CN108333349A - A kind of 4- methyl methcathinone artificial antigen and preparation method and utilization - Google Patents
A kind of 4- methyl methcathinone artificial antigen and preparation method and utilization Download PDFInfo
- Publication number
- CN108333349A CN108333349A CN201710038362.3A CN201710038362A CN108333349A CN 108333349 A CN108333349 A CN 108333349A CN 201710038362 A CN201710038362 A CN 201710038362A CN 108333349 A CN108333349 A CN 108333349A
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- CN
- China
- Prior art keywords
- methyl
- methcathinone
- haptens
- ketone
- reagent
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
The present invention provides a kind of new 4 methyl methcathinone artificial antigens and preparation method, this method to include:Haptens is prepared first:Bromoethane Grignard Reagent is prepared in anhydrous ether as raw material using bromoethane and magnesium metal, again using bromoethane Grignard Reagent with and 4 methoxy benzonitriles be raw material through carbonylation, bromination, methylamino and acidolysis, the 4 hydroxyl α methylamino propiophenones for carrying hydroxyl on phenyl ring are prepared, finally use succinic anhydride that the haptens containing long-armed carboxyl is made with hydroxyl reaction on phenyl ring.Haptens synthesizes artificial antigen with albumen coupling:Haptens and bovine serum albumin coupling are prepared 4 methyl methcathinone artificial antigens by the active ester technique being catalyzed by carbon imidodicarbonic diamide.Prepared artificial antigen can carry out animal immune, obtain corresponding antibody, the research for all kinds of methcathinone immunoassays and the critical materials as the production of methcathinone immune chromatography reagent kit.
Description
Technical field
The invention belongs to biochemical industry and technical field of immunoassay, and in particular to a kind of 4- methyl methcathinone manually resists
Former preparation method
Background technology
4- methyl methcathinones, also known as metamfepyramone are a kind of artificial synthesized excitant based on Cathinone, molecule
Formula:C11H15NO;Molecular weight:177;English name:4-methylmethcathinone and Mephedrone, foreign countries are commonly called as
" Meow Meow (mew mew) ", " MCAT ", " 4-MMC " and " Bubbles ", China is commonly called as " native ice ".4- methyl methcathinones are micro-
The principal mode of the liquid of yellow, abuse is hydrochloride, usually the powdered object of white or off-white color, and bitter, smelling has
Typical letones sweet taste, easy moisture absorption caking, structural formula are:
4- methyl methcathinone and methylene dioxypyrrole pentanone are two kinds of novel drug " bath salt " (Bath Salts)
Main component is chemical synthesis central nervous excitation agent, has comparable harm to human body." bath salt " does not imply that certain is specific
Drugs, but a kind of " designer drug (designer drug) " or be synthesis excitant, come out by Illegal fabrication, use
Has the effects that the synthetic drug of psychotropic activity with simulation.Under international community is to the anti-depressant strict supervision of tradition, contain
The hallucinogenic of crystal methamphetamine and MDMA ingredients is just being gradually backed out drugs consumption market, and cassie ketone and synthesis cannabis etc. are novel
Drugs are just becoming new lover when misuser's amusement.
On May 26th, 2012, one nude man of Midland, MI attacks a wanderer on highway causeway, and gnaws
His half face.The police said assailant may suck the novel street drug of a kind of entitled " bath salt " at that time.This emerging drugs
" bath salt " appearance and the bath salt of bathing seem identical, are easy that eater is easily allowed to hide police's inspection.In America, Europe, Australia
Some countries of continent and Southeast Asia, " bath salt " often with " ivory white wave ", " purple wave ", " vanilla sky ", " mercy ",
" drone ", " energy 1 ", " red pigeon ", " snow leopard etc. is sold with epigamic trade name, has certain lure to teenager
Puzzled power, the teenager's number for sucking " bath salt " in some razzles, bar and dance hall in recent years are in rising trend.
The abuse route of 4- methyl methcathinones is snuffing and intravenous injection, can produce the effect of similar amphetamine, MDMA
Fruit, long-term or a large amount of take can form dependence.4- methyl methcathinone is as a kind of dopamine reuptake inhibitor, medicine
Rationality matter is similar to crystal methamphetamine, easily causes the glad and excited of human body, including:Glad, alertness rising, pupil
Amplify, be short of breath, heart rate increases, speech is spoken unceasingly, links up desire increases, sexual desire improves and Cognitive function damage etc..
Meanwhile the abuse of methcathinone can be with the generation of a series of side effects, and generate undesirable effect:Vain hope, illusion, coke
Worry, vibrations, insomnia, malnutrition, weight loss, dehydration, sweating, abdominal pain, nose is bleeding and overall pain, are excessively used 4- methyl
Methcathinone can cause myocardial inflammation, cause to die suddenly.
Nineteen twenty-nine, Sanchez has synthesized 4- methyl methcathinones for the first time, but does not cause the concern of people at that time,
It was rediscovered until 2003.4- methyl methcathinone first enters European market in 2007, is sold, is arrived on network
In many countries in Europe, especially Britain is very popular within 2009.On April 16th, 2010, Britain arranged 4- methyl methcathinones
Enter B grades of controlled substances, the same year many European countries provide that it is illegal, and December, European Union provided it European illegal.It is beautiful in addition to Europe
4- methyl methcathinones are classified as controlled drug by the country such as state, Canada, Australia, New Zealand, Israel in succession.2010
August 2 days, State Food and Drug Administration of China, the Ministry of Public Security, the Ministry of Public Health, which combine, to issue a public proclamation, and determines from the l of in September, 2010
Day rises, and by 4- methyl methcathinone row people's the first kind of spiritual drugs control, without approval, any entity or individual must not carry out 4-
Experiment, research, production, operation, use, storage, transport and the inlet and outlet of methyl methcathinone etc. are movable.
Currently, the analysis detection of 4- methyl methcathinones relies primarily on high performance liquid chromatography (HPLC), gas-chromatography
(GC), the large-sized analytic instruments such as mass spectrum (MS).Agilent company of the U.S. develops the liquid chromatography-tandedm mass spectro-metry side LC/MS/MS
Analysis inspection of the method for 4 methylene dioxypyrrole pentanone (MDPV) of urine " bath salt " ingredient and 4- methyl methcathinone (MCAT)
It surveys;The special test of drug law enforcement administration of United States Justice Department also establishes gas chromatography-mass spectrography (GC/MS) with research laboratory
" bath salt " ingredient standard analysis method;Domestic many drugs and drugs mechanism also develop MCAT analyzing detecting methods, substantially all
It is the instrument analytical method of gas-chromatography and liquid chromatogram and mass spectrum linkage.
Above-mentioned analysis method is there are expensive equipment, when check fee, needs professional technician to operate, test sample needs numerous
The shortcomings of living together reason.Immunoassay can overcome the defect of the above method, it have specificity it is strong, it is easy to operate, detection when
Between it is short, do not need professional's operation and the advantages that suitable for extensive detection, have been widely used for various diseases, drug abuse
With the quick detection of the projects such as food security.Immunoassay is a kind of various using the detection of antigen and antibody specific association reaction
The analysis method of substance (drug, hormone, protein, microorganism etc.), the premise of this method are exactly to need to provide the anti-of specificity
Former and antibody.In addition, the market demand rapid development of the lateral flow immunochromatography kit of current 4- methyl methcathinones, and give birth to
The critical materials for producing its detection kit are 4- methyl methcathinone artificial antigens, therefore develop and provide a kind of effective 4- first
The preparation method of base methcathinone artificial antigen has become the task of top priority.
Invention content
The object of the present invention is to provide a kind of preparation method of 4- methyl methcathinone artificial antigen, prepared 4- methyl
Methcathinone artificial antigen can carry out animal immune, obtain corresponding 4- methyl methcathinone antibody, be used for all kinds of 4- methyl first
The research of Cathinone immunoassay and the critical materials produced as 4- methyl methcathinone immune chromatography reagent kits.
In the first aspect of the present invention, a kind of haptens of 4- methyl methcathinone, the following general formula of structure are provided
(1):
Wherein, Z is hydrogen atom or carbonylic oxygen atom, and m is 1-4 carbon atoms.In preferred mode, 2,3 or 4 carbon of m
Atom.More preferably, m is the straight chain of 1-4 carbon atoms.
In the second aspect of the present invention, a kind of artificial antigen of 4- methyl methcathinone is provided, structure is general formula (2)
Shown in structure:
Wherein, Z is hydrogen atom or carbonylic oxygen atom, and m is 1-4 carbon atoms;P is carrier protein.
In some preferred modes, carrier protein is selected from bovine serum albumin (BSA), ox gamma Globulin (BGG), ox first
One kind in shape gland globulin (BTG), keyhole limpet hemocyanin (KLH) and chicken egg white (OVA).
Third aspect of the present invention, the present invention provide a kind of haptens preparing 4- methyl methcathinones or artificial antigen
Method, wherein this method includes the synthesis of intermediate methylene dioxy pentanone, wherein the conjunction of intermediate methylene dioxy pentanone
Start at using 4- methoxy benzonitriles as starting material.
In some preferred methods, the method further comprises:It is standby go out alkyl halide lattice reagent, then with alkyl halide lattice
Reagent, by carbonylation, halogenation and clemastine base, prepares the 4- first that hydroxyl is connected on phenyl ring with 4- methoxy benzonitriles
Base methcathinone finally uses succinic anhydride that the haptens containing long-armed carboxyl is made with hydroxyl reaction on phenyl ring.
In some preferred methods, the preparation method of alkyl halide magnesium Grignard Reagent includes:In absolute ether medium, metal
Magnesium chips is with alkyl halide under the conditions of completely cutting off atmospheric moisture, and back flow reaction is completed under absolute ether boiling point.In some preferred methods
In, absolute ether be diethyl ether, dipropyl ether, butyl oxide, tetrahydrofuran and dioxane etc., one kind of the kinds such as non-aromatics alkyl halide or
Person is several.In some preferred methods, reaction medium is done using acyclic ether.In some preferred methods, diethyl ether conduct
Dry ether reaction medium.In some preferred methods, alkyl halide can be include one kind in alkyl chloride, bromoalkane or idoalkane
Or it is several.In some preferred methods, halogenated alkane reagent of the bromoalkane as Grignard Reagent.Preferably, magnesium metal with
The molar ratio of bromoethane is generally 1:1-1:1.5, the molar ratio of the preferred magnesium metal of the present invention and bromoethane is 1:1.2.
In some preferred methods, 4- methoxy benzonitriles and bromoethane magnesium Grignard Reagent (alkyl halide magnesium Grignard Reagent
In one kind) prepare the synthetic method of 1- (4- methoxyphenyls) propane -1- ketone through carbonylation and include the following steps:In high temperature item
Under part, in absolute ether medium, 4- methoxy benzonitriles and bromoethane magnesium Grignard Reagent be carbonylated instead.It is preferred at some
It, since the boiling point of ether is relatively low, is needing that higher boiling solution raising back flow reaction temperature is added in mode.Aromatic hydrocarbons higher boiling is molten
Agent includes one or several kinds of mixing in chlorobenzene, toluene, ethylbenzene and dimethylbenzene.It is integrated from reactionlessness, boiling point and toxicity
Consider that the present invention preferably toluene is reaction medium.In preferred method, specially:Ether is steamed first, then in toluene boiling point
Back flow reaction under (110 DEG C).Preferably, it is inorganic that hydrochloric acid, nitric acid, phosphoric acid and sulfuric acid etc. generally can be used in the acidification of reaction mixture
Formic acid, the organic acids such as acetic acid and propionic acid can also be used in acid.The preferred concentrated sulfuric acid of the present invention is hydrolysis acid.Hydrolysis is needed relatively
It is carried out at low temperature.
In some preferred methods, 1- (4- methoxyphenyls) propane -1- ketone prepares 1- (4- methoxybenzenes through bromination
Base) methods of -2- bromopropane-1-ketones includes the following steps:1- (4- methoxybenzenes are carried out in the solvent medium existing for the acid of Louis
Base) bromination reaction of the propane -1- ketone at a.Wherein, preferred solvent includes that dichloromethane, chloroform, tetrahydrofuran etc. are organic molten
Agent.Preferably, Louis acid catalysis.The present invention selects acetic acid as reaction medium, both can be as the solvent of reactant, itself is again
With acidity, solvent effect and catalytic effect are preferable, and reaction can carry out at room temperature.
In some preferred methods, 1- (4- methoxyphenyls) -2- bromopropane-1-ketones prepare 1- (4- through methylaminoization
Methoxyphenyl) -2- methylamino propane -1- ketone methods include the following steps:1- (4- methoxyphenyls) -2- bromopropane-1-ketones
It is reacted in inert organic solvents medium with methylamine water solution.Preferably, inert organic solvents medium includes dichloroethanes, trichlorine
Ethane, N,N-dimethylformamide and tetrahydrofuran.Preferably, wherein tetrahydrofuran is to 1- (4- methoxyphenyls) -2- bromines third
Alkane -1- ketone and methylamine water solution have good dissolubility, and boiling point is less than n,N-Dimethylformamide, react later stage solvent
It is easy to remove, so, the preferred tetrahydrofuran of the present invention is that 1- (4- methoxyphenyls) -2- bromopropane-1-ketones are molten through methylaminoization
Agent.In some preferred methods, 1- (4- methoxyphenyls) -2- methylaminos propane -1- ketone prepares 1- (4- hydroxyls through acidolysis
Phenyl) method of -2- methylamino propane -1- ketone includes the following steps:In order in MCAT molecule construction carboxyls, present invention selection pair
The benzonitrile with methoxyl group is the initial reaction raw material of carbonylation on position, and methoxyl group is under certain temperature and certain acid condition
Acidolysis occurs and generates hydroxyl.Acidolysis generally uses inorganic acid, including sulfuric acid, hydrochloric acid, nitric acid, hydrobromic acid and hydroiodic acid.The present invention
It is preferred that 48% hydrobromic acid is acidolysis medium.Acidolysis generally requires to be carried out at a warm condition, and temperature range is generally in 80-150
Degree, the preferred methoxyl group dissociation temperature of the present invention are 110 degree.
In some preferred methods, 1- (4- hydroxy phenyls) -2- methylaminos propane -1- ketone prepares 1- through amido protection
The method of (4- hydroxy phenyls) -2- trifluoroacetyl methylamino propane -1- ketone includes the following steps:In 1- (4- hydroxy phenyls) -2-
Before methylamino propane -1- ketone carboxylated, the amido of 1- (4- hydroxy phenyls) -2- methylamino propane -1- ketone must protect.
Because this methylamino activity is greatly, it is very easy to react with succinic anhydride.Amido protecting agent generally commonly uses tertiary fourth oxygen
Carbonyl (Boc), tablet held before the breast by officials methoxycarbonyl group (Fmoc), trifluoroacetic anhydride and trifluoroacetic acid diethylester.Because of 1- (4- hydroxy phenyls) -2- first
Contain hydroxyl in amido propane -1- ketone molecules, reaction medium is METHANOL MEDIUM, so the preferred trifluoroacetic acid diethylester of the present invention is
Methylamino protects reagent.
In some preferred methods, 1- (4- hydroxy phenyls) -2- trifluoroacetyl methylamino propane -1- ketone is through carboxylated system
Include the following steps for the method for going out the 4- methyl methcathinone haptens with carboxyl:1- (4- hydroxy phenyls) -2- trifluoro second
The esterification of acyl methylamino propane -1- ketone and succinic anhydride carries out in having solvent medium existing for the alkali of Louis.Wherein,
Lewis base includes triethylamine, tri-n-butylamine, pyrroles and pyridine etc., and solvent medium includes dichloromethane, chloroform, toluene and tetrahydrochysene furan
It mutters and waits organic solvents.The present invention selects pyridine as reaction medium, both can be as the solvent of reactant, itself has alkali again
Property, solvent effect and catalytic effect are preferable.Wherein, long-chain halogenated carboxylic ester includes bromo butyric acid methyl ester, bromobutyrate, bromine penta
Sour methyl esters, bromine valeric acid ethyl ester, bromocaproic acid methyl esters and bromocaproic acid ethyl ester etc., the carboxylate of introducing generates carboxyl after hydrolysis.Carboxylic acid
Acid anhydride includes ethanedioic acid acid anhydride, malonic anhydride, succinic anhydride and glutaric anhydride etc..The preferred bromobutyrate of the present invention or succinic anhydride are made
For carboxylated reagent.1- (4- hydroxy phenyls) -2- trifluoroacetyl methylamino propane -1- ketone and succinic anhydride are under nitrogen protection
100 degree are reacted 5 hours, and the molar ratio of 1- (4- hydroxy phenyls) -2- trifluoroacetyl methylamino propane -1- ketone and succinic anhydride is preferred
It is 1:1.45.
The fourth aspect of the present invention provides a kind of preparation method of the artificial antigen of 4- methyl methcathinone, this method packet
It includes:On the basis of synthesizing 4- methyl methcathinone haptens, the artificial antigen of 4- methyl methcathinones is prepared.4- methyl first cards
Western ketone is a small molecule, since its molecular mass is small (177 dalton), only has immunoreactivity, does not have immunogenicity,
It must be attached on protein macromolecule, antibody specificity could be generated by the t cell epitope stimulation body of macromolecular
Immune response.The specific method of preparation is:4- methyl methcathinone haptens is in N, N- dimethyl formyl media, with N- hydroxyls
Succinimide and N, active ester is prepared in reaction to N- cyclohexyl carbon imidodicarbonic diamide at room temperature under nitrogen protection, and then it is lived
Property ester in sodium carbonate-bicarbonate buffer solution, coupling reaction occurs with albumen sloughs trifluoroacetyl protecting group simultaneously, finally
To 4- methyl methcathinone artificial antigens.
The fifth aspect of the present invention, the present invention provide a kind of antibody, the antibody be in aforementioned arbitrary scheme haptens or
Antigen-immunized animal and obtain.
In the sixth aspect of the present invention, provide in a kind of detection sample whether contain Cathinone and its analogue matter
Kit, wherein the kit includes haptens in aforementioned arbitrary concrete scheme either antigen or aforementioned arbitrary scheme
The antibody of acquisition.Cathinone and its analogue matter should be special including many different types of substances, such as Chinese invention
Those structures and 4- methcathinones and 4- methoxy methyl Cathinones disclosed in the disclosed CN105954511A of profit application.
In the seventh aspect of the present invention, provide it is a kind of utilizing colloidal gold, but the immunochromatography technique for being not limited to colloidal gold is fast
Speed detection 4- methyl methcathinones kit, including outer packing, detection kit it is characterized in that:The detection kit
It is made of four parts, is successively:The anti-4- of the sample pad, colloidal gold or colored latex nanoparticle label that are handled through special solution
The label pad of methyl methcathinone antibody is coated with 4- methyl methcathinone-BSA and (quality control region C) in coating region (detection zone T)
It is coated with nitrocellulose filter, the water sucting plate of polyclonal antibody.
In some preferred technical solutions, the 4- methyl methcathinone antibody is present invention any way above-mentioned
What the 4- methyl methcathinone antigen immunising mammals of middle preparation obtained.In some preferred technical solutions, the 4-
Methyl methcathinone-BSA is that the haptens prepared in the aforementioned any way of the present invention connects the antigen obtained after protein carrier.
In some preferred technical solutions, the preparation process for quickly detecting the kit of 4- methyl methcathinones includes such as
Lower step:T line solution is configured with 4- methyl methcathinones-BSA, polyclonal antibody configures C line solution, then they are distinguished
It is coated with T lines on nitrocellulose filter, C lines region, the immune colloid gold marked with anti-4- methyl methcathinone antibody
Or in immune colored latex nanoparticle solution coating to glass fibre or harmless cloth, sample pad is handled with special solution.It will be upper
It states material and pastes sequentially in either order and combine to form reagent strip with water sucting plate again on PVC offset plates, strip may be separately formed examination
Agent item can also be assembled into reagent strip in plastic housing for detecting and form the detection of cassette kit.
In some preferred technical solutions, the preparation process of the kit of 4- methyl methcathinones is quickly detected, it is special
Sign is:Sample pad is handled with special solution, to improve product specificity and sensitivity.
In some preferred technical solutions, the preparation process of the kit of 4- methyl methcathinones is quickly detected, it is special
Sign is that sample pad special solution main component is constituted by containing surfactant, bovine serum albumin(BSA), PBS solution.
In some preferred technical solutions, the preparation process of 4- methyl methcathinone kits, feature are quickly detected
It is:It can also be polyclonal antibody that anti-4- methyl methcathinone antibody for label, which can be monoclonal antibody,.
In some preferred technical solutions, the preparation process of 4- methyl methcathinone kits, feature are quickly detected
It is:It is polyclonal antibody, preferably sheep anti-mouse antibody or goat anti-rabbit antibody for coated nature controlling line.
In some preferred technical solutions, the preparation process of 4- methyl methcathinone kits, feature are quickly detected
It is:Can also be that colored latex nanometer is micro- for marking the marker of anti-4- methyl methcathinone antibody that can be colloidal gold
Ball, fluorescent nanometer microsphere.
In some preferred technical solutions, the preparation process of 4- methyl methcathinone kits, feature are quickly detected
It is:The nitrocellulose filter being coated with is pasted on PVC bottom plates, the immune glue of 4- methyl methcathinone antibody will be coated with
The glass pricker dimension (label pad) of body gold or colored latex nanoparticle overlaps the nitrocellulose filter 0.5mm being coated with, and will contain
There is the sample pad of special solution to overlap to the 1/2 of label pad, the upper part for the nitrocellulose filter that water sucting plate overlap joint has been coated with.
Advantageous effect
The present invention provides the synthetic route of a kind of new 4- methyl methcathinone haptens or antigen, overcomes existing biography
The deficiency of system technology, in addition for detection sample in whether new approach is provided containing Cathinone.
Description of the drawings
Fig. 1:4- methyl methcathinone antigens in the present invention prepare chemical reaction process flow diagram.
Fig. 2:4- methyl methcathinone-BSA albumen attachment UV scanning figures in the present invention.
Fig. 3:4- methyl methcathinone hydroxy derivatives mass spectrograms in the present invention.
Fig. 4:4- methyl methcathinone detection kit structural schematic diagrams in the present invention.
Fig. 5:4- methyl methcathinone test result schematic diagrames in the present invention.
It is described in detail
Detection
Detection indicates chemical examination or tests a kind of substance or material whether there is, for example, but it is not limited to this, chemical substance,
Organic compound, inorganic compound, metabolism product, drug or drug metabolite, organic organization or organic organization generation
Thank object, nucleic acid, protein or polymer.In addition, detection indicates test substances or the quantity of material.Furtherly, table is gone back in chemical examination
Show immune detection, chemical detection, enzyme detection etc..
The synthesis of the haptens of 4- methyl methcathinones
The synthesis of the haptens of 4- methyl methcathinones relates generally to two key factors, and one is intermediate 1- (4- first
Phenyl) propane -1- ketone synthesis, the other is introducing carboxyl in 4- methyl methcathinone molecular structures.
The synthesis of 4- methyl methcathinones generally uses ephedrine oxidation technology or the aminated technique of propiophenone
(US3492351).Ephedrine belongs to national management and control class drug, and propiophenone belongs to national management and control class industrial chemicals, if with these
National management and control goods and materials are that raw material produces antigen, and production management and inventory management procedures become complicated, and management and control substance also readily flows to
Society is utilized by criminal.
The present invention, which is reacted using methoxy benzonitrile with alkyl halide magnesium Grignard Reagent, prepares 4- methyl methcathinone derivatives,
Its carbonylating process than it is traditional pay-gram carbonylation is more advantageous, reaction process condition is mild, reaction yield height, and avoids
Use the propionyl chloride raw material being more toxic.Pay-a gram acylation uses aluminum trichloride (anhydrous) for catalyst, carbonylation process conditions are severe
It carves, reaction is violent, and industrial manufacture process control difficulty is big;And the bromoalkane Grignard Reagent carbonylating process that the present invention uses avoids
Above-mentioned disadvantage.
The synthesis of semiantigen of 4- methyl methcathinones is as follows:Prepare the alkyl halide and magnesium metal of Grignard Reagent.With halogen
Be raw material for alkane and magnesium metal, prepare alkyl halide lattice reagent in anhydrous ether is medium, then with alkyl halide lattice reagent with and
4- methoxy benzonitriles are that raw material passes through carbonylation, halogenation and clemastine base, prepare the 4- first that hydroxyl is connected on phenyl ring
Base methcathinone finally uses succinic anhydride that the haptens containing long-armed carboxyl is made with hydroxyl reaction on phenyl ring.
In the above-mentioned methods, alkyl halide magnesium Grignard Reagent preparation method is as follows:Alkyl halide magnesium Grignard Reagent is in absolute ether
In medium, metal magnesium chips is with alkyl halide under the conditions of completely cutting off atmospheric moisture, and back flow reaction is completed under absolute ether boiling point.At some
In preferred mode, the absolute ether as medium can be diethyl ether, dipropyl ether, butyl oxide, tetrahydrofuran and dioxane etc.,
One or several kinds in non-aromatics alkyl halide etc..Reaction medium, the preferred diethyl ether conduct of the present invention are generally done using acyclic ether
Dry ether reaction medium.In some preferred modes, the alkyl halide for preparing Grignard Reagent can be include alkyl chloride, bromoalkane or
One or several kinds in idoalkane, their reactivity sequence is idoalkane>Bromoalkane>Alkyl chloride;Idoalkane is of high cost,
Alkyl chloride reactivity is relatively low.Halogenated alkane reagent of the preferred bromoalkane of the present invention as Grignard Reagent.Preferably, metal
The molar ratio of magnesium and bromoethane is generally 1:1-1:1.5, the molar ratio of the preferred magnesium metal of the present invention and bromoethane is 1:
1.2。
The synthesis of antigen and the preparation of corresponding antibody
In some preferred embodiments, haptens of the invention is activated by the coupling with carrier protein, is carried
The modified antigen of carrier protein has stronger immunocompetence, because individually small molecule itself does not have immunocompetence usually
Or immunocompetence is very low.The carrier protein of coupling can be, but be not limited to hemocyanin (Keyhole limpet
Hemocyanin, KLH), bovine serum albumin (Bovine Serum Albumin, BSA), ovalbumin (Ovalbumin, OVA),
Bovine hemoglobin (Bovine gamma globulin, BGG), bovine thyroid albumen (Bovine Thyroglobulin, BTG),
Sperm whale myoglobin (Sperm Whale Myoglobin, SWM), tetanus toxoid (Tetanus Toxoid, TT), methyl
Bovine serum albumin (Methylated Bovine Serum Albumin, mBSA), the human immunoglobulin(HIg) IgG or IgA of change
The immunogen protein etc. of (Human immunoglobulins IgG, IgA) or other prior arts.
It can be immunized animal, such as mouse with the small molecule for being coupled active group above, rabbit or other mammals come
Polyclonal antibody is produced, monoclonal antibody can also be generated with hybridoma, these methods are that this field is conventionally known
Technology, details are not described herein, may refer to some textbooks or immune handbook to obtain the antibody or antibody fragment (system of the present invention
For and using antibody-application manual (Making AND Using Antibodies-A practical handbook) .CRC
Press,2007).Certainly, antibody can also pass through nature and generate, and can also be by artificial synthesized antibody or antibody fragment.
These antibody can be with specific recognition artificial antigen either haptens or small-molecule substance, and cannot identify other unrelated antigens
Or small-molecule substance.Here so-called " specificity " refer to the antibody be only capable of identify or combine a certain certain types of antigen, and
Nonrecognition combines other kinds of antigen.In the present invention, prepared antibody only identifies 4- methyl methcathinones;4- first
Base methcathinone haptens, 4- methyl methcathinone artificial antigens, and cannot identify other small molecule chemicals.
As combination or identification methcathinone and its analogue matter, such as 4- methyl methcathinone, 4- methyl first cards
Western ketone haptens or 4- methyl methcathinone artificial antigens, antibody can be the present invention antibody.Antibody can be immune
The immunoglobulin molecules of globulin molecule or incomplete antigen specific site, such as those molecule energy with antigen binding site
Enough special (by being immunized) combines analyte, the mimicry substance or ligand of analyte.Antibody also includes artificial synthesized
Hybrid antibody or the antibody or Antibody molecule fragments for passing through modified, include, but are not limited to, antibody fragment and Fv segments.
With some segments on abiogenous antibody on antibody with combination antigen function.One binding fragment or antibody fragment
Include, but are not limited to, (i) Fab segments, it includes VL, VH, the regions CL and CH1;(ii) Fd segments, it includes the areas VH and CH1
Domain (iii) Fv segments, it includes the regions VL and VH on one of antibody single-stranded;(iv) regions dAb (Ward et al.,
Nature 341:544-546 (1989), it includes the regions VH;(v) an independent determinant (CDR);(vi) F (ab')
2 segments, a bivalent fragment include two Fab segments connected in hinge area by disulphide.In addition, though this Fv segment
On two regions be different gene code and determined, artificial synthesized connection reagent can allow them to form single albumen
Chain (known single Fv (scFv) chain) (Bird et al., Science 242:423-426(1988);With Huston et al.,
PNAS85:5879-5883(1988)).Protein fragments include the segment of those target antigens that can be crosslinked in conjunction with them, such as
Bivalent fragment, such as 2 segments of F (ab').Optionally, those cannot the protein fragments of self crosslinking combining target antigen also can be with the
Two antibody combining target antigen together.
Haptens, artificial antigen, the application of antibody
4- methyl methcathinone haptens that the present invention synthesizes or 4- methyl methcathinone artificial antigens and utilize him
The antibody (monoclonal is polyclonal) that generates can be used to prepare and whether contained to detect in sample using immunization method
Cathinone and methcathinone analogue, for example, 4- methyl methcathinone, methcathinone, Cathinone, 4- methcathinones
And 4- methoxy methyls Cathinone).Here sample is usually mammal or the body fluid sample of people, such as saliva, urine, sweat
The tissues such as the tissue samples of liquid or mammal or people, such as liver, spleen.Here so-called " immunologic detection method " one
As be using the combination of antibody and antigen principle carry out immunoassay or detection method, this immunization method include enzyme-linked
Immune (ELSA) method, also includes the method for lateral flow (lateral flow) test paper, method may include competition side
Method.
It can be made lateral flow test paper of water suction or unwetted property material, and a test-strips can use more
Kind material, is used for the transmission of liquid.A kind of material of test-strips can be superimposed upon on another test strip material, for example, filter paper
Superposition is on nitrocellulose.Alternatively, a region at least containing a kind of material in test-strips, which is located at another kind, at least contains one
After the region of kind different materials.In this case, liquid is circulation between each region, can be overlapped mutually between them
Or it is not superimposed.Material in test-strips can be fixed on such as support of plastics liner or hard surface, to reinforce
Test-strips can holding force.Each region of test-strips can be arranged as follows:Sample application zone, at least one reagent area, at least one testing result
Area, at least one control zone, at least one detection of adulterations area and liquid absorption area.If detection zone includes a control zone, excellent
Selected control area is located at after the analyte detection zone of test results zone.A kind of material can contained in all these areas or combinations thereof
In the single test strips of material.In addition, these areas are made from a variety of materials, and link together by the direction that liquid transmits.Example
Such as, different zones can directly or indirectly carry out liquid transmission.In the present example, different areas can be along the side that liquid transmits
Terminad is connected with end, or is overlapped mutually along liquid direction of transfer, or is connected by other materials, such as connects medium
Material (preferably water-absorbing material such as filter paper, glass fibre, non-woven fabrics or nitrocellulose).When with connecting material, connection
Material can be such that material, the end comprising each region and the joining distal ends but liquid of the end comprising each region and joining distal ends do not flow
Logical material or the material not circulated comprising each region overlapped (being such as, but not limited to overlapped from the beginning to the end) but liquid, shape
At liquid communication.
In addition to this, antibody prepared by the antigen of the present invention can also be utilized for immunization therapy because of 4- methyl first cassies
Various diseases caused by ketone, such as a kind of immunoreagent for treating 4- methyl methcathinone habituation.Thus it is used as one kind
Pharmaceutical agent uses.
Detect the immune chromatography reagent kit of the methcathinone in human urine
The object of the present invention is to provide the Cathinones and its analogue in a kind of detection human urine, such as 4- methyl
Methcathinone, immune chromatography reagent kit preparation method, its step are as follows:Test strips are made of four parts, are successively:Sample
Pad, label pad (glass fibre for being coated with the 4- methyl methcathinone antibody of colloid gold label or colored latex microsphere label),
It is coated with 4- methyl methcathinone-BSA in T lines region (detection zone) and has been coated with polyclonal antibody in C lines region (quality control region)
Nitrocellulose filter, water sucting plate.
It is prepared by 4- methyl methcathinone 4- methyl methcathinone detection kits:
1) main material 1. 4- methyl methcathinone-BSA compounds (of the invention) 2. anti-4- methyl methcathinone Dan Ke
Grand antibody or polyclonal antibody (commercially available) (3. polyclonal antibody 4. nitrocellulose filter 5. immune colloid gold solution 6. plastics
Shell (being made of upper and lower cover) 7. water sucting plate 8. PVC bottom plates
2) preparation of nitrocellulose filter:4- methyl methcathinone-BSA compounds configure T line solution, and polyclonal antibody is matched
C line solution is set, then they are coated with respectively detection line T lines on nitrocellulose filter, nature controlling line C line region;
3) preparation of colloidal gold:0.01%HAuCl4 aqueous solution 100ml are taken, 1% citric acid, three sodium water solution is added
15min~30min is boiled in 0.75ml, heating, until color reddens.Stop heating, it is cooling for use.
4) prepared by immune colloid gold:The combination of colloidal gold and protein (IgG):By colloidal gold solution 0.1Mol/L
Anti- 4- methyl methcathinone antibody-solutions are added in K2CO3 tune pH to 9.0, electromagnetic agitation colloidal gold solution, continue to stir 10min,
A certain amount of stabilizer is added to precipitate to prevent antibody protein from colloidal gold polymerizeing, immune colloid is obtained by centrifugal purification
Golden marker.
5) preparation of colloidal gold pad:In the immune colloid gold solution coating to glass fibre of deployed debita spissitudo.It dries
It is dry.
6) preparation of sample pad:Glass fibre is immersed in sample pad solution, is dried.
7) prepared by kit:1. the composition of kit:Since detection kit of the present invention be successively being loaded end:Sample
Pad, has been coated with 4- methyl methcathinone-BSA in T lines region (detection zone) and has been coated in C lines region (quality control region) label pad
Polyclonal antibody is preferably four nitrocellulose filter of sheep anti mouse or goat anti-rabbit antibody, water sucting plate parts.This four parts are equal
It pastes successively on PVC bottom plates, is then cut into required width, form reagent strip.It can also be packed by plastic housing size
In designed plastic housing.Form cassette kit.
This component is characterized in:Present invention selection handles sample pad using special reagent, it may ensure that the stabilization of product
Property and repeatability.Non-specific background can significantly be reduced.Improve the uniform characteristic of testing result.It is apparent overcome due to
False positive caused by nonspecific reaction, therefore, the present invention improve the sensitivity and specificity of detection.
The formula of sample pad processing solution:0.1%BSA, 0.03M PBS, 0.01% surfactant.
Specific embodiment
How thought in order to illustrate the present invention is realized, only illustrates how the present invention realizes by way of example, this
A little realize is only the specific limited explanation under the marrow without prejudice to the present invention, does not constitute the specific limit to the present invention
System.
Examples of implementation 1:The synthesis of bromoethane magnesium Grignard Reagent
250 milliliters of drying four-hole bottle, installation nitrogen snorkel, upper end are inserted with the reflux condensation mode of anhydrous calcium chloride drying tube
Device and the constant pressure funnel for being connected with anhydrous calcium chloride drying tube.Equipped with 40 milliliters of adiabatic drying ether and 5.75 grams in funnel
Bromoethane.40 milliliters of adiabatic drying ether, 1.1 grams of metal magnesium chips and two iodine grains are added in four-hole bottle.Start stirring, nitrogen
After displacement, bromoethane diethyl ether solution is added in four-hole bottle, half an hour is added dropwise, temperature rising reflux reaction 3 hours, then temperature
It is down to room temperature, prepares bromoethane magnesium Grignard Reagent.
Examples of implementation 2:The synthesis of 1- (4- methoxyphenyls) propane -1- ketone
By 5.33 grams of 4- methoxy benzonitriles and 50 milliliters of toluene from being added drop-wise in constant pressure funnel in examples of implementation 1
In the Grignard Reagent of preparation, it is added dropwise within 1 hour.After heating steams ether, 30 milliliters of toluene are added, temperature rising reflux reacts, instead
Process is answered to be monitored by thin-layer chromatography.Back flow reaction is reacted after 3 hours to be terminated, and room temperature is down to, and 40 milliliters of mixture of ice and water and 8 are added
The milliliter concentrated sulfuric acid, after hydrolysis, liquid separation, water phase is extracted twice with 100 milliliters of ether, merges organic phase, with 100 milliliters of saturations
Salt washing is primary, and deionization is washed twice, and anhydrous sodium sulfate is added and is dried overnight.Rotary evaporation obtains an oily except solvent
Object crude product, by sudden strain of a muscle chromatographic isolation (the silicagel column elution agent of this crude product:Ethyl acetate:Petroleum ether=1:7.5) 5.89 grams are obtained
(yield 67%) object 1- (4- methoxyphenyls) propane -1- ketone.
Examples of implementation 3:The synthesis of 1- (4- methoxyphenyls) -2- bromopropane-1-ketones
5.65 grams of 1- (4- methoxyphenyls) propane -1- ketone and 30 milliliters of acetic acid are added in 100 milliliters of there-necked flasks;It will
6.15 grams of bromines being dissolved in 30 milliliters of acetic acid are added in dropping funel.By the liquid in funnel under room temperature and magnetic agitation
Bromoacetic acid solution is added drop-wise in there-necked flask, is added dropwise within 30 minutes, and room temperature continues to be stirred to react, the monitoring reaction of thin-layer chromatography contact plate
Process.It reacts and completes after three hours, rotary evaporation removes acetic acid, and 50 milliliters of water are added and are dispersed with stirring, are then transferred to separatory funnel
In, it is extracted twice with 50 milliliters of chloroforms, merges organic phase, washed once respectively with 30 milliliters of saturated solution of sodium bicarbonate, 30 millis
It rises deionized water washed once, 30 milliliters of saturated salt solutions washed once, and anhydrous sodium sulfate is then added and is dried overnight.Rotation is steamed
Hair removes chloroform, sudden strain of a muscle chromatographic isolation (the silicagel column elution agent of gained crude product:Ethyl acetate:Petroleum ether=1:15) 6.55 grams are obtained
(yield 85%) object 1- (4- methoxyphenyls) -2- bromopropane-1-ketones
Examples of implementation 4:The synthesis of 1- (4- methoxyphenyls) -2- methylamino propane -1- ketone
In 100 milliliters of dry single port bottles, 5.8 grams of 1- (4- methoxyphenyls) -2- bromopropane-1-ketones and 60 milliliters are added
Tetrahydrofuran is stirred 15 minutes.5.55 gram of 40% methylamine solution is fitted into dropping funel, is added dropwise in there-necked flask
Reaction mixture in, half an hour drips, and is warming up to 50 degree, continues to be stirred to react, thin-layer chromatography contact plate monitor reaction process.
Reaction terminates after 3 hours, by the reaction mixture rotary evaporation in there-necked flask, removes solvent and obtains crude product, dodge chromatographic isolation (silicon
Rubber column gel column eluant, eluent:Methanol:Chloroform=1:9) 3.48 grams of (yield 75%) object 1- (4- methoxyphenyls) -2- methylaminos are obtained
Propane -1- ketone
Examples of implementation 5:The synthesis of 1- (4- hydroxy phenyls) -2- methylamino propane -1- ketone
In 100 milliliters of single port bottles, 3.2 grams of 1- (4- methoxyphenyls) -2- methylaminos propane -1- ketone and 65 millis are added
48% hydrobromic acid is brought rapidly up after being stirred to 110 degree, and the reaction was continued 30 minutes at a temperature of this.30 degree are cooled to, it will be above-mentioned
Reaction mixture is added in 120 ml deionized waters, and 60 milliliters of ether are added after being uniformly mixed and are extracted twice, are associated with
Machine phase, it is primary with 60 milliliters of saturated common salt washings, then it is dried overnight with anhydrous sodium sulfate.Rotary evaporation is removed solvent and is obtained slightly
Product, sudden strain of a muscle chromatographic isolation (the silicagel column elution agent of gained crude product:Ethyl acetate:Hexane=4:6) it obtains 2.47 grams (yields 78%)
Object 1- (4- hydroxy phenyls) -2- methylamino propane -1- ketone.
Examples of implementation 6:The synthesis of 1- (4- hydroxy phenyls) -2- trifluoroacetyl methylamino propane -1- ketone
In 100 milliliters of single port bottles, 2.25 grams of 1- (4- hydroxy phenyls) -2- methylamino propane -1- ketone, 45 milliliters of first are added
Alcohol and 1.49 grams of triethylamines are stirred.2.85 grams of Trifluoroacetic Acid Ethyl Esters are added dropwise from dropping funel at room temperature to reaction system
In, half an hour drips off, and the reaction was continued at room temperature 5 hours.Above-mentioned reaction mixture is added in 50 milliliters of 6M hydrochloric acid, stirring is mixed
It closes, 60 milliliters of ethyl acetate is then added and are extracted twice, merge organic phase, it is primary with 60 milliliters of saturated common salt washings, then use
Anhydrous sodium sulfate is dried overnight.Rotary evaporation removes solvent and obtains 2.76 grams of object 1- (4- hydroxy phenyls) -2- trifluoroacetyls
Methylamino propane -1- ketone.
Examples of implementation 7:The synthesis of haptens
In 100 milliliters of dry single port bottles, 2.50 grams of 1- (4- hydroxy phenyls) -2- trifluoroacetyl methylaminos propane-are added
1- ketone, 60 milliliters of anhydrous pyridines and 1.42 grams of succinic anhydrides are stirred to react for 100 degree under nitrogen protection, the monitoring of thin-layer chromatography contact plate
Reaction process.Reaction terminates after 5 hours, and rotary evaporation removes pyridine and obtains dark brown mucus, this crude product sudden strain of a muscle chromatographic isolation
(silicagel column elution agent:Methanol:Chloroform=1:5) 2.26 grams of haptens (yield 66%) are obtained.
Examples of implementation 8:Haptens activity Lipase absobed
In 25 milliliters of dry single port bottles, be added 110 milligrams of haptens, 86.87 milligrams of n-hydroxysuccinimides,
196.42 milligrams of N, N- cyclohexyl carbon imidodicarbonic diamide base and 10 milliliters of anhydrous N,N-dimethylformamides, under nitrogen protection room temperature
It is stirred overnight.After the completion of reaction, reaction mixture is divided in 7 1.5 milliliters of centrifuge tube, 10000rpm centrifuges 10 points
Clock, by supernatant, hermetically storing is spare in 7 1.5 milliliters of centrifuge tube.
The synthesis of examples of implementation 9.. artificial antigens (methcathinone-BSA conjugate)
Take 100 milligrams of BSA in 25 milliliters of single port bottles, the 100mM sodium carbonate-bicarbonates that 7.2 milliliters of PH8.6 are added are slow
Fliud flushing and 2.5 milliliters of DMF stirring and dissolvings, make the temperature of reaction mixture maintain 4 degree or so with ice-water bath, by 2.6 milliliters of work
Property ester solution be slowly added drop-wise in BSA solution, adjust the pH value of reaction solution at any time with 0.5N sodium hydroxide solutions, maintain mixing
The pH value of liquid is 8.5 or so.After being added dropwise, reaction bulb is put into 4 degree of cold houses, is stirred to react overnight.It after reaction, will be anti-
It answers mixture to be transferred in bag filter, dialyses two days to the PBS buffer solution of 0.1M PH7.4, be then lyophilized, obtain target antigen
(this antigen is used as envelope antigen).
The synthesis of examples of implementation 10.. artificial antigens (methcathinone-KLH conjugates)
Take 100 milligrams of KLH in 25 milliliters of single port bottles, the 100mM sodium carbonate-bicarbonates that 8.6 milliliters of PH8.6 are added are slow
Fliud flushing and 3.5 milliliters of DMF stirring and dissolvings, make the temperature of reaction mixture maintain 4 degree or so with ice-water bath, by 3.7 milliliters of work
Property ester solution be slowly added drop-wise in BSA solution, adjust the pH value of reaction solution at any time with 0.5N sodium hydroxide solutions, maintain mixing
The pH value of liquid is 8.5 or so.After being added dropwise, reaction bulb is put into 4 degree of cold houses, is stirred to react overnight.It after reaction, will be anti-
It answers mixture to be transferred in bag filter, dialyses two days to the PBS buffer solution of 0.1M PH7.4, be then lyophilized, obtain target antigen
(this antigen is used as immunizing antigen).
Examples of implementation 11:Artificial antigen is identified
The 4- methyl methcathinone artificial antigens that the present invention is prepared can identify by the following method
Coupling ratio measures:The combination ratio of conjugate refers to the ratio between the Molecules of haptens and carrier protein in conjugate,
Spectrophotometry measurement can be used in coupling ratio.It is in proportionate relationship to the absorption of light and its concentration that spectrophotometry, which is using substance,
Principle measures two kinds of molecular concentrations being coupled respectively.In macromolecular and small molecule conjugate, two kinds of molecules have respectively not
Same ultraviolet scanning spectrum, and show the property of spectrum superposition.The combination ratio of conjugate refers to haptens and load in conjugate
The ratio between Molecules of body protein.
The molar absorption coefficient (£) of carrier protein, haptens, conjugate under a certain wavelength is measured respectively, according to spectrum
The additive property principle of absorbance in analysis estimates the molal quantity of haptens coupled on every mole of carrier protein according to following formula
(N).According to uv scan as a result, the ultra-violet absorption spectrum feature of control three can carry out qualitative parsing.
ε=(εConjugate-εCarrier protein)/εHaptens
4- methyl methcathinone haptens (MCAT), bovine serum albumin and 4- methyl methcathinone-bovine serum albumin coupling
The UV scanning spectrogram of object is shown in attached drawing 2:
4- methyl methcathinone (MCAT) haptens has a typical absorption at 285nm and 345nm, BSA in 275nm and
There is maximum absorption band at 280nm, and the maximum absorption band of MCAT-BSA conjugates is then located at 274nm and 278nm, this explanation
MCAT artificial antigen conjugate maximum absorption bands move to left, and have different characteristic ultraviolet absorptions from its precursor substance, show 4- first
Base methcathinone haptens is coupled clearly success with carrier proteins Bovine, and 4- methyl methcathinones MCAT is calculated according to above-mentioned formula
It is combined with the molecule of bovine serum albumin BSA than being 16: 1
Examples of implementation 12:The cross-flow type test-strips of detection 4- methyl methcathinones are prepared using the artificial antigen of the present invention
1:It is prepared by sample pad:Glass fibre is immersed in sample pad processing solution, is then dried, sample pad processing solution
Formula:0.1%BSA, 0.03M PBS, 0.01% surfactant.
2:It is prepared by gold-labelled pad:0.01%HAuCl4 aqueous solution 100ml are taken, 1% citric acid, three sodium water solution 0.75ml is added,
15min~30min is boiled in heating, until color reddens.Stop heating, it is cooling for use.By colloidal gold solution with 0.1Mol/L
K2CO3 tune pH to 9.0, electromagnetic agitation colloidal gold solution, be added anti-4- methyl methcathinone monoclonal antibody (Eastcoast HM457) after
Continuous stirring 10min, is added a certain amount of stabilizer and is precipitated with preventing antibody protein from polymerizeing with colloidal gold, pass through centrifugal purification
Obtain immuno-gold labeling object.In the immune colloid gold solution coating to glass fibre of deployed debita spissitudo, drying is standby
With.
3:Nitrocellulose film preparation:4- methyl methcathinone-BSA compounds (present invention) configure T line solution, sheep anti mouse
IgG antibody configures C line solution, then they is coated with respectively T lines on nitrocellulose filter, C lines region;
Examples of implementation 13:Detect the immunochromatography diagnostic kit of 4- methyl methcathinones in human urine
The immunochromatography diagnostic kit of 4- methyl methcathinones in human urine is detected, the kit includes 1:Sample
Pad, 2:Label pad prepared by antibody label colloidal gold, 3:Nitrocellulose filter, 4:Water sucting plate and 5:PVC bottom plates.Pasting board mode
As shown in Figure 4:Nitrocellulose filter detection line (T lines) direction linkage flag pad, nature controlling line (C lines) direction connect water sucting plate.Gold
Mark pad pressure nitrocellulose filter 0.5mm.Sample pad is connected with gold mark label pad at pressure gold-labelled pad 1/2, and lower section is neat with bottom plate.Water suction
Plate presses nitrocellulose filter 1mm, upper end neat with bottom plate.T lines coating 4- methyl methcathinones-BSA, C on the nitrocellulose membrane
Line is coated with sheep anti-mouse antibody.The gold mark label pad marks the anti-4- methyl methcathinone-BSA monoclonal antibodies of mouse.Test sample to be checked
Product are added drop-wise at sample pad, and sample is chromatographed upwards using capillary effect, and sample is along sample pad-label pad-nitrocellulose filter-suction
Water plate direction is moved, and reaction process is completed.
Examples of implementation 14:Detect the immunochromatography diagnostic kit of 4- methyl methcathinones in human urine
Example 14 does not exist together with example 13 for the gold-labelled pad label is rabbit-anti 4- methyl methcathinones-BSA mostly anti-.
Coated nature controlling line is goat anti-rabbit antibody.
Examples of implementation 15:Detect the immunochromatography diagnostic kit of 4- methyl methcathinones in human urine
Example 15 does not exist together with example 13 for the gold pad is the 4- methyl methcathinones of colored latex nanoparticle label
Antibody.Testing result judges as shown 5.
Urine sample 100ul to be detected is taken, the sample pipetting volume area of strip is added in, after sample is added drop-wise in sample pad, urine will
It is analysed because capillary chromatography effect is acted on to water sucting plate end layer, if without methcathinone and analogue in urine, (for sample
In, any methcathinone and analogue can antibody or antigen cooperation using the present invention detect), such as
4- methyl methcathinones, the colloid gold particle of anti-4- methyl methcathinone antibody, which will be marked, to be run to film in company with urine sample solution
After T line positions, the 4- methyl methcathinone carrier conjugates being coated with T lines occur that association reaction is immunized, and colloid gold particle is in T
The accumulation of line position to show a macroscopic pink lines in T lines, this is negative findings.If contained in urine
There are 4- methyl methcathinones, they will compete limited antibody bound site with the coated 4- methyl methcathinone-BSA of T line positions
Point, when the 4- methyl methcathinone concentration in patient urine reaches a certain amount of, it will occupy the institute marked on colloid gold particle
Have antibody combining site, thus prevent on T line positions coated 4- methyl methcathinone-BSA and its antibody colloidal gold
In conjunction with, in this way, T lines area occurs without lines, indicate that result is positive, no matter 4- methyl methcathinones whether there is in urine sample, and one
Red stripes all can be at quality control region (C lines), and red stripes are to determine whether there is enough measuring samples, and chromatography process is
It is no to meet normal standard, while also as the inner quality standard of kit.Generally, positive findings:In the Quality Control of kit viewing area
There are a red stripes in line.Negative findings:Respectively there are a red stripes in kit viewing area detection line and nature controlling line.It loses
Lose result:It is shown in kit viewing area detection line and the equal redfree band of nature controlling line or only a red bar occurs in detection line
Band.
It is described herein or record article, patent, patent application and every other document and can electronically obtain
The content of information include herein by reference, just as each individual publication by specific and single in full to a certain extent
Solely point out by reference.Applicant retains from any of any this article, patent, patent application or other documents
And all material and information are incorporated into.Right in the application.The all patents and publications mentioned in description of the invention is all
Indicate that these are the public technologies of this field, the present invention can use.All patents referred to herein and publication are all same
Sample is listed in bibliography, being specifically individually referenced with each publication.The present invention described here can be with
It is realized in the case where lacking any type element or a kind of limitation of multiple element or a variety of limitations, this limitation here does not have
It illustrates.Such as term "comprising" in each example here, " essence by ... form " and " by ... form " can use
Remaining 2 term of one of both replace.Here describing mode carried out by the terms and expressions mode used, and be not limited except as,
Here also without any intention any equivalent feature is eliminated to indicate these terms of this book description and explain, but can be with
Know, any suitable be altered or modified can be done in the present invention and the scope of the claims.It is appreciated that the present invention is retouched
The examples of implementation stated all are some preferred embodiments and feature, and any those of ordinary skill in the art can be according to this
It invents and does some changes and variation under the marrow of description, these changes and variation are recognized as belonging to the scope of the present invention and independence
The range that claim and appended claims are limited.
Claims (28)
1. a kind of haptens of 4- methyl methcathinone, the following general formula of structure (1):
Wherein, Z is hydrogen atom or carbonylic oxygen atom, and m is 1-4 carbon atoms.
2. the haptens according to claim, wherein 2,3 or 4 carbon atoms of m.
3. the haptens according to claim, wherein m is the linear carbon chain of 1-4 carbon atoms.
4. a kind of artificial antigen of 4- methyl methcathinone, structure is structure shown in general formula (2):
Wherein, Z is hydrogen atom or carbonylic oxygen atom, and m is 1-4 carbon atoms;P is carrier protein.
5. the antigen according to claim, carrier protein is selected from bovine serum albumin (BSA), ox gamma Globulin (BGG), ox
One kind in thyroglobulin (BTG), keyhole limpet hemocyanin (KLH) and chicken egg white (OVA).
6. a kind of method for the haptens preparing 4- methyl methcathinones, wherein this method includes intermediate methylene dioxy penta
The synthesis of ketone, wherein the synthesis of intermediate methylene dioxy pentanone starts using 4- methoxy benzonitriles as starting material.
7. according to the method described in claim 6, wherein, this method further comprises the preparation of alkyl halide lattice reagent, then with halogen
For alkane lattice reagent and 4- methoxy benzonitriles by carbonylation, halogenation and clemastine base, prepares and be connected with hydroxyl on phenyl ring
4- methyl methcathinones.
8. according to the method described in claim 7, wherein, the preparation method of alkyl halide magnesium Grignard Reagent includes:It is situated between in absolute ether
In matter, metal magnesium chips is with alkyl halide under the conditions of completely cutting off atmospheric moisture, and back flow reaction is completed under absolute ether boiling point.
9. according to the method described in claim 8, wherein, the absolute ether as medium is diethyl ether, dipropyl ether, butyl oxide, four
One or several kinds in hydrogen furans, dioxane, non-aromatics alkyl halide.
10. according to the method described in claim 8, wherein, using acyclic ether as absolute ether reaction medium.
11. according to the method described in claim 10, wherein, the acyclic ether is diethyl ether.
12. according to the method described in one of claim 7-11, wherein alkyl halide can be include alkyl chloride, bromoalkane or iodine
For the one or several kinds in alkane.
13. according to the method for claim 12, wherein halogenated alkane reagent of the bromoalkane as Grignard Reagent.
14. according to the method for claim 13, wherein the molar ratio of magnesium metal and bromoethane is 1:1-1:1.5.
15. according to the method described in one of claim 7-14, wherein 4- methoxy benzonitriles and bromoethane magnesium Grignard Reagent
(synthetic method that 1- (4- methoxyphenyls) propane -1- ketone is prepared through carbonylation includes the following steps:Under the high temperature conditions, exist
In absolute ether medium, 4- methoxy benzonitriles and bromoethane magnesium Grignard Reagent be carbonylated instead.
16. according to the method for claim 15, wherein 1- (4- methoxyphenyls) propane -1- ketone prepares 1- (4- through bromination
Methoxyphenyl) methods of -2- bromopropane-1-ketones includes the following steps:1- (4- are carried out in the solvent medium existing for the acid of Louis
Methoxyphenyl) bromination reaction of the propane -1- ketone at a.
17. according to the method for claim 16, wherein 1- (4- methoxyphenyls) -2- bromopropane-1-ketones are through methylamino
1- (4- methoxyphenyls) -2- methylamino propane -1- ketone methods are prepared to include the following steps:1- (4- methoxyphenyls) -2- bromines
Propane -1- ketone is reacted with methylamine water solution in inert organic solvents medium.
18. according to the method for claim 17, wherein 1- (4- methoxyphenyls) -2- methylamino propane -1- ketone is through acidolysis
The method for preparing 1- (4- hydroxy phenyls) -2- methylamino propane -1- ketone includes the following steps:It selects and carries methoxyl group in contraposition
Benzonitrile be carbonylation initial reaction raw material, acidolysis occurs under methoxyl group suitable temperature and suitable acid condition and generates hydroxyl
Base.
19. according to the method for claim 18, wherein protect 1- (4- hydroxy phenyls) -2- first using amido protection reagent
1- (4- hydroxy phenyls) -2- trifluoroacetyl methylamino propane -1- ketone is prepared under conditions of amido in amido propane -1- ketone.
20. according to the method for claim 19, wherein the Dun amido protecting agents are tertbutyloxycarbonyl (Boc), tablet held before the breast by officials first
The one or several kinds of oxygen carbonyl (Fmoc), trifluoroacetic anhydride and trifluoroacetic acid diethylester kind.
21. it is trifluoroacetic acid diethylester that according to the method for claim 20, methylamino, which protects reagent,.
22. according to the method for claim 20, wherein this method further includes 1- (4- hydroxy phenyls) -2- trifluoroacetyl first
Amido propane -1- ketone prepares the step of 4- methyl methcathinone haptens with carboxyl through carboxylated.
23. a kind of detection sample whether the reagent containing Cathinone or its analogue, wherein the reagent include as weigh
Profit requires the artificial antigen described in one of 4-5 that the antibody that animal obtains is immunized, or is made with the haptens described in 6-22 methods
Standby artificial antigen and antibody that immune animal obtains.
24. reagent according to claim 23, wherein the Cathinone or its analogue include:4- methyl
One kind or concentration in methcathinone, methcathinone, Cathinone, 4- methcathinones or 4- methoxy methyl Cathinones.
25. in a kind of detection sample whether the kit containing Cathinone or its analogue, wherein the kit includes
The artificial antigen of haptens, 4-5 as described in claim 1-3, or the people that is prepared with the haptens described in 6-22 methods
Work antigen.
26. kit according to claim 24, wherein the kit includes that lactation is immunized with the artificial antigen of 4-5 to move
Object and the antibody obtained.
27. kit according to claim 24, wherein the kit includes immunoassay item.
28. according to the kit described in one of claim 25-27, wherein the methyl methcathinone or its structure class
Include like object:4- methyl methcathinone, methcathinone, Cathinone, 4- methcathinones and 4- methoxy methyl Cathinones.
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