. 0.05% Tween ^™ 20: Add 25 mL Tween ^™ 20 to a 100 mL Erlenmeyer flask containing 50 mL water. 2.5N HCl: 41.75 mL of 12N HCl / 200 mL. 250mL
Save in glass bottle. Caffeine-HRP conjugate: Add 9 mL Buffer C to a 15 mL glass tube. Add 90 μL of caffeine-HRP stock solution. 1,7-DMX-HRP conjugate: Add 9 mL of Buffer C to a 15 mL glass tube. Add 90 μL of 1,7-DMX-HRP stock solution. 1,7-DMU-HRP conjugate: Add 9 mL of Buffer C to a 15 mL glass tube. Add 90 μL of 1,7-DMU-HRP stock solution. Buffer E-H ₂ O _2: 1 vials E- substrate water solubility / 50 mL The contents of the. 2
Add 5 μL of 30% H ₂ O ₂ solution (freshly prepared). Table 10 Caffeine, 1,7-DMX and 1,7-DMU standard solutions (diluted in buffer B)

[0091]

[Table 10]

ELISA conditions Starting from the last row, add 50 μL of caffeine-HRP (1,7-DMX-HRP or 1,7-DMU-HRP) conjugate solution to the wells. Well # 9
Starting from 6 (see Table 11), 50 μL of diluted urine samples (in duplicate), standards, blanks are added to the wells with a micropipette (0-200 μL). Cover the plate and mix gently by stirring for a few seconds. The plate is left at room temperature for 3 hours. 100 μL / well of Buffer C using a microtiter plate washer
And wash three times. Wash three times with 100 μL / well of 0.05% Tween ^™ 20. Add 150 μL / well of buffer E-H ₂ O ₂ (prepared just before pipetting into microtiter plate wells). Shake for 20-30 minutes at room temperature using an orbital shaker. 2.5N HCl solution at 5 per well
Add 0 μL. Shake for 3 minutes at room temperature using an orbital shaker. Read the absorbance of the wells at 490 nm using a microplate reader. Print the data sheet and paste it appropriately.

[Caffeine], [1,7-DMX] and [1,7-
DMU] Calculation Table 11 is drawn with a computer. Using a microplate reader data sheet, record in Table 11 the average absorbance of blanks, controls (no free hapten), standards and samples. Sigma-plot (or other software)
Use to draw a calibration curve in a semilogarithmic plot. Find [AAMU] (or [1X]) in the microtiter well of the unknown sample from the calibration curve and enter the data in Table 12. The dilution ratio was determined using the unknown sample [caffeine] ([1,7-DMX] or [
1,7-DMU]) and multiply the result in the corresponding cell of Table 12. Table 11 Average absorbance values of samples in microtiter plate

[0094]

[Table 11]

Table 12 Caffeine, 1,7-DMX and 1,7-DMU concentrations in urine samples

[0096]

[Table 12]

Table 13 Composition of different buffers

[0098]

[Table 13]

Although the invention has been described with reference to specific embodiments thereof, further modifications are possible and the application generally encompasses any variation, use or application of the invention in accordance with the principles of the invention. It is intended that, and such new developments from the present disclosure, be within the known or customary practice of the technology to which this invention pertains,
It will be understood that it is included as an adaptation of the essential features set forth above and in accordance with the appended claims.

[Brief description of the drawings]

FIG. 1 shows p-aminosalicylic acid derivatives for NAT1 phenotyping by ELISA.

FIG. 2 shows a caffeine derivative for CYP1A2 phenotyping by ELISA.

FIG. 3 shows the 1,7-type for CYP1A2 phenotyping by ELISA.
Shows a dimethylxanthine derivative.

FIG. 4 shows the 1,7-type for CYP1A2 phenotyping by ELISA.
The dimethyluric acid derivative is shown.

FIG. 5 shows dextromethorphan derivatives for CYP2D6 phenotyping by ELISA.

FIG. 6 shows chlorzoxazone derivatives for CYP2E1 phenotyping by ELISA.

FIG. 7 shows dextromethorphan derivatives for CYP3A4 phenotyping by ELISA.

FIG. 8 shows a synthetic route for the production of caffeine and 1,7-dimethylxanthine derivatives for CYP1A2 phenotyping according to one embodiment of the present invention.

FIG. 9 illustrates a synthetic route for the production of caffeine and 1,7-dimethyluric acid derivatives for CYP1A2 phenotyping according to one embodiment of the present invention.

FIG. 10 shows the pattern of the sample to be pipetted into a Falcon 96-well microtest tissue culture plate.

[Procedural Amendment] Submission of translation of Article 34 Amendment

[Submission date] April 9, 2001 (2001.4.9)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Contents of correction]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU , ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Wong, Pierre Quebec Earth, Canada 2ix 2b4, Montreal, Eirume 3415, Apartment 4

Claims (80)

[Claims] 1. A method for determining the CYP1A2 phenotype of an individual, said method comprising the steps of: drinking at least three antibodies specific for caffeine or different caffeine metabolites; Measuring the molar ratios of caffeine and the first and second different metabolites of caffeine in the target sample,
Wherein the molar ratio of 4 is indicative of a slow intermediate, 12 is indicative of a fast CYP1A2 metabolite, and the molar ratio is indicative of the CYP1A2 phenotype of the individual. 2. The metabolite of the first caffeine is selected from the group consisting of 1,7-dimethylxanthine (1,7-DMX) and those exemplified in FIG. 3; The product is selected from the group consisting of 1,7-dimethyluric acid (1,7-DMU) and those exemplified in FIG. 4; and wherein the third metabolite is 1,
2. The method of claim 1, wherein the method is selected from the group consisting of 3,7-trimethylxanthine (caffeine) and those illustrated in FIG. 3. The method according to claim 2, wherein said biological sample is a urine sample. 4. The method of claim 3, wherein the determined CYP1A2 phenotype of the individual allows a physician to predict susceptibility to a carcinogen-induced disease and / or to personalize drug treatment. 5. A competitive enzyme-linked immunosorbent assay (ELISA) method for determining a CYP1A2 phenotype, wherein the caffeine or a different caffeine in a biological sample of an individual after drinking a caffeine solution. Using at least three antibodies each specific to them to determine the molar ratio of the metabolites,
Where the molar ratio of 4 is an indicator of a slow intermediate, 12 is an indicator of a fast CYP1A2 metabolite, and the molar ratio is an indicator of the individual's CYP1A2 phenotype, EL
ISA method. 6. The metabolite of the first caffeine is selected from the group consisting of 1,7-dimethylxanthine (1,7-DMX) and those exemplified in FIG. 3; The product is selected from the group consisting of 1,7-dimethyluric acid (1,7-DMU) and those exemplified in FIG. 4; and wherein the third metabolite is 1,
The ELISA method according to claim 5, wherein the ELISA method is selected from the group consisting of 3,7-trimethylxanthine (caffeine) and those exemplified in FIG. 7. The ELISA according to claim 6, wherein said biological sample is a urine sample.
Law. 8. The ELISA method according to claim 7, wherein the determined CYP1A2 phenotype of the individual allows a physician to predict susceptibility to a carcinogen-induced disease and / or to personalize drug treatment. . 9. A competitive enzyme-linked immunosorbent assay (ELISA) kit for determining the CYP1A2 phenotype, which measures their molar ratio in a biological sample of an individual after drinking a caffeine solution. Thus, each contains at least three antibodies specific to them, where a molar ratio of 4 is indicative of a slow intermediate, 12 is indicative of a fast CYP1A2 metabolite, and the molar ratio is CYP1A2
Kit, a phenotypic indicator. 10. A plate coated with a) a first antibody specific to caffeine; b) a second antibody specific to a first metabolite of caffeine; c) a second antibody specific to caffeine. A) a known amount of caffeine-horseradish peroxidase conjugate for which a standard calibration curve is obtained; e) a known amount of 1,7-dimethylxanthine-for which a standard calibration curve is obtained. 10. A competitive ELISA kit according to claim 9, comprising a horseradish peroxidase conjugate; and f) a known amount of a 1,7-dimethyluric acid-horseradish peroxidase conjugate for which a standard calibration curve is obtained. 11. The method according to claim 1, wherein said specific antibody is a polyclonal or monoclonal antibody. 12. The method according to claim 1, wherein said specific antibody is a polyclonal antibody. 13. The competitive antigen-enzyme-linked immunosorbent assay (ELISA) according to claim 5, wherein said specific antibody is a polyclonal or monoclonal antibody. 14. The competitive antigen-enzyme-linked immunosorbent assay (ELISA) according to claim 5, wherein the specific antibody is a polyclonal antibody. 15. The competitive ELISA kit according to claim 10, wherein said specific antibody is a polyclonal or monoclonal antibody. 16. The competitive ELISA kit according to claim 10, wherein said specific antibody is a polyclonal antibody. 17. A method for determining the NAT1 phenotype of an individual, the method comprising the steps of:
At least two antibodies specific for aminosalicylic acid or different metabolites of p-aminosalicylic acid, comprising determining the molar ratio of p-aminosalicylic acid in the biological sample of the individual after consuming p-aminosalicylic acid. And the molar ratio is indicative of the NAT1 phenotype of the individual. 18. The first p-aminosalicylic acid metabolite is selected from the group consisting of 4-oxomethyl-aminosalicylic acid and those exemplified in FIG. 1, wherein p-aminosalicylic acid is selected and exemplified in FIG. The method of claim 17, wherein 19. The method according to claim 18, wherein said biological sample is a urine sample. 20. The method of claim 19, wherein said determined NAT1 phenotype of said individual allows a physician to predict susceptibility to a carcinogen-induced disease and / or to individualize drug treatment. 21. A competitive enzyme-linked immunosorbent assay (ELISA) method for determining the NAT1 phenotype, comprising the steps of: p-aminosalicylic acid or p-aminosalicylic acid or p-aminosalicylic acid in a biological sample of an individual after consuming p-aminosalicylic acid. Using at least two antibodies, each specific for them, to determine the molar ratio of aminosalicylic acid metabolites, and wherein said molar ratio is an indicator of the NAT1 phenotype of said individual,
LISA method. 22. The first p-aminosalicylic acid metabolite is selected from the group consisting of 4-oxomethyl-aminosalicylic acid and those exemplified in FIG. 1, wherein p-aminosalicylic acid is selected and exemplified in FIG. 22. The E of claim 21, wherein
LISA method. 23. The ELI of claim 22, wherein said biological sample is a urine sample.
SA method. 24. The ELISA method of claim 23, wherein the determined NAT1 phenotype of the individual allows a physician to predict susceptibility to a carcinogen-induced disease and / or to personalize drug treatment. . 25. A competitive enzyme-linked immunosorbent assay (ELISA) kit for determining the NAT1 phenotype, comprising: p-aminosalicylic acid or p-aminosalicylic acid or p-aminosalicylic acid in a biological sample of an individual after consuming p-aminosalicylic acid. A kit comprising at least two antibodies each specific for them for determining the molar ratio of aminosalicylic acid metabolites, and wherein said molar ratio is indicative of the NAT1 phenotype of said individual. 26. A plate further coated with a) a first antibody specific for p-aminosalicylic acid; b) a second antibody specific for a first metabolite of p-aminosalicylic acid; c) a standard 26. The method of claim 25, comprising a known amount of p-aminosalicylic acid-horseradish peroxidase conjugate from which a calibration curve is obtained; d) a known amount of p-aminosalicylic acid metabolite-horseradish peroxidase conjugate from which a standard calibration curve is obtained. Competitive ELISA kit. 27. The method according to claim 17, wherein said specific antibody is a polyclonal or monoclonal antibody. 28. The method according to claim 17, wherein said specific antibody is a polyclonal antibody. 29. The competitive antigen-enzyme-linked immunosorbent assay (ELISA) according to claim 21, wherein said specific antibody is a polyclonal or monoclonal antibody.
. 30. The competitive antigen-enzyme-linked immunosorbent assay (ELISA) according to claim 21, wherein the specific antibody is a polyclonal antibody. 31. The competitive ELISA kit according to claim 26, wherein said specific antibody is a polyclonal or monoclonal antibody. 32. The competitive ELISA kit according to claim 26, wherein said specific antibody is a polyclonal antibody. 33. A method for determining a CYP2D6 phenotype of an individual, wherein the at least two antibodies specific for dextromethorphan or a metabolite are present in a biological sample of the individual after consuming dextromethorphan. Measuring the molar ratios of the different metabolites of the first and second dextromethorphans, wherein a molar ratio> 1 is an indicator of a slow intermediate and a molar ratio <1 is an indicator of a fast CYP2D6 metabolite. And the molar ratio is indicative of the CYP2D6 phenotype of the individual. 34. The first dextromethorphan metabolite is selected from the group consisting of 3-hydroxy-17-methylmorphinan and those illustrated in FIG. 5, and dextromethorphan is selected and illustrated in FIG. 34. The method of claim 33, wherein 35. The method according to claim 34, wherein said biological sample is a urine sample. 36. The method of claim 35, wherein the determined CYP2D6 phenotype of the individual allows a physician to predict susceptibility to a carcinogen-induced disease and / or individualize drug treatment. 37. A competitive enzyme-linked immunosorbent assay (ELISA) method for determining a CYP2D6 phenotype, comprising dextromethorphan or dextromethorphan in a biological sample of an individual after consuming dextromethorphan. Consists of using at least two antibodies, each specific for them, to determine the molar ratio of the metabolites, wherein a molar ratio> 1 is a slow indicator and a molar ratio <1 is a fast CYP2D6 metabolite And the molar ratio is the CYP of the individual.
ELISA, an indicator of the 2D6 phenotype. 38. A dextromethorphan selected from the group consisting of 3-hydroxy-17-methylmorphinan and those exemplified in FIG. 5;
The ELISA method of claim 37, as illustrated in FIG. 5. 39. The ELI of claim 38, wherein said biological sample is a urine sample.
SA method. 40. The ELISA method of claim 38, wherein the determined CYP2D6 phenotype of the individual allows a physician to predict susceptibility to a carcinogen-induced disease and / or individualize drug treatment. . 41. A competitive enzyme-linked immunosorbent assay (ELISA) kit for determining the CYP2D6 phenotype, comprising different metabolism of dextromethorphan in a biological sample of an individual after consuming dextromethorphan. To determine the molar ratio of the products, each comprising at least two antibodies specific to them, wherein a molar ratio> 1 is a slow indicator and a molar ratio <1 is an indicator of a fast CYP2D6 metabolite, and The kit, wherein the molar ratio is an indicator of the CYP2D6 phenotype of the individual. 42. Further, a) a plate coated with a first antibody specific for dextromethorphan; b) a second antibody specific for a first metabolite of dextromethorphan; c) a standard calibration curve. 42. A competitive ELISA kit according to claim 41 comprising a known amount of dextromethorphan-horseradish peroxidase conjugate from which is obtained; d) a known amount of dextromethorphan metabolite-horseradish peroxidase conjugate from which a standard calibration curve is obtained. . 43. The method according to claim 33, wherein said specific antibody is a polyclonal or monoclonal antibody. 44. The method according to claim 33, wherein said specific antibody is a polyclonal antibody. 45. The competitive enzyme-linked immunosorbent assay (ELISA) according to claim 37, wherein said specific antibody is a polyclonal or monoclonal antibody. 46. The competitive enzyme-linked immunosorbent assay (ELISA) according to claim 37, wherein said specific antibody is a polyclonal antibody. 47. The competitive ELISA kit according to claim 42, wherein said specific antibody is a polyclonal or monoclonal antibody. 48. The competitive ELISA kit according to claim 42, wherein said specific antibody is a polyclonal antibody. 49. A method for determining the CYP2E1 phenotype of an individual, wherein the at least two antibodies each specific for a different metabolite of chlorzoxazone are used in a biological sample of the individual after chlorzoxazone has been consumed. A method comprising measuring the molar ratio of different metabolites of the first and second chlorzoxazones, wherein the molar ratio is indicative of the CYP2E1 phenotype of the individual. 50. The first chlorzoxazone metabolite is selected from the group consisting of 5-chloro-6-hydroxy-benzoxazole and those illustrated in FIG. 6, wherein chlorzoxazone is selected and illustrated in FIG. 50. The method of claim 49. 51. The method of claim 50, wherein said biological sample is a urine sample. 52. The method of claim 51, wherein said determined CYP2E1 phenotype of said individual allows a physician to predict susceptibility to a carcinogen-induced disease and / or to personalize drug treatment. 53. A competitive enzyme-linked immunosorbent assay (ELISA) kit for determining the CYP2E1 phenotype, comprising the molar ratio of different metabolites of chlorzoxazone in a biological sample of an individual after consuming chlorzoxazone. A kit comprising at least two antibodies each specific for measuring the CYP2E1 phenotype of the individual. 54. The first chlorzoxazone metabolite is selected from the group consisting of 5-chloro-6-hydroxy-benzoxazole and those illustrated in FIG. 6, and chlorzoxazone is selected and illustrated in FIG. An ELISA method according to claim 53. 55. The ELI of claim 53, wherein said biological sample is a urine sample.
SA method. 56. The ELISA method of claim 53, wherein the determined CYP2E1 phenotype of the individual allows a physician to predict susceptibility to a carcinogen-induced disease and / or to personalize drug treatment. . 57. A competitive enzyme-linked immunosorbent assay (ELISA) kit for determining the CYP2E1 phenotype, comprising the molar ratio of different metabolites of chlorzoxazone in a biological sample of an individual after consuming chlorzoxazone. A kit comprising at least two antibodies each specific for measuring the CYP2E1 phenotype of the individual. 58. Further, a) a plate coated with a first antibody specific for chlorzoxazone; b) a second antibody specific for a first metabolite of chlorzoxazone; c) a known standard curve obtained. 58. A competitive ELISA kit according to claim 57 comprising an amount of a chlorzoxazone-horseradish peroxidase conjugate; d) a known amount of a chlorzoxazone metabolite-horseradish peroxidase conjugate for which a standard calibration curve is obtained. 59. The method according to claim 49, wherein said specific antibody is a polyclonal or monoclonal antibody. 60. The method according to claim 49, wherein said specific antibody is a polyclonal antibody. 61. The competitive antigen-enzyme-linked immunosorbent assay (ELISA) of claim 53, wherein said specific antibody is a polyclonal or monoclonal antibody.
. 62. The competitive antigen-enzyme-linked immunosorbent assay (ELISA) of claim 53, wherein said specific antibody is a polyclonal antibody. 63. The competitive ELISA kit according to claim 58, wherein said specific antibody is a polyclonal or monoclonal antibody. 64. The competitive ELISA kit according to claim 58, wherein said specific antibody is a polyclonal antibody. 65. A method for determining the CYP3A4 phenotype of an individual, wherein the organism of the individual after dextromethorphan is consumed with at least two antibodies specific for dextromethorphan or a metabolite of dextromethorphan, respectively. Measuring the molar ratio of the first and second different metabolites of dextromethorphan in a biological sample, wherein the molar ratio is indicative of the CYP3A4 phenotype of the individual. 66. The method wherein the first dextromethorphan metabolite is 3-methoxy-
66. The method of claim 65, wherein the method is selected from the group consisting of morphinans and those illustrated in Figure 7, and dextromethorphan is selected and illustrated in Figure 7. 67. The method of claim 66, wherein said biological sample is a urine sample. 68. The method of claim 67, wherein said determined CYP3A4 phenotype of said individual allows a physician to predict susceptibility to a carcinogen-induced disease and / or individualize drug treatment. 69. A competitive enzyme-linked immunosorbent assay (ELISA) kit for determining the CYP3A4 phenotype, comprising different metabolism of dextromethorphan in a biological sample of an individual after consuming dextromethorphan. A kit, comprising at least two antibodies each specific for them to determine the molar ratio of the products, wherein the molar ratio is indicative of the CYP3A4 phenotype of the individual. 70. The method wherein the first dextromethorphan metabolite is 3-methoxy-
70. The EL of claim 69, wherein the EL is selected from the group consisting of morphinans and those illustrated in FIG. 7, and dextromethorphan is selected and illustrated in FIG.
ISA method. 71. The ELI of claim 69, wherein said biological sample is a urine sample.
SA method. 72. The ELISA method of claim 69, wherein the determined CYP3A4 phenotype of the individual allows a physician to predict susceptibility to a carcinogen-induced disease and / or individualize drug treatment. . 73. A competitive enzyme-linked immunosorbent assay (ELISA) kit for determining the CYP3A4 phenotype, comprising different metabolism of dextromethorphan in a biological sample of an individual after consuming dextromethorphan. A kit, comprising at least two antibodies, each specific for them, for determining the molar ratio of the products, wherein the molar ratio is indicative of the CYP3A4 phenotype of the individual. 74. Further, a) a plate coated with a first antibody specific for dextromethorphan; b) a second antibody specific for a first metabolite of dextromethorphan; c) a standard calibration curve. 74. A competitive ELISA kit according to claim 73 comprising a known amount of dextromethorphan-horseradish peroxidase conjugate from which is obtained; d) a known amount of dextromethorphan metabolite-horseradish peroxidase conjugate from which a standard calibration curve is obtained. . 75. The method of claim 65, wherein said specific antibody is a polyclonal or monoclonal antibody. 76. The method according to claim 65, wherein said specific antibody is a polyclonal antibody. 77. The competitive antigen-enzyme-linked immunosorbent assay (ELISA) according to claim 69, wherein said specific antibody is a polyclonal or monoclonal antibody.
. 78. The competitive antigen-enzyme-linked immunosorbent assay (ELISA) of claim 69, wherein said specific antibody is a polyclonal antibody. 79. The competitive ELISA kit according to claim 74, wherein said specific antibody is a polyclonal or monoclonal antibody. 80. The competitive ELISA kit according to claim 74, wherein said specific antibody is a polyclonal antibody.