CN106749622A - A kind of 4 methyl methcathinone antigen and preparation method and its application in collaurum detection - Google Patents
A kind of 4 methyl methcathinone antigen and preparation method and its application in collaurum detection Download PDFInfo
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Abstract
The present invention relates to a kind of 4 methyl methcathinone antigen, it is characterised in that its general structure is as follows：Wherein, X=CH2, C2H4, C3H6, C4H8, C5H10.The invention further relates to the preparation method of 4 methyl methcathinone antigens and its application.Material and catalyst used in synthesis and preparation process of the invention are mill run, therefore more practicality, are suitable to industrial production and can produce economic worth；The present invention does not use chloroform, and toxicity is relatively low, with more security.
Description
Technical field
The present invention relates to technical field of immunoassay；Particularly relate to chemical synthesis process synthesis 4- methyl methcathinones
Derivative, then be applied in colloidal-gold detecting-card with high molecular weight protein coupling.
Background technology
Following background technology is used to help reader and understands the present invention, and is not construed as prior art.
4- methyl methcathinone (4-MMC), the methedrone that is otherwise known as (Mephedrone), others are called to be included,
Drone [1], MCAT [2], white magic (white magic) or mew mew (meow meow).4-MMC is in I claps tea
It was found that Cathinone artificial synthesized analog.The main mode of sucking is swallowed, and takes out food and injection etc..
Earliest 4-MMC was synthesized in nineteen twenty-nine.But until 2003, most of country did not all have for 4-MMC in the world
One clearly recognizes.4-MMC re-recognized be in 2003, at that time on a shared website user name
The anonymous chemist of " Kinetic " mentions him and attempts synthesizing 2- methyl methcathinone and 4- methyl methcathinones, and after
Description has strong happiness, and no any medicine can bring so sensation before this.At that time, 4-MMC is also
Sale and purchase that can be legal on the internet.Until 2008, law enforcement agency just recognized that 4-MMC is a kind of drugs.It is first
First in 2008, Israel represents that the transaction of 4-MMC is illegal, and later Sweden also illustrates that 4-MMC by management and control.But arrive
2010 before, and most of Europe area particularly Britain, 4-MMC is especially popular.In December, 2010, European Union announces that 4-MMC is disobeyed
Method；Announce to carry out control to it in the next year September U.S..In July, 2012, the U.S. is become a full member of prevention synthetic drug abuse bill
(SDAPA).It is now verified its almost without medical value, every country in the world has all been listed in the row of tubing products
Row.
After sucking 4-MMC, drug addict will produce：Excitement, stimulates, and increases appreciation of music, improves mood, reduces enemy
Meaning and appropriate sexual stimulus.These effects are similar to cocaine, amphetamine and head-shaking pill etc..Once there is investigation 70 to suck
The Dutchman of 4-MMC, wherein 58 people are described as being an experience for pleasant that 12 people are described as offending warp
Go through.The side effect of 4-MMC is sucked also including as follows：Pupil amplifies, and concentrated force is poor, grinds one's teeth in sleep, and eyeball focus cannot be concentrated, short-term note
Recall forfeiture, mirage, vain hope and behavior are unstable.Also have and suck person chart and reveal：Temperature changing, heart rate increases, expiratory dyspnea, food
It is intended to depressed, night sweat, anxiety, the side effect such as bigoted and depressed.
Shown to be expected, in 50-100 μ g/L, to be more than in the human blood and blood plasma for typically suck 4-MMC according to current research
100 μ g/L and intoxicating phenomenon can occur when being less than 500 μ g/L, then there is acute poisoning phenomenon during more than 500 μ g/L.In Sweden,
The ground such as Britain and the U.S., has several 4-MMC poisonings to cause the Case Report of death.
Therefore the exploitation for carrying out the related reagent of 4- methyl methcathinone Misuse detections and products thereof just turns into needs.
The content of the invention
The present invention is directly synthesized the partly anti-of the N crosslinking arms of atom upper band with methylbenzene raw material by Friedel-Crafts reaction and affine substitution
Original, then by the protein molecule for being crosslinked arm He have immunogenicity, is obtained with reactionogenicity and immunogenicity with this
Comlete antigen.In order to obtain the specific antibody for haptens, crosslinking arm will typically follow two principles：1. try not tool
Standby complicated structure, is typically preferred with hydrocarbon chain, so can as far as possible avoid producing the antibody of anti-crosslinking arm；2. to possess certain
Length, generally 1-10 atom, haptens can be so exposed to the appearance of protein, rather than quilt by preferably 1-6
Protein hides.Therefore synthesis immunizing antigen selection crosslinking agent be usually bromoacetate, bromobutyrate or bromine oneself
Acetoacetic ester etc., then reacted with the amino on protein by taking off ethyl ester, its crosslinking arm is the hydrocarbon chain of simple structure, and length is 4-8
Individual atom.
Specifically, a kind of 4- methyl methcathinone antigen that the present invention is provided, its general structure is as follows：
Wherein, X=CH2, C2H4, C3H6, C4H8, C5H10。
On the other hand, the present invention also provides a kind of method for preparing 4- methyl methcathinone antigens, wherein, with 2- bromine propionyl
Chlorine and toluene are raw material, first synthesize the derivative of 4- methyl methcathinones, then the carboxyl and macromolecular egg by being crosslinked on arm
The white covalent coupling that carries out prepares 4- methyl methcathinone antigens, and its reaction equation is as follows：
Reaction equation one：
Reaction equation two：
Wherein,
1：2- bromo propionyl chloros
2：Toluene
3：The bromo- 4- methyl phenyl ketones of 2-
4：First ammonia
5：3- bromo-propionic acids
6：3- aminomethyl propionic acid
7：3- (methyl (1- ketone -1- (p-methylphenyl) -2-) amino) propionic acid
8：4- methyl methcathinone antigens
Additionally, in preparing the method for 4- methyl methcathinone antigens, specific preparation process is as follows：
(1) under argon gas protection, anhydrous Aluminum chloride is dissolved in and adds 2- bromo propionyl chloros, stirring in anhydrous methylene chloride afterwards
Dissolving；Toluene is added, is added dropwise to complete in 30min；Solution is cooled to room temperature after flowing back 2 hours；Solution at room temperature is poured into frozen water
In, dichloromethane and hydrochloric acid are added, acutely rock rear static layering；The dichloromethane phase of lower floor is left after layering, chlorination is used
Hydrogen washing dichloromethane phase 2 times, then dried with anhydrous magnesium sulfate, filtering, revolving obtains clear oil material；Add in transparency material
Ether dissolution, is then slowly added dropwise petroleum ether to slightly muddiness, and quiescent crystallization obtains transparent crystal product 3；Wherein, 2- bromines third
The mol ratio 1 of acyl chlorides, toluene and alchlor：1：3~6；5~20ml dichloromethane is added to carry out per 10mmol alchlors molten
Solution；
(2) under argon gas protection, by dichloromethane, methylamine hydrochloride and triethylamine are slowly added dropwise bromine third after stirring
Acid, was added dropwise to complete in 2 hours；It is stirred at room temperature 8-16 hours；With 0.5M NaOH aqueous solution extractions 3 times；Rotated after extraction and removed
Solvent obtains weak yellow liquid, as product 6；Wherein, methylamine hydrochloride and bromo-propionic acid mol ratio are 1~1.2：1；Dichloromethane
Alkane, triethylamine and methylamine hydrochloride ratio are 30~80ml：0.5~1.5ml：1mmol；
(3) whole products 6 that the whole products 3 for obtaining (1) and (2) obtain are dissolved in three second of rear addition in dichloromethane
Amine, is stirred at room temperature 8-16 hours；TLC detection reactions,；After the completion of reaction, reaction solution Rotary Evaporators are spin-dried for obtaining yellow
Liquid；Yellow liquid is crossed into silicagel column, faint yellow solid as product 7 is obtained；Wherein, the ratio of product 6 and product 3 is
1mmol：1-6mmol, the ratio of dichloromethane, triethylamine and product 6 is 75~125ml：2~4ml：1g；
(4) by product 7,1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride (EDC), N- hydroxysuccinimidyl acyls
Imines (NHS) is dissolved in N,N-Dimethylformamide (DMF), and 8H is stirred at room temperature.Then instill in protein solution, be stirred overnight
Obtain product 8；Wherein, product 7, DEC and NHS ratios are 1:1.2~1.5:1.2~1.5；The DMF solution of product 7 is 50gm/
ml；Protein solution is 5mg/ml, and solvent is the phosphate buffer of pH=7.4 10mM；Product 7 and albumen quality ratio are 1g:2-
10g。
Some preferred embodiment in.High molecular weight protein be bovine serum albumin(BSA) (BSA), bovineγ-globulin (BGG) or
Chicken egg white (OVA).
The present invention also provides a kind of method of 4- methyl methcathinone collaurum detection, comprises the following steps：
S1. antibody is prepared using 4- methyl methcathinone antigen-immunized animals；
S2. collaurum is prepared with the chlorauride of reduction of sodium citrate four, then by colloid gold label to anti-4- methyl first cassie
On the antibody of ketone, gold labeling antibody is formed；
S3. gold labeling antibody is equably sprayed in label pad；
S4.4- methyl methcathinone antigens spray to the detection line on nitrocellulose filter, while in nitrocellulose filter
On control line at the corresponding immune animal of spraying antigen；
S5. by sample pad, label pad is assembled into test strips on nitrocellulose filter with blotting paper；
S6. detect.
Beneficial effect
The invention has the advantages that：
1st, the material and catalyst used in 4- methyl methcathinone antigen synthesis and preparation process of the invention are general
Logical product, therefore more practicality, are suitable to industrial production and can produce economic worth.
2nd, the present invention does not use chloroform, and toxicity is relatively low, with more security.
3rd, 4- methyl methcathinone antigen of the invention is applied to ELISA test strip, accuracy of reading and convenient and swift.
Brief description of the drawings
Fig. 1 is the general structure of 4- methyl methcathinone antigens；
Fig. 2 is the structural representation of colloidal-gold detecting-card；
Fig. 3 is colour atla schematic diagram；
Fig. 4 is 4- methyl methcathinone antigens for small molecule testing result figure；
Fig. 5 is the accelerated stability result figure of antigen；
Reference：
Sample pad 201, gold standard pad 202, nitrocellulose filter 203, blotting paper 204, kilocalorie 205, test strip 200,
Detection line (T lines) 206, control line (C lines) 207
Specific embodiment
The present invention is described in detail below in conjunction with the accompanying drawings.
Embodiment 1：The preparation of 4- methyl methcathinones antigen (Fig. 1)
1. the preparation of 4- methyl methcathinone haptens
(1) under argon gas protection, to (4g, 30mmol) anhydrous Aluminum chloride is added in reaction bulb, it is subsequently adding anhydrous dichloro
Methane 20ml, stirring dissolves it.(1.7g, 10mmol) 2- bromo propionyl chloros are added after dissolving, continues to stir 20min.Then
(0.92g, 10mmol) toluene is added, is added dropwise to complete in 30min.After being added dropwise to complete, flow back 2H, and backflow is cooled to after completing
Room temperature.Solution is poured into 40ml frozen water after cooling, and adds 20ml dichloromethane and 20ml 6N hydrochloric acid, acutely rocked
Later static layering.The dichloromethane phase of lower floor is left after layering, after washing dichloromethane phase 2 times with 1N HCL, then with nothing
Water magnesium sulfate is dried, and filtering, revolving obtains clear oil material.Transparency material is placed in crystallising dish and the dissolving that adds diethyl ether, so
After be slowly added dropwise petroleum ether to slightly muddiness, add a cover and press from both sides a piece of paper piece and be still in fume hood and crystallize, obtain 1.37g products
3 transparent crystals.Yield 60.6%
13CNMR(DMSO)δ:192.562,144.674,134.845,129.242,129.025,41.009,22.502,
20.963
ESI m/z=227 (M+1)
(2) under argon gas protection, 60ml dichloromethane is first added in 250ml flasks, is subsequently adding (1.3g, 2mmol)
Methylamine hydrochloride, 1ml triethylamines, stirs.Bromo-propionic acid (3g, 2mmol) is slowly added dropwise after the completion of stirring, was dripped in 2 hours
Add into.After completion feeds intake, it is stirred overnight at room temperature (i.e. 8-16 hours).Then 0.5M NaOH aqueous solution extractions are used 3 times, every time
All remove upper strata aqueous phase.Removing solvent is rotated after extraction and obtains product 6 (yield 73.3%), be directly used in next step reaction, no
Need purifying.
(3) product 3 (103mg, 1mmol) and product 6 (227mg, 1mmol) for obtaining first two steps are dissolved in 17ml dichloros
Methane, is subsequently adding 0.46ml triethylamines, is stirred at room temperature 8-16 hours.TLC detection reactions, after the completion of reaction, reaction solution rotation
Turn evaporimeter to be spin-dried for obtaining yellow liquid.Yellow liquid is crossed into silicagel column, 163mg faint yellow solids as final product 7 is obtained.
Yield 65.5%
13CNMR(DMSO)δ:195.564,176.029,143.454,133.809,129.075,77.657,52.699,
42.876,36.885,22.102,14.967
ESI m/z=249 (M+1)
2. product 7 (4- methyl methcathinones haptens) coupling carrier albumen
1. take 60mg products 7 and be dissolved in 2ml DMF, concentration is 30mg/ml.Configure the PBS-BSA albumen of 10mg/ml.
2. 40mg EDC, 25mg NHS are added in 2ml × 30mg/ml products 7,8H is stirred at room temperature.
3. the PBS-BAS protein solution 30ml of 1 step are taken, the product 7 for activating then is instilled, room temperature reaction overnight obtains end
Product 8.PBS 3 days, measures concentration for 6.62mg/ml.
Embodiment 2：4- methyl methcathinones haptens (product 7) is prepared using different material quantities
Compared with Example 1, the usage amount of part raw material is different, remaining step all same.
(1) under argon gas protection, anhydrous Aluminum chloride (6g, 45mmol) is dissolved in anhydrous methylene chloride (55ml) and is added afterwards
Enter 2- bromo propionyl chloros (1.72g, 10mmol), stirring and dissolving；Toluene (0.92g, 10mmol) is added, is added dropwise to complete in 30min；
Solution is cooled to room temperature after flowing back 2 hours；Solution at room temperature is poured into frozen water, dichloromethane and hydrochloric acid is added, after acutely rocking
Static layering；The dichloromethane phase of lower floor is left after layering, dichloromethane phase is washed with hydrogen chloride 2 times, then use anhydrous slufuric acid
Magnesium is dried, and filtering, revolving obtains clear oil material；Add diethyl ether dissolving in transparency material, is then slowly added dropwise petroleum ether to slightly
There is muddiness, quiescent crystallization obtains 1.50g transparent crystals product 3 (yield 64.5%)；
(2) under argon gas protection, by dichloromethane (115ml), methylamine hydrochloride (1.5g, 2.3mmol) and triethylamine
(2.8ml) is slowly added dropwise bromo-propionic acid (3g, 2mmol) after stirring, be added dropwise to complete in 2 hours；It is stirred at room temperature 8-16 hours；
Then 0.5M NaOH aqueous solution extractions are used 3 times, upper strata aqueous phase is removed every time.Rotated after extraction and remove solvent to obtain 1.60g light
Yellow liquid, as product 6 (yield 77.7%) are directly used in next step reaction, it is not necessary to purify.
(3) product 3 (412mg, 4mmol) and product 6 (227mg, 1mmol) that will be obtained are dissolved in dichloromethane
Triethylamine (0.68ml) is added after in (22.7ml), is stirred at room temperature 8-16 hours；TLC detection reactions；After the completion of reaction, will react
Liquid Rotary Evaporators are spin-dried for obtaining yellow liquid；Yellow liquid is crossed into silicagel column, 152mg faint yellow solids is obtained and is finally
Product 7 (yield 61.1%)；
Embodiment 3：Another different material quantity prepares 4- methyl methcathinones haptens (product 7)
Compared with Example 1, the usage amount of part raw material is different, remaining step all same.
(1) under argon gas protection, after anhydrous Aluminum chloride (8g, 60mmol) is dissolved in anhydrous methylene chloride (120ml)
Add 2- bromo propionyl chloros (1.72g, 10mmol), stirring and dissolving；Toluene (0.92g, 10mmol) is added, is dripped in 30min
Into；Solution is cooled to room temperature after flowing back 2 hours；Solution at room temperature is poured into frozen water, dichloromethane and hydrochloric acid is added, acutely rocked
Static layering afterwards；The dichloromethane phase of lower floor is left after layering, dichloromethane phase is washed with hydrogen chloride 2 times, then use anhydrous sulphur
Sour magnesium is dried, and filtering, revolving obtains clear oil material；Add diethyl ether dissolving in transparency material, is then slowly added dropwise petroleum ether extremely
Slightly muddy, quiescent crystallization obtains 1.2g transparent crystals product 3 (yield 53.1%)
(2) under argon gas protection, by dichloromethane (190ml), methylamine hydrochloride (1.6g, 2.4mmol) and triethylamine
(3.6ml) is slowly added dropwise bromo-propionic acid (3g, 2mmol) after stirring, be added dropwise to complete in 2 hours；It is stirred at room temperature 8-16 hours；
Then 0.5M NaOH aqueous solution extractions are used 3 times, upper strata aqueous phase is removed every time.Rotated after extraction and remove solvent to obtain 1.49g light
Yellow liquid, as product 6 (yield 72.4%) are directly used in next step reaction, it is not necessary to purify.
(3) product 3 (620mg, 6mmol) and product 6 (227mg, 1mmol) that will be obtained are dissolved in dichloromethane (28ml)
In after add triethylamine (0.9ml), be stirred at room temperature 8-16 hours；TLC detection reactions；After the completion of reaction, by reaction solution rotation
Evaporimeter is spin-dried for obtaining yellow liquid；Yellow liquid is crossed into silicagel column, 140mg faint yellow solids, as final product 7 is obtained
(yield 56.3%)；
Embodiment 4：Application of the antigen in colloidal gold colloidal gold detection test paper strip
It is anti-using 4- methyl methcathinone in preparing antibody, this experiment using 4- methyl methcathinone antigen-immunized animals
Former immunized mice prepares antibody.
Collaurum of the diameter in 20-40nm is prepared with the chlorauride of reduction of sodium citrate four, then by colloid gold label to anti-
The antibody of 4- methyl methcathinones represents the concentration of gold labeling antibody with the light absorption value (OD) of λ max to upper；
Gold labeling antibody is loaded on golden mark machine, the gold labeling antibody of certain OD values is equably sprayed into polyester film (gold standard pad
202) 37 degree of baking ovens are put on, after spray is good and dry 8h；Suitable size is cut into, is put into the aluminium foil bag equipped with drying machine, room temperature is deposited
Put standby；
By 4- methyl methcathinone antigen diluents to suitable concn, with corresponding T on a film machine point to nitrocellulose filter 203
Line position 206, the corresponding position of C lines 207 (is used to detect the inspection plus corresponding mouse antigen equally on nitrocellulose filter 203
Whether effective survey card), 37 degree of drying 12h；
Sample pad 201--- glass fibres by buffer solution and surface-active treatment, 37 degree drying 10h it is standby；
By sample pad 201, gold standard pad 202, nitrocellulose filter 203, blotting paper 204, kilocalorie 205 is assembled into collaurum inspection
Test paper card 200, is shown in Fig. 2；
The small molecule solution of respective concentration is prepared, is added in sample pad 201, result is read after 5min；
Requirement of the product to the Cutoff values of 4- methyl methcathinones is 300ng/ml, i.e., -50% is 150ng/ml ,+
50% is 450ng/ml.
Remarks 1：All small molecules to be measured are dissolved in negative urine；
Remarks 2：Standard color card:The ratio color range of standard color card between 0-10, i.e. G1-G10, color from scratch, face
Color depth is incremented by successively, as shown in Figure 3.
Remarks 3：Examination criteria：The detection T lines 206 of test strip are contrasted with standard color card：
Line depth represents negative when being more than G8；
In the case of -50%cutoff, line depth ﹥ G4 are qualified；
Line depth ﹤ G3.5 are qualified in the case of+50%cutoff
Table one：Three kinds of antigens are shown in Fig. 4 to the detection sensitivity result of 4- methyl methcathinone small molecules, from left to right according to
Secondary is feminine gender, -50% ,+50%
Table two:Accelerated stability (the detection card that will be assembled 55 DEG C dry 72 hours) result of antigen, is shown in Fig. 5, from a left side to
The right side is followed successively by feminine gender, and -50% ,+50%
From table one, table two：Product meets QC standards, and heat endurance is good, and detection antigen is qualified.
Claims (5)
1. a kind of 4- methyl methcathinone antigen, it is characterised in that its general structure is as follows：
Wherein, X=CH2, C2H4, C3H6, C4H8, C5H10。
2. a kind of method for preparing 4- methyl methcathinone antigen as claimed in claim 1, it is characterised in that with 2- bromine propionyl
Chlorine and toluene are raw material, first synthesize the derivative of 4- methyl methcathinones, then the carboxyl and macromolecular egg by being crosslinked on arm
The white covalent coupling that carries out prepares 4- methyl methcathinone antigens, and its reaction equation is as follows：
Reaction equation one：
Reaction equation two：
Wherein,
1：2- bromo propionyl chloros
2：Toluene
3：The bromo- 4- methyl phenyl ketones of 2-
4：First ammonia
5：3- bromo-propionic acids
6：3- aminomethyl propionic acid
7：3- (methyl (1- ketone -1- (p-methylphenyl) -2-) amino) propionic acid
8：4- methyl methcathinone antigens.
3. a kind of method for preparing 4- methyl methcathinone antigen as claimed in claim 1, it is characterised in that specific to prepare step
It is rapid as follows：
(1) under argon gas protection, anhydrous Aluminum chloride is dissolved in anhydrous methylene chloride and adds 2- bromo propionyl chloros afterwards, stirred molten
Solution；Toluene is added, is added dropwise to complete in 30min；Solution is cooled to room temperature after flowing back 2 hours；Solution at room temperature is poured into frozen water,
Dichloromethane and hydrochloric acid are added, rear static layering is acutely rocked；The dichloromethane phase of lower floor is left after layering, is washed with hydrogen chloride
Wash dichloromethane phase 2 times, then dried with anhydrous magnesium sulfate, filtering, revolving obtains clear oil material；Added diethyl ether in transparency material
Dissolving, is then slowly added dropwise petroleum ether to slightly muddiness, and quiescent crystallization obtains transparent crystal product 3；Wherein, 2- bromo propionyl chloros,
The mol ratio 1 of toluene and alchlor：1：3~6；5~20ml dichloromethane is added to be dissolved per 10mmol alchlors；
(2) under argon gas protection, by dichloromethane, methylamine hydrochloride and triethylamine are slowly added dropwise bromo-propionic acid after stirring, 2
It is added dropwise to complete in hour；It is stirred at room temperature 8-16 hours；With 0.5M NaOH aqueous solution extractions 3 times；Removing solvent is rotated after extraction to obtain
To weak yellow liquid, as product 6；Wherein, methylamine hydrochloride and bromo-propionic acid mol ratio are 1~1.2：1；Dichloromethane, three second
Amine and methylamine hydrochloride ratio are 30~80ml：0.5~1.5ml：1mmol；
(3) whole products 6 that the whole products 3 for obtaining (1) and (2) obtain are dissolved in rear addition triethylamine in dichloromethane,
It is stirred at room temperature 8-16 hours；TLC detection reactions,；After the completion of reaction, reaction solution Rotary Evaporators are spin-dried for obtain yellow liquid
Body；Yellow liquid is crossed into silicagel column, faint yellow solid as product 7 is obtained；Wherein, the ratio of product 6 and product 3 is 1mmol：
1-6mmol, the ratio of dichloromethane, triethylamine and product 6 is 75~125ml：2~4ml：1g；
(4) by product 7,1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride (EDC), N-hydroxy-succinamide
(NHS) it is dissolved in N,N-Dimethylformamide (DMF), 8H is stirred at room temperature.Then instill in protein solution, be stirred overnight and obtain
Product 8；Wherein, product 7, DEC and NHS ratios are 1:1.2~1.5:1.2~1.5；The DMF solution of product 7 is 50gm/ml；Egg
White solution is 5mg/ml, and solvent is the phosphate buffer of pH=7.4 10mM；Product 7 and albumen quality ratio are 1g:2-10g.
4. according to the 4- methyl methcathinone antigens that one of claim 1-3 is described, it is characterised in that high molecular weight protein is ox blood
Pure albumen (BSA), bovineγ-globulin (BGG) or chicken egg white (OVA).
5. a kind of method that 4- methyl methcathinone collaurum is detected, it is characterised in that comprise the following steps：
S1. antibody is prepared using 4- methyl methcathinone antigen-immunized animals；
S2. collaurum is prepared with the chlorauride of reduction of sodium citrate four, then by colloid gold label to anti-4- methyl methcathinone
On antibody, gold labeling antibody is formed；
S3. gold labeling antibody is equably sprayed in label pad；
S4.4- methyl methcathinone antigens spray to the detection line on nitrocellulose filter, while on nitrocellulose filter
The antigen of corresponding immune animal is sprayed at control line；
S5. by sample pad, label pad is assembled into test strips on nitrocellulose filter with blotting paper；
S6. detect.
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