TW420717B - Monoclonal antibody against marijuna metabolites, hybridoma producing the antibody and the use thereof in the detection of the drug abuse of marijuna - Google Patents

Abstract

The invention provides a monoclonal antibody specific to the major metabolite of marijuna, tetrahydrocannabinol (THC-COOH) and analogs thereof, hybridoma producing the antibody, and kit containing the same. The antibody is useful for the detection of the drug abuse of marijuna.

Description

420717 A7 B7 V. Description of the Invention (i) Field of the Invention The present invention relates to a single antibody specific to cannabis metabolites, a fusion tumor producing the antibody, and its use for detecting abuse of cannabis. BACKGROUND OF THE INVENTION Cannabis mainly acts on the central nervous system and the cardiovascular system. The main active ingredient is d-9-THC-carboxylic acid. Its impact on the central Shenpan system will cause indirect memory impairment, reduce the ability to perform nerve-consuming tasks, and may also result in a divisive situation. In addition, on the cardiovascular system, it can cause rapid heartbeat, prone to angina pectoris, angina, and decreased immunity. Cannabis drug resistance is not significant at low doses and infrequent use, but it occurs at high doses and continuous, long-term use, and may result in abstinence due to sudden suspension, and most The drug resistance is due to the functional and pharmacological adjustment of the central nervous system, not the phenomenon of accelerated metabolism. Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs (please read the notes on the back before filling this page). The three richest components of the cannabis plant include Cannabinol CBN and Cannabiadiol (CBD) and four Tetrahydrocannabinol (THC-carboxylic acid) isomer. Among the isomers of tetra-cannabinol, the chemical component that has most of the divine effect of cannabis is β-9-THC-carboxylic acid, also known as L-β-9-THC-carboxylic acid. The component β-8-THC has a similar effect, but it has a small content. The effect of β-9-THC is about 20 times that of β-8-THC. β-9-THC-carboxylic acid will soon be transformed into the still active metabolite 11-hydroxy-d-9-THC in the body, which has a similar effect to β-9-THC-carboxylic acid and further metabolizes It becomes a polarized and inactive metabolite and is eliminated through urine and feces. The more common metabolite is 11 -hydroxy-9-THC-9 -carboxy_47492-l.DOC_ ~ 4 ~; _ This paper size applies to China National Standard (CNS) Α4 size (210 X 297 mm) A7 420717 B7 V. Description of the invention (2) Acid, which is a metabolite excreted by the bile, and may be reabsorbed. However, there is still a trace amount of unmetabolized Θ-9-THC-carboxylic acid that can be found in urine. β-9-THC-carboxylic acid and metabolite 11-hydroxy-d-9-THC decreased rapidly after reaching a peak in blood concentration, and quickly distributed to peripheral adipose tissue for slow metabolism. c?-9-THC-carboxylic acids and metabolites can be maintained in human blood for several days or even one week. This is consistent with the actual situation that can be measured in urine. The result is α f. After long-term use, although it will not surface the disorder like other addictive drugs, and its incidence is low, once it is unconscious and abused, it will easily become the drug of other addictive drugs, laying a mixture of multiple drugs Based on abuse, it is necessary to develop test reagents that can detect cannabis. '' Drug abuse screening is divided into two major steps: preliminary screening and further confirmation (Drug of Abuse Testing Market, Theta Corp. 1991). Preliminary screening needs to be highly sensitive, easy to use, and suitable for handling a large number of samples. The positive samples after screening are then tested by methods with high specificity and accuracy, such as gas chromatography / mass spectrometry (J. Chromatography, 617: 265, 1993). Analytical methods for screening for overuse of cannabis drugs include thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas-mass spectrometry (GC / MS), and immunoassay (Immmioassay). Among them, the immunoreagent developed by using multiple or single antibodies is preferred. In addition to high specificity and sensitivity, it is easy to use and responds quickly, and the sample does not need to undergo any pre-treatment steps, which facilitates large-scale screening. The present invention provides a monoclonal antibody with high specificity against cannabis and its metabolites, and develops and develops test reagents. _47492-l.DOC_ ~ 5 ~ _; _ This paper size applies to China National Standard (CNS) A4 (210X297 mm) ---- ^ ------------- 1T (Please First read the notes on the reverse side and then fill out this page) Printed by the Consumers' Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs — 420717 V. Description of the invention (

Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. 6-Overview of Ai-Meng One object of the present invention is to provide a monoclonal antibody specific to cannabis and its metabolites. Another object of the present invention is to provide a fusion that secretes the monoclonal antibody. The present invention has another object to provide a method and kit for detecting cannabis abuse. The objects, advantages, and characteristics of the present invention can be understood from the following description. Brief description of the formula · Figure 1 shows the standard inhibition curve of the cannabis direct competition ELISA. Figure 2 shows the results of cannabis simple chromatography immunoassay test on different concentrations of cannabis. DETAILED DESCRIPTION OF THE INVENTION The antibody specific to cannabis metabolites of the present invention is an antibody produced by a fusion tumor cell line. According to the present invention, a b-cell fusion tumor can be prepared in a b-cell fusion manner by using a conventional technique, using a main metabolite of marijuana d_9_THC-chinic acid as an antigen. In particular, the method includes using a known cell fusion agent, such as alcohol (PEG), to fuse a mouse myeloma cell line with B lymphocytes producing anti-marijuana antibodies, screening the fused tumor cell line with HAT, and then using indirect Enzyme-linked enzyme-linked immunosorbent assay (ELISA) and competitive ELISA to analyze the specificity of antibodies in the fusion tumor culture medium, and then select a single fusion tumor cell line specific for & _ 9 _ THC _ carboxylic acid . This fusion tumor cell line was then injected into the abdominal cavity of a mouse to produce ascites, which proliferated specific antibodies.

47492-l.DOC The standard of this paper is General Ningguo National Standard (CNS) A4 (21〇 × 297 mm) {Please read the precautions on the back before filling this page)

420717 A7 B7 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs

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5. Description of the invention (4) The specificity and sensitivity of the immunoassay depend on the characteristics of the antibody, and whether or not it can produce specific antibodies. The design and manufacture of the immunogen is the main key. Especially drugs with molecular weight less than 1000 Daltons such as fuma, because of their small molecular weight, belong to haptens on immunogens, and will not stimulate immunity when they exist alone. The epidemic system produces an immune response, generally small haptens and A large molecular weight immunogenic carrier protein binds to help the hapten to develop immunity through the carrier's immunogenicity. Therefore, the design, binding and preparation of small molecule haptens and carrier conjugates will affect the quality and specificity of antibodies. In the present invention, a conjugate of a β-9-THC-carboxylic acid derivative and a carrier protein is used as an immunogen. The d-9-THC-leptic acid derivative has the following general formula: X = 0 or N; η = 2-4; and m = 1-3. 474QP-1.nor-Ί-This paper size applies to Chinese National Standard (CNS) A4 (210X29? Mm) --------- r, i installed ------ 1T (Please read first Note on the back, please fill out this page again.) A7 B7 420717 V. Description of the invention (5) The carrier protein can be, for example, albumin, globulin, hemocyanin, iron protein, etc. In one embodiment of the present invention, it is applied "-9-THC-COO-Nitrosinocyclobutanediamine and bovine serum albumin as a combination of immunogens. The single antibody produced by the present invention can be developed into an enzyme immunoassay kit and / or method for detecting cannabis abuse. For example, direct or indirect competitive enzyme immunoassay kits and / or methods, especially One Step CJiromatographic Dip Stick, have high convenience. The direct competitive immunoassay method is to bind the combination of β-9-THC-COO-nitrohydroxylcyclobutanediamine and bovine hemoglobin to the surface of a solid support. When testing, different standard concentrations of-9-THC-carboxylic acid or sample to be tested, and a single antibody against d-9-THC-carboxylic acid to which signals (such as enzymes, fluorescent agents, radioactive elements, etc.) have been connected The complex is put into the reaction together. Because β-9-THC-carboxylic acid will compete with the limited β-9-THC-COO-nitrohydrocyclobutanediamine and bovine hemoglobin combination and resist β-9-THC- Carboxylic acid antibody-signal complexes are combined, so a standard curve is established based on the signal strength of a known standard concentration of β -9-THC -carboxylic acid, which can detect the β-9-THC -carboxylic acid of a urine sample to be tested by tT ' Presence and content. Specific examples of direct-competitive immunoassay kits include tritium solid-phase support substances, ⑵-9-THC-carboxylic acid derivatives and carriers, ⑶β-9-THC-carboxylic acid standards, and anti-β- 9-THC-carboxylic acid monoclonal antibody-signal complex, and tritium coloring substances. The solid support and support materials can be microtiter plates, beads, and paper, and are composed of materials suitable for protein immobilization. Suitable materials can be, for example, polyethylene, polystyrene, etc. 47492-l.DOC _ ； _ " 8 ~ _; _ This paper size is applicable to Chinese National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page) W_ Order at the center of the Ministry of Economic Affairs Printed by the Consumer Cooperatives of the Standards Bureau 420727 Printed by the Consumer Cooperatives of the Central Procurement Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (6) Women, nitrocellulose, Nylon, etc. Signals can use, for example, fluorescent materials, such as lanthanide-labour materials; luminescents, such as biological luminescent indicators and luminescent indicators; radioactive elements; and enzymes, such as hydrogen peroxide enzymes, alkaline phosphoric acid Esterase, galactosidase, etc. The analytical method and reaction conditions are based on conventional techniques (Antibody: A Laboratory Manual Harlow &

Dayid Lane, 1988). A preferred specific example for implementing the present invention is a simple chromatographic immunoassay test sheet 'which may include: a sample selective area, anti-d _ 9 _ THC-a few acids ·-indicator area, ⑶ reaction test area, and Liquid recovery area. The test piece is composed of a porous carrier material that can provide capillary phenomenon and absorbs liquid, such as filter paper, nitrocellulose membrane, dragon film, and broken glass fiber membrane. The indicator can use, for example, colored latex particles, such as polymers of materials such as polystyrene, polyethylene, toluene, polystyrene, acrylic acid, and polyacryl; metal colloidal solutions, such as silicone solutions, and dye colloidal solutions. Other raw materials are commercial products. The sample contact area is located at one end of the test piece, and is in contact with the sample to be tested, and the adsorbed sample is moved toward the other end according to the capillary phenomenon, so that the entire test piece is wetted. Refers to the combination of the TF agent area close to the sample contact area. This area is a dry form of a combination of monoclonal antibodies against d-9-THC-metanoic acid and an indicator that has high stability and does not require special equipment and can be interpreted with the naked eye. body. The test area is located above the indicator area. The distant area is fixed with a chromogenic reactant, such as ^ -carboxylic acid-carrier antigen conjugate, which can react with the anti-4_9_THC-carboxylic acid antibody-indicator and show — Changes in the signal interpreted by the naked eye. During the reaction test, the end of the sample contact area of the test piece and the known standard concentration-47 states-·) .. _ once applicable to China ---- ------------- -—1T (Please read the precautions on the back before filling out this page) 4 20717 A7 B7 Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. of the invention description (7) < 5 · _ 9-THC-Lean aciduria The liquid and the sample to be tested are in contact. Urine is moisturized according to the appearance of the capillary, and the antibody-indicator conjugate is dissolved back. If the urine contains J _ 9 _ THC _ lean acid, the shellfish will compete with the J _ 9 THC _ renegadenic acid-carrier antigen on the test area with limited anti-β-9-THC-carboxylic acid indicator combination. Therefore, the higher the content of β-9-THC-carboxylic acid in the sample, the weaker the positive signal in the test zone. From the presence and absence of the reaction in the test zone, it can be determined whether d-9-THC-.carboxylic acid and its Content ► In order to make the purpose, method, characteristics and principle of the present invention clearer, the following examples are described in detail in conjunction with the drawings, but these examples are not intended to limit the scope of the present invention. The nest term is defined as follows: HAT: Hypoxanthine / Aminopterin / Thymidine.

Tg: bovine thyroglobulin FCS: fetal bovine serum 'DMSO: dimethyl sulfoxide PBS: phosphate buffer solution TMB: tetramethyl benzidine BSA: bovine serum albumin Example 1: fusion tumor (3402 -13.9) Production Θ-9-THC-COO-Nitrosinocyclobutanediamine-bovine serum albumin Preparation method 14 mg of d-9-THC-COO-Nitrosinocyclo Succinimide is dissolved in 2.8 ml of DMSO, and then 2.55 ml of the solution is added to 20 ml of bovine serum albumin _47492-l.DOC _- 10 __ · _ This paper size applies the Chinese National Standard (CMS) A4 specification ( 210X297 mm) --------- i —----— IT ../IV L (Please read the notes on the back before filling out this page) Printed by the Central Standards Bureau, Ministry of Economic Affairs, Consumer Cooperatives 420717 at _____B7 V. Description of the invention (8) (100 mg / 0.05 M sodium carbonate solution), stir the reaction in an ice bath. After overnight, the reaction was sufficiently dialyzed against 50 mM (pH 7.4) phosphate buffer solution, and frozen crystals were stored. The preparation method of fusion tumor is a combination of d-9-THC-COO-nitrohydrocyclobutanediamine and bovine serum albumin (-9-THC-BSA, Molar number ratio 30) and Freund's. Adjuvant (full adjuvant was used for the first immunization, and incomplete adjuvant was used for subsequent immunizations). A 1: 1 (v / v) emulsion was injected subcutaneously into BALB / C mice ( Use 125 " g β-9-THC-BSA each, once a week for 4 times in a row. Then strengthen it once every four weeks (g β-9-THC- BSA every 40 days) for a total of 5 times. Three days before cell fusion, an additional intravenous injection (without adjuvant) was performed. Three days later, the spleen cells were fused with a mouse-derived myeloma cell FO cell line (ATCC CRL 1646) using 50% polyethylene glycol-400 (PEG 400) in a 1: 3 case. After fusion, the cells were suspended in RPMI medium (HAT, 20% FCS), diluted to 1 X 105 FO cells, and implanted in 96-well culture plates (0.2 ml / well). After 10 days, the cell culture supernatant was tested in an ELISA plate with β-9-THC-carboxylic acid-burdock protein conjugate (d-9-THC-COOH-Tg, Molar ratio 30) adsorbed. Fusion tumors with β-9 -THC -carboxylic acid specificity were selected and singulated by limiting dilution method. A mouse-derived fusion cell line 3402-13.9 of a single cell was selected by this method, and the single antibody secreted by the mouse was determined to be IgG2a and Kappa light keys. This fusion tumor (in 10% DMSO and 90% FCS) can be stored at -70 ° C and in liquid nitrogen, and it can be supplemented with 200 using standard mammalian cell culture techniques (RPMI 1640 with 10% fetal bovine serum) mM glutamine and 50 μM 2 -thioethanol) were given 47492 ~ 1.DOC__ _- 11 -___ This paper applies the national standard (CNS) Α4 specification (210 × 297ϋ) (Please read the precautions on the back before filling this page ), Installed. -Order 17 A7 B7 V. Description of the invention (9) Culture. This fusion tumor has been deposited at the Strain Preservation and Research Center of the Food Industry Development Research Institute on July 9th, 1996. CCRC 960065. Example 2: Preparation of a direct competition ELISA method for detecting cannabis abuse. An antigen attachment plate is prepared by d-9-9THC-COO-nitrosinocyclobutanediamine-bovine serum albumin antigen with carbonate. Diluent (pH = 9 · 6) diluted 2.5 pg / ml. Take 200 microliters and attach. Place on the surface of each well of a 96-well polystyrene microplate and incubate at 4 ° C overnight. Use 0.05% Tween 20-PBS Rinse 3 times, then pat dry. Add 250 μΐ of 0.1% skimmed milk powder-PBS to each microwell. Block overnight at ° C. Wash twice with 0.05% Tween 20-PBS and pat dry before use. Preparation method of anti-d-9-THC carboxylic acid monoclonal antibody-hydrogenase enzyme conjugate Enzyme (1.0 mg / ml) was dissolved in deionized water and oxidized with 100 mM peracid steel at room temperature for 20 minutes. Then 50 mM steel acetate solution (pH 4.5) was dialyzed to remove sodium periodate. Also prepared 0.5 mg of anti-d-9-THC-carboxylic acid monoclonal antibody, which was dialyzed against a 0.2 M sodium carbonate buffer solution. 1.0 mg of a hydrogen peroxide enzyme treated with sodium peroxidase was added, and 0.5 mg of the antibody was added. d-9-THC -carboxylic acid monoclonal antibody (molar ratio of hydrogen peroxide enzyme to monoclonal antibody 2 to 1) After reacting at room temperature for 2 hours, then add 20 mM sodium borohydride and overnight at 4 ° C Reduction. The reactants were dialyzed against 0.1 M sodium acetate solution (pH = 8.2) for 24 hours, and the dialysis solution was changed every 8 hours, and stored at -20 ° C for later use. During the test, do not take 50 μΐ to prepare negative urine. Β-9-THC -carboxylic acid 47492-l.DOC-12-in the liquid. This paper size applies to China National Standard (CNS) A4 (210X 297 mm) ---------------— Order (Please read the notes on the back before filling this page) Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 420 717 B7 V. Description of the invention (10 ) (Please read the notes on the back before filling this page) Standards (〇, 25, 50, 75 and 100 ng / ml) and the drugs listed in Tables 2 and 3 (see Tables 2 and 3 for the concentration), Add d-9-THC-carboxylic acid-bovine serum albumin to the microwells, and add 150 μΐ of anti-d-9-THC-carboxylic acid monoclonal antibody-hydrogenase conjugate. After mixing, the reaction was shaken at room temperature for 10 minutes. The reaction was then discarded, filled with 0.05% Tween 20-PBS, and discarded. After repeated washing 5 times, add 100 # 1 peroxidase enzyme TMB,

After 10 minutes of reaction at room temperature, 100 A 1 2N HC1 was added to stop the reaction. Determination of 〇450 / 650. Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economics. Table 1 shows the absorbance of the standard concentration of β-9-THC-carboxylic acid, and the coefficient of variation (cv) and inhibition percentage of 6 repeated tests. It can be seen that the cv at each concentration is less than 10%. The percentage of inhibition represents the relative sensitivity of the antibody to a specific substance, which is calculated as '(1-0D test / ODthccooh)' where ODTHeeQQH is the OD of the negative control solution without β-9-THC -carboxylic acid, and the OD test is OD of drugs containing different concentrations of THC-COOH or other non-β-9-THC-carboxylic acids (see Tables 2 and 3 for details). Figure 1 is a standard inhibition curve for cannabis using the direct competition ELISA of the present invention. If using a β-9-THC-carboxylic acid negative control group with a standard deviation of 10% of the standard deviation of 2% > 20% represents a significant difference, then β-9-THC-carboxylic acid directly competes for the sensitivity of the immune set Up to 2.5 ng / ml. None of the 118 non-d-9-THC-carboxylic acid drugs listed in Table II produced > 20% inhibition when tested at 100 pg / ml. When different concentrations of the drugs listed in Table 3 were added, the cross-reaction percentage was calculated from the measured concentrations. It was found that in addition to β-9-THC-carboxylic acid, 3402-13.9 single textiles could also detect β-8-THC and Others cannabis (see Table III). _47492-l.DOC_ ~ 13 ~ _; _ This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) 420717 A7 B7 V. Description of the invention (u) I68 tests using the direct competition ELISA kit of the present invention Of the samples, 44 of them were confirmed as positive by gas chromatography / mass spectrometry, and the rest were confirmed as positive by ELISA at 250ng / ml, and the results obtained by this reagent and the gas chromatography / mass spectrometer were accurate. 98.81%, sensitivity 10 () () ΰ / 98.39% of the failure, the results are shown in Table 4 and details (please read the weeping matters on the back and fill in the purchase) Central Office of Standards, Ministry of Economic Affairs Consumer Cooperatives Printed on / in —--------- 1) 1 ---- ^ —— K ----! 47492-l.DOC-14-This paper size applies to Chinese National Standard (CNS) A4 Specifications (2 丨 0 'dog 297 mm) Printed by the Consumers Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 420717 at ^ _-_ V. Description of the invention (12) Table 1 &y; 9-THC-carboxylic acid standard is different Repeated test of six concentrated ng / ml OD average% Β / Βο CV 250 0.106 0.102 0.105 0.107 0.103 0.127 0.108 4.5 8.6 100 0.439 0.417 0.417 0.470 0.537 0.469 0.458 19: 1 9.9 5 0 1.809 1.936 1.688 1.650 1.862 1.784 1.788 74.6 6.0 25 2.314 2.324 2.092 1.999 2.232 2.194 2.193 91.5 5.8 10 2.454. 2.464 2.251 2.192 2.347 2.297 2.334 97.4 4.7 5 2.501 2.605 2.434 2.205 2.365 2.344 2.409 100.5 5.7 0 2.563 2.675 2.305 2.232 2.326 2.279 2.397 100.0 7.5 --------- 11 (please read 意 意 意 事 3 on the back!-Then fill out this page)-order .doc 15 this paper is more suitable for money _ house standard (CNS) A4 size (2lGx297 Male thin) 420 '7 A7 B7 V. Description of the invention (13) Table 2 Drugs that do not cross-react with the cannabis direct competition-type enzyme immunoassay reagent of the present invention --- Drugs with a percent inhibition of less than 20% (iopg / ml ) Acetaminophen ♦ cimetidine ----------) ulI —.- I—IT (Please read the precautions on the back before filling this page) Staff Consumption of Central Bureau of Standards, Ministry of Economic Affairs Cooperative print acetylmorphine aminephyline aminotriplyline amphetamine ascorbic acid aspartic acid atropine aspartam aspartam apomorphi ne) Barbital benzoic acid, buprenorphine, caffeine, cannabidiol, carbinoxamine, chloramphenicol, chlordiazepoxide, chloramphetamine Chlorpheniramine maleate, chlorpromazine, citric acid, cocaine, deoxyephedrine, demipramine, and desiramine (desmethyldiazepam) dextromethorphan diethylpropion dihydrocodeine diphenhydramine diphenhydramine dirprenorphine ephedrine epinephrine erythromycin Ethyl-p-aminobenzoate EDTA disodium salt fenoprofen gentamycin D-(+)-glucose (D- (+)-glucose) 16

47492-1.DOC This paper scale is applicable to Chinese National Standard (CNS) A4 specification (210X 297 mm) 420717 A7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs B7 5. Description of the invention (14) r] ♦ Guaifenesin (guaiphenesin) ♦ 晞 丙 吗 ># (nalorphine) 1 ♦ homotropine ♦ naloxone 1 I ♦-# is methyl methyl amphetamine ♦ naltrexioiie Please read the back 1 1 (p-hydoxymethamphetamine ) ♦ Nicotinamide 1 1 ♦ Hydrocodon ♦ (+) Nicotine (X +) nicotin Note-I ♦ Hydromorphone ♦ Nornorphine Meaning 1 1 ♦ ♦ haloperidol hydroxyphenol ethylamine ♦ ♦ nortriptyline noscapine; * Please fill out this page / 、 (, install ♦ ibuprofen ♦ • papaverine 1 I ♦ propyl Throat HCl (imipramine HC1) ♦ penicillin G I let p lead p multiply indomethacin ♦ pentazocine I ♦ isoxsuprine ♦ p-ethylammonium phenyl ether ( phenacetin) ΊΤ ♦ Ketamine ♦ phencyclidine 1 ♦ labetalol ♦ phenelzine 1 ♦ lidocain ♦ phenobarbital 1 1 ♦ Carbonate) ♦ phentermine .1 1 ♦ lorazepam ♦ phenylephrine I ♦ 1-methyl amphetamine ♦ phenylethylamine I (1-methamphetamine) ♦ Phenylpropanolamine 1. ♦ mephentermine ♦ prazosin 1 ♦ oxymorphone ♦ prednisone «1 | ♦ (+) methadone ((+) methadone) ♦ Primidone 1 1 ♦ methapyrilene HC1 ♦ procainamide 1 1 ♦ methylephedrin ♦ procaine 1 1 47492-l.DOC -17 — 1 This paper size applies the Chinese National Standard (CNS) A4 (210X 297 mm) Printed by the consumer cooperation of the Central Bureau of Standards of the Ministry of Economic Affairs 42701? 5. Description of the invention (15) Isopropyl " ^ (promethazine) poxyphene) ♦ pseudoephedrine ♦ quinine ♦ ranitidine ♦ salicylic acid ♦ streptomycin ♦ trychnine ♦ inexpensive Sugar (sucrose) ♦ Ethylamine · sulfacetamide ♦ amine disulfide (sulfadiazine) ♦ amine dimethoxine (sulfadimethoxine) ♦ amine sulfamerazine (sulfamerazine). ♦ amine dimethyl p dense lake ( sulfamethazine) ♦ sulfamethizole ♦ sulfamethoxazole ♦ sulfamethpyridazine ♦ sulfamonomethoxine ♦ sulfanilamide ♦ sulfanilamide ♦ _ Sulfanitran ♦ _ ^ '^' ^^ (sulfaquinoxaline)

_47492-1.DOC A7 B7___A amine sulfathiazole ♦ sulfinpyrazone ♦ sulfisoxazole ♦ terbutaline ♦ tetracycline tetracycline) ♦ 四 氲 峻 '^ (tetrahydrozoline) ♦ theophylline ♦ tripleleimamine ♦ norcodeine ♦ tyramine ♦ (+) brompheniramine maleate (( +) bromphenkainine maleeate) ♦ (+) 2,5-dimethoxy-4-methylamphetamine ((+) 2,5-dimethoxy-4-methylamphetamine) ♦ 4-dimethylaminoantiprme ♦ DL-b-hydroxyethylamine ( DL-b-hydroxyphenethylamine) ♦ (+) 3,4-Methylene dioxyamphetamine ((+) 3, '4-methylenedioxy-amphetamine) -18------------)! / # ------- tr (Please read the notes on the back first to fill in the original text) The paper size applies to the Chinese National Standard (CNS) A4 specification (2 丨 0 > < 297 mm) 420717 A7 __B7_ V. Invention Explanation (16) Table III crosses the direct cannabis-enzyme immunoassay reagent of the present invention. Reactive drugs

Test compound added concentration (ng / ml) Tested concentration% Cross-reactivity 8-THC 1,000.0 2343.2 234.3 500.0 1178.7 235.7 250.0 780.9 312.4 125.0 451.4 361.1 62.5 275.0 440.0 31.3 219.0 699.7 20.0 44.0 220.0 10.0 149.2 1492.4 8-THC-C00H 1,000.0 436.7 43.7 500.0 247.3 49.5 250.0 100.3 40.1 125.0 20.7 16.6 8yS- < y 9-THC 1,000.0 237.3 23.7 500.0 42.3 8.5 250.0 17.2 6.9 8 ^ -ll-: .- d9-THC 1,000.0 537.3 53.7 500.0 48.3 9.7 11--hydroxy- d 9-THC 1,000.0 137.5 13.8 500.0 117.2 23.4 250.0 30.8 12.3 cy 9-THC (Please read the notes on the back before filling this page) 1,000.0 584.3 58.4 500.0 326.6 65.3 250.0 255.2 102.1 125.0 162.6 13.0 62.5 102.9 164.6 31.3 68.6 219.2 20.0 44.1 220.5 10.0 28.4 '284.0 19-

47492-1.DOC This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) Hemp 100,000.0 37.3 50,000.0 '28 .3 25,000.0 20.3 ii ο · ο * ο * Α7 Β7 420717 V. Description of the invention (17) ( Please read the notes on the back before filling out this page) Table 4 Comparison results of marijuana GC / MS test results with marijuana direct competition E LI SA Qualitative test number: 168 > ELISA + _ GC / MS + 44 0 2 122 Sensitivity: 100.00% Specificity: 98.39% Accuracy: 98.81% Example 3: Simple Chromatographic Immunoassay for Detection of Marijuana Abuse Anti-β-9-THC-Carboxylic Acid Antibody (3402-13.9)-Colloidal Gold Conjugate Prepared by the printing method of the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, 240 ml of colloidal gold was placed in a clean 500 ml serum bottle, and the pH was adjusted to 7.5 with 20 mM Na2C03. Add 0.72 ml of anti-Θ-9-THC-carboxylic acid antibody (3402-13.9), and stir for 3 minutes. Add 24 ml of 10% BSA_phosphate buffer solution, and stir the reaction for 5 minutes, then let it stand at room temperature for 2 hours. Add 12 grams of trehalose, mix and dissolve at room temperature, then add 0.24 ml of 10% Triton X-100 and mix well to complete the anti-d-9-THC -carboxylic acid antibody 47492-l.DOC-20. Size of this paper Applicable to Chinese National Standard (CNS) M specifications (210 X 297 mm) 5. Description of the invention (18) (3402-13.9)-Preparation of colloidal gold conjugates. Store at 4 ° C. Preparation of simple chromatographic immunoassay. Test sheet preparation Nitrocellulose (Schleider & Schnell) with a pore size of 5 μm was rewarded as a sheet with a width of 30 cm x 2.0 cm. At a distance of about 0.75 cm (at the control zone) and 1.25 cm (at the test zone) from the top of the wide side (2.0 cm), use a spray gun (36 psi pressure) to separate the difficult anti-mouse IgG antibody (1 mg / ml) and β-9-THC-carboxylic acid-bovine serum albumin (p.3 mg / ml) was sprayed on the two places to form a line having a width of about 0.8 mm. After air-drying at room temperature for 30 minutes, it was placed in blocking buffer (0.25% casein, 0.01% Triton X-100, 3% Trehalose-PB (20 mM)). Block 30 at room temperature. After 15 minutes, take out and dry in a constant temperature and humidity room at 20 ° C and 30% humidity for 30 minutes. Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page) Cut out a length and width 30 cm x 7 cm, plastic backing sheet with glue on one side. At the distance of 4.2 cm to 6.2 cm from the top of the wide side (7 cm), the prepared nitrocellulose membrane is attached. From the top to 3 cm, stick With 30cm X 2.4cm filter paper (Whatman). At the bottom of the sample contact area, attach a 30cm X 1cm anti-marijuana antibody-colloid gold conjugate strip, each covering a layer of 4.2cm X 30cm along the lower edge of the back plate Fold the glass fiber membrane in half. After drying overnight in a constant temperature (20 ° C) constant temperature (30%) room, cut into small slices of 0-8 cm x 7 cm. During the test, soak the contact area of the sample at about 200 〃 1 containing d-9 -THC-carboxylic acid concentration in urine of 0, 25, 50, 75 and 100 ng / ml The urine without d-9-THC-carboxylic acid was used as a control group. After 3 to 5 minutes, if the control area and the test area showed red signals, the response was negative, indicating that the sample did not contain the marijuana to be detected: If only the control area shows a red signal, but the test area does not show a red signal, the response is positive, indicating that the sample contains _47492-l.DOC ____ ~ 2 1 ~ _ This paper size applies the Chinese National Standard (CNS) A4 specification ( 210X 297 mm) 420717 Μ B7 V. Description of the invention (19 tested cannabis. The test results are shown in Figure 2 at 50ng / ml. Using the simple cannabis simple tomography test set of the present invention, 168 samples were tested, of which 44 gas The phase chromatography / mass spectrometer was confirmed to be positive, and the rest were confirmed to be negative. The accuracy of the results obtained by this reagent and the gas chromatography / mass spectrometer was 9S.S1%, the sensitivity was 100%, and the specificity was 98 39 %, The results are shown in the table for details. The sensitivity of this test sheet is five. Read the first. Note on the back * Note *. * (1), Table 5 ::: = Test ... Qualitative page with simple cannabis immune test sheet. Economic Printed inspection by the Consumer Standards Cooperative of the Ministry of Standards Body number: 168

Specificity Accuracy The present invention can be modified or changed in various forms as required, which is also included in the scope of the present invention. The present invention covers all modifications, similarities and changes within the spirit and scope of the present invention as defined by the appended claims.

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Claims (1)

Ο λ, ^ ry heart υ Patent No. 17447 Chinese Patent Application Amendment (September 88) Scope of patent application (please read the precautions on the back and then this page) 1. A fusion tumor that produces antibodies specific to the cannabis metabolite 5-9-THC-carboxylic acid derivative, which is a mouse-derived fusion tumor cell line 3402-13.9 was deposited at the Strain Preservation and Research Center of the Food Industry Development Research Institute on July 9, 1986. The deposit number is CCRC960065. 2. The fusion tumor according to item 1 of the scope of patent application, wherein the 5-9-THC -acid derivative has the following general formula COR C5H11 where R table -〇-〇 or -X-(CH2) n- X-C (O)-(CH2) m- C (O)-printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economy X = O or N; n = 2-4; and m = 1-3. 3. The fusion tumor according to item 2 of the scope of patent application, wherein the (5-9-THC-carboxylic acid derivative 6-9-THC-COO-nitrohydrocyclobutanediamine). 4 An anti 5-9-THC-carboxylic acid monoclonal antibody, which is made by fusion tumor according to item 1 of the scope of patent application.]: \ 47 \ 47492-lD〇C This paper applies Chinese National Standard (CNS) A4 Specifications (210X297 mm) i2 Α8 Β8 C8 D8 κ, patent application scope 5. According to the single antibody of the patent application scope item 1, it can be used to detect cannabis and its metabolites. 6.-Detection of suspected cannabis samples A method for marijuana content, comprising the following steps: (1) providing an anti-5_9_THC_carboxylic acid monoclonal antibody secreted by fusion tumor 3402_139: (b) providing a signal-generating substance which is operative and resistant to anti-^ _ 9 _ THC -carboxylic acid antibody binds to generate a signal; (c) d-9-THC -carboxylic acid antigen is adhered to the support substance to form an antigen-support substance conjugate; (d) the sample or d-9- THC-carboxylic acid standard solution competes with antigen-supporting substance and with 5-9 THC-carboxylic acid monoclonal antibody— Conjugate is conjugated, and the signal generated by the substance produced by the signal is measured. 7- The method according to item 6 of the patent scope of the application, wherein the conjugate of the _ 9 _ THC _ renegadenic acid derivative-antigen has The following general formula is 3 _ 9 _ THC _ Metabolic acid derivatives: (Please read the precautions on the back first and then 1 ^ \ this page) Dingquan-Ministry of Economic Affairs, Prospective Bureau Member, H Consumer Cooperative, printed COR 1 C5H11 Among them: _ 0: \ 47 \ 47492-l.DOC The secret of the national standard (CNS) of the country (21〇) & 297 public 4 9η Scope of application for patents: Printing of Form R or -X-(CH2) n- X-C (0)-(CH2) m- C (〇)-jx = 0 or N; = 2 -4; JL m = 1-3. 8. The method according to item 7 of the scope of the patent application, wherein the -carboxylic acid derivative is 9_THC-C00_azahydroxocyclobutanediamine. 9. The method according to item 6 of the scope of patent application, wherein the protein m used to screen the fusion is selected from albumin, globulin, hemocyanin, and ferritin. According to the method of the patent application No. 6 in the central patent application, the myeloma cell used to prepare the fusion tumor is a mouse myeloma cell F0 cell line, and the B cell line is derived from a hemp derivative 5_ 9 _ THC_c 〇〇_ N-Hydroxycyclobutanediamine-bovine serum albumin immunized rats. U. A kit for the detection of cannabis and its metabolites, which includes a monoclonal antibody according to item 4 of the scope of the patent application. ! 2_ The kit according to item 11 of the scope of patent application, which is a direct competition ELISA kit. 13. The kit according to item 12 of the scope of patent application, which includes: ⑴solid phase support substance, and anti-5 _ 9-THC-carboxylic acid monoclonal antibody-signal complex according to item 4 of patent scope, ⑶5 _ 9 _ THC _ carboxylic acid standard, 品 < 5-9-THC-carboxylic acid derivative-antigen conjugate, and ⑸ coloring substance. 14 The set according to item 13 of the patent application park, where the solid support O: \ 47 \ 47492-l.DOC 〇 This paper size applies Chinese National Standard (CNS) A4 specification (210X297 public epidemic) f Please Please read the precautions on the back first, then this page.] 7Ψ. C books 1 2 1 1. Ά petition application patent park A8 Β8 C8 D8 The standard of the Ministry of Economic Affairs is employee consumers. The print quality of the company is micro-disc, micro Sphere or paper. 15. The set according to item 13 of the scope of application for a patent, wherein the signal is selected from the group consisting of a glorious object cold light marker, a radioactive element, and an enzyme. 16. According to the set of item 15 in the scope of the hsing patent, the enzyme is selected from the group consisting of a hydrogen peroxide enzyme, an alkaline phosphate enzyme, and a galactose enzyme. 17. According to the set of the patent claim (1), it is a chromatographic immunoassay tablet. i8 • The kit according to item 17 of the scope of the patent application, which includes a sample contact area, an individual antibody according to the scope of the patent application, the indicator ⑤, a reaction test area, and a liquid recovery area. 19. The kit according to item 17 of the patent application park, wherein the test piece is composed of a porous carrier material that can provide hair cell phenomenon and adsorb liquid. 20_ According to the scope of patent application! The set of 9 items, wherein the test piece is selected from the group consisting of application paper, nitrocellulose film, Shilong film, and glass fiber film. 21. The set according to item 8 of the patent application park, wherein the indicator is selected from the group consisting of colored latex particles, metallic colloidal solution, non-metallic colloidal solution, and dye colloidal solution. 22. The set according to item 2 of the patent application park, the granule is a polymer selected from the group consisting of polystyrene and polyethylene acetone 'polyacrylic acid. 23. Set according to the scope of application for patent No. 21 Liquid-based gold colloidal solution. 24. According to the 21st set of the scope of the patent application _ £: \ 47 \ 47492-l.nnr 'where the colored milk wins polyethene' -propionic acid and ^ where the metal colloid is dissolved in the non-metal Gelatin (please read the precautions on the back first, and then "quotation" on this page) J—It ·, 1Τ -Cambodia • -------------- 4-This' '' Zhang scale is applicable to China National Standard (CNS) A4 see (210 × 25 »7 mm A8 420717 S D8 χ, the patent application range solution is a silicone solution. 25. According to the 18th item of the patent application range A kit, wherein the reaction test area contains a cannabis metabolite (5-9THC-carboxylic acid derivative-carrier antigen conjugate. 26. The kit according to item 25 of the patent application scope, wherein the carrier antigen is albumin , Globulin, hemocyanin, and ferritin. 27. A method for detecting cannabis and its metabolites 5-9-THC-carboxylic acid in urine, this method uses a single strain according to item 8 of the scope of patent application Antibodies or urine according to the set of any of the claims 1 to 26 of the scope of the patent application. (Please read the precautions on the back before reading this page) , Va • Quan Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs O: \ 47 \ 47492-1.DOC-J-This paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm)