CN108732345A - The Test paper and preparation method of mitragynine and its derivative and metabolin in a kind of detection human urine - Google Patents
The Test paper and preparation method of mitragynine and its derivative and metabolin in a kind of detection human urine Download PDFInfo
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- CN108732345A CN108732345A CN201810347872.3A CN201810347872A CN108732345A CN 108732345 A CN108732345 A CN 108732345A CN 201810347872 A CN201810347872 A CN 201810347872A CN 108732345 A CN108732345 A CN 108732345A
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- 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
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- 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
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Abstract
The present invention relates to the Test papers and preparation method of mitragynine and its derivative and metabolin in a kind of detection human urine, belong to technical field of biological.The Test paper of mitragynine and its derivative and metabolin in the detection human urine of the present invention, including sample pad, gold-labelled pad, NC films, blotting paper, bottom plate, the sample pad, gold-labelled pad, NC films and blotting paper are pasted onto on bottom plate successively, it is coated with mitragynine antibody-colloidal gold marker and rabbit igg colloid gold label object in the gold-labelled pad, is coated with mitragynine-BSA haptens, goat anti-rabbit igg on the NC films respectively.The Test paper of the present invention, using urine sample pad complete urine acquisition and conveying, using colloidal gold technique and immunochromatography principle preparation test strip, the two is implemented in combination with one-step method and quickly detects mitragynine (Mitragynine) in urine and its derivative and metabolin.
Description
Technical field
The present invention relates to the Test papers and preparation of mitragynine and its derivative and metabolin in a kind of detection human urine
Method belongs to technical field of biological.
Background technology
Card pain (kratom), Rubiaceae, large-scale greenery arbor are distributed in Southeast Asia.Traditionally, fresh or dry
Tea is chewed or be made to kratom leaves, but seldom is used to suck.The kratom of low dosage has excitatory effects, can eliminate
Work long hours fatigue；When high dose, then there is calmness-anesthetic effect.It also can be by the replacement as traditional drugs and opium
Product.Research is found after taking several cured leafs, and salubrious effect and pleasure, and sustainable one to one can be experienced in 10 minutes
Half an hour.Although the person's of swallowing ability to work, alertness, sociability etc. increased, and the weightlessness generated therewith is tired, just
Secret and cheek pigmentation is then its significant side effect.It is found in human clinical trial, takes orally the cap spar of 50 milligrams of dosage
Alkali (the important spiritual active constituent of kratom) can generate locomotor stimulatory, and the thing followed is dizziness, and sports coordination is lost (grand
The front test of Burger) and four limbs and face tremble.Continue to increase mitragynine to being equivalent to 10-25 grams of kratom cured leaf
Contained dosage, subject may initially show as perspiring, dizzy, nausea and have the fidgets, but these effects will soon be by
Calm, glad and fantasy experience is replaced, and the duration is six hours, and their pupil has showing for diminution
As.Kratom, which is used for a long time, can generate dependence, may occur in which weakness, and drowsiness, anxiety is had the fidgets, runny nose, myalgia, and nausea goes out
Sweat, myalgia, four limbs are stiff, tremble and sleep disturbance and illusion.But the withrawal symptom of the mankind is relatively light, generally one
It can be reduced in week.
From card pain tree each position detach phytochemicals include the relevant alkaloid of more than 40 kinds structures and
Several flavonoids, terpene saponin(e, polyphenol and various glucosides.Main psychoactive compositions are mitragynine (I institutes of formula among these
Show) and 7- hydroxyls mitragynine (shown in formula II).Content of the two in blade is influenced by varying environment and kind.In Thailand
In state's kind, mitragynine rich content the 66% of total alkaloid (up to), and to be then accessory constituent account for 7- hydroxyls mitragynine
The 2% of total alkaloid content).In Malay kratom kinds, the concentration of mitragynine is relatively low (to account for total alkaloid
12%).
In Thailand, " national household survey " provides the information of state's drug abuse popularity.Investigation in 2007 shows,
In 26633 12-65 Sui interviewee, kratom and 1 year in the past, a month ratio difference using kratom is used for a long time
It is 2.32%, 0.81% and 0.57%.In addition to the kratom crowd of long-time service, other two group's ratio is apparently higher than hemp,
This makes kratom become the most popular illicit drug of the state.In Thailand, 13 to 16 in 2002,2003 and 2004
The ratio that kratom is used for a long time in year high school student (n=8708-12148) increases to 9.43% by 3.97%, and hemp sucks ratio
Example rises to 6.75% from 4.44%, and then falls to 2.32% by 2.79% using the ratio of amphetamine.The past 1 year and one
A month kratom sucks crowd's ratio and similar trend also occurs.
It can be detected in urine through metabolin mitragynine after body intake kratom.Cap column in domestic market
It is significant to develop a kind of detection reagent detecting mitragynine in urine also in the blank phase for the wooden alkali detection.
Invention content
The purpose of the present invention is to provide the detections of mitragynine and its derivative and metabolin in a kind of detection human urine
Test paper, can one-step method realize human urine in mitragynine and its derivative and metabolin quick detection.
Second object of the present invention is to provide mitragynine and its derivative and generation in a kind of above-mentioned detection human urine
Thank to the preparation method of the Test paper of object.
Third object of the present invention is to provide mitragynine and its derivative and metabolin in a kind of detection human urine
Detection kit.
In order to achieve the above object, the technical scheme is that：
The Test paper of mitragynine and its derivative and metabolin in a kind of detection human urine, including sample pad, Jin Biao
Pad, NC films, blotting paper, bottom plate, the sample pad, gold-labelled pad, NC films and blotting paper are pasted onto on bottom plate successively, the gold-labelled pad
On be coated with mitragynine antibody-colloidal gold marker and rabbit igg colloid gold label object, be coated with cap column respectively on the NC films
Wooden alkali-BSA the haptens, goat anti-rabbit igg.
Above-mentioned bottom plate is PVC board.
The derivative of above-mentioned mitragynine includes 7- hydroxyl mitragynines.
The Test paper of mitragynine and its derivative and metabolin in the detection human urine of the present invention is pressed down using competitiveness
Colloidal gold immunochromatography technique processed marks analyte detection mitragynine and its derivative and generation using mitragynine antibody-colloidal gold
Thank to object, when the mitragynine concentration contained in sample to be tested is prescribed a time limit equal to or higher than lowest detection, mitragynine and colloidal gold mark
The specific antibody of note combines, and is moved forward along nitrocellulose membrane under chromatography effect, inhibits specific antibody and detection zone
(areas T) pre-coated mitragynine-BSA haptens combines, and finally can not form macroscopic red response line in the areas T；Phase
Instead, when prescribing a time limit less than lowest detection without mitragynine or its concentration in sample to be tested, mitragynine antibody-colloidal gold marks
Object can be combined with pre-coated mitragynine-BSA haptens, and form naked eyes red color visible response line in the areas T.The depth of color
There is negative correlation with the content of mitragynine in sample, mitragynine concentration is higher in sample, and T lines (detection line) color is more shallow.
Quality control region (C) is coated with goat anti-rabbit igg polyclonal antibody, no matter whether there is mitragynine in sample, rabbit igg can be more with the areas C
Anti-reflective should form red response line (nature controlling line).
The Test paper of mitragynine and its derivative and metabolin in the detection human urine of the present invention, using urine sample
The acquisition and conveying of pad completion urine prepare test strip using colloidal gold technique and immunochromatography principle, and the two is combined
Realize that one-step method quickly detects the mitragynine in urine and its derivative and metabolin.
The Test paper of the present invention, can quickly detect mitragynine and its derivative and metabolism in human urine with one-step method
Outturn sample pad need to only be inserted into urine when the Test paper detects, place 15-30 seconds by object, until the visible urine sample of naked eyes
Climb up detection zone, you can take out, start to detect after waiting for five minutes so that entire detection process is more convenient, without considering not
The difference brought with detection people.The Test paper of mitragynine and its derivative and metabolin in the detection human urine of the present invention,
Have many advantages, such as convenient, safety and sanitation of detection, noninvasive painless.
The Test paper of the present invention can be widely used for law enforcement agency and try the mitragynine primary dcreening operation that doubtful drug addict carries out
It tests, it can also be used to mitragynine Primary Screening Test when joining the army.
Mitragynine-BSA the haptens is made by the preparation method included the following steps：
A) mitragynine and succinic anhydride are dissolved in dichloro methanol solution mixing, are cooled to 0 DEG C, tri-chlorination is then added
Aluminium reacts 2h, obtains reaction solution；Gained reaction solution is added dropwise in cryosel acid solution, is filtered, precipitation is collected, washs, is dry, i.e.,
Obtain mitragynine hemisuccinate derivative；The mass ratio of the mitragynine and succinic anhydride is 2.5:1；
B) the mitragynine hemisuccinate derivative obtained by step a) is dissolved in DMF, then be added 2-morpholine ethane sulfonic acid,
N- hydroxy thiosuccinimides react at room temperature 2 hours, obtain the mitragynine of activation, the mitragynine of activation is added to BSA
In solution, 4 DEG C of reactions overnight, are dialysed with PBS solution later to obtain the final product；The mitragynine hemisuccinate derivative, 2- morpholines
Ethanesulfonic acid, N- hydroxy thiosuccinimides mass ratio be 10:10:5.
The mass ratio of mitragynine and alchlor is 5 in step a):1.
A concentration of 10~20mg/ml of BSA solution, volume 2ml in above-mentioned steps b).
The mitragynine antibody-colloidal gold marker is made by the preparation method included the following steps：By mitragynine
Antibody is added in colloidal gold solution, stirring, and the closing of BSA solution is added later, is then centrifuged for, and discards supernatant liquid, precipitation buffering
Liquid be resuspended to get.
The mitragynine antibody is made by the preparation method included the following steps：It is injected with mitragynine-BSA haptens
Immune mouse, largely screened by cell fusion and to monoclonal cell to get.
It is above-mentioned that mitragynine antibody is added in colloidal gold solution to a concentration of 4~12 μ g/ml for making mitragynine antibody.
The mass fraction of above-mentioned addition BSA solution closing, BSA solution is 10%, off-period 30min.
Above-mentioned centrifugation is to be centrifuged 30~45 minutes with the rotating speed of 12000~15000r/min.
The buffer solution is composed of the following components：The borate of a concentration of 5-10mmol/L, mass fraction 0.1-0.3%
Tween80；The pH of the buffer solution is 8.0.
Above-mentioned mitragynine antibody-colloidal gold marker is saved backup in 4 DEG C.
Above-mentioned rabbit igg colloid gold label object is made by the preparation method included the following steps：It is molten that colloidal gold is added in rabbit igg
In liquid, stirring is added the closing of BSA solution, is then centrifuged for, discards supernatant liquid later, precipitation buffer solution be resuspended to get.
It is above-mentioned that rabbit igg is added in colloidal gold solution to a concentration of 4~12 μ g/ml for making rabbit igg.
The mass fraction of above-mentioned addition BSA solution closing, BSA solution is 10%, off-period 30min.
Above-mentioned centrifugation is to be centrifuged 30~45 minutes with the rotating speed of 12000~15000r/min.
The buffer solution is composed of the following components：The borate of a concentration of 5-10mmol/L, mass fraction 0.1-0.3%
Tween80；The pH of the buffer solution is 8.0.
Above-mentioned colloidal gold solution is made by the preparation method included the following steps：1L ultra-pure waters are heated to boiling, to boiling water
The middle chlorauric acid solution that 1ml mass fractions are added and are 10%, rapidly joins the citric acid that 1.2~1.5ml mass fractions are 10%
Three sodium solutions, stir and evenly mix；Solution boiling is kept, is to stop heating until liquid is in claret, mistake is supplied after being cooled to room temperature
Water.
The preparation method of the Test paper of mitragynine and its derivative and metabolin in above-mentioned detection human urine, including with
Lower step：
1) mitragynine antibody-colloidal gold marker and rabbit igg colloid gold label object is used to impregnate glass fibre, Zhi Hougan
It is dry, obtain gold-labelled pad；
2) mitragynine-BSA haptens, goat anti-rabbit igg are coated with respectively on NC films, it is spare after dry；
3) sample pad, gold-labelled pad, NC films, blotting paper are pasted on bottom plate successively to obtain the final product.
The mitragynine antibody-colloidal gold marker is made by the preparation method included the following steps：By mitragynine
Antibody is added in colloidal gold solution, stirring, and the closing of BSA solution is added later, is then centrifuged for, and discards supernatant liquid, precipitation buffering
Liquid be resuspended to get.
The mitragynine antibody is made by the preparation method included the following steps：It is injected with mitragynine-BSA haptens
Immune mouse, largely screened by cell fusion and to monoclonal cell to get.
Above-mentioned centrifugation is to be centrifuged 30~45 minutes with the rotating speed of 12000~15000r/min.
The buffer solution is composed of the following components：The borate of a concentration of 5-10mmol/L, mass fraction 0.1-0.3%
Tween80；The pH of the buffer solution is 8.0.
It is above-mentioned that mitragynine antibody is added in colloidal gold solution to a concentration of 4~12 μ g/ml for making mitragynine antibody.
The mass fraction of above-mentioned addition BSA solution closing, BSA solution is 10%, off-period 30min.
Above-mentioned colloidal gold solution is made by the preparation method included the following steps：1L ultra-pure waters are heated to boiling, to boiling water
The middle chlorauric acid solution that 1ml mass fractions are added and are 10%, it is molten to rapidly join the trisodium citrate that 1.5ml mass fractions are 10%
Liquid stirs and evenly mixs；Solution boiling is kept, is to stop heating until liquid is in claret, is cooled to room temperature.
A concentration of 0.2~1.5mg/ml of mitragynine-BSA haptens in step 2), the goat anti-rabbit igg it is a concentration of
0.5~1.0mg/ml.
Above-mentioned mitragynine-BSA haptens is made by the preparation method included the following steps：
A) mitragynine and succinic anhydride are dissolved in dichloro methanol solution mixing, are cooled to 0 DEG C, tri-chlorination is then added
Aluminium reacts 2h, obtains reaction solution；Gained reaction solution is added dropwise in cryosel acid solution, is filtered, precipitation is collected, washs, is dry, i.e.,
Obtain mitragynine hemisuccinate derivative；The mass ratio of the mitragynine and succinic anhydride is 2.5:1；
B) the mitragynine hemisuccinate derivative obtained by step a) is dissolved in DMF, then be added 2-morpholine ethane sulfonic acid,
N- hydroxy thiosuccinimides react at room temperature 2 hours, obtain the mitragynine of activation, the mitragynine of activation is added to BSA
In solution, 4 DEG C of reactions overnight, are dialysed with PBS solution later to obtain the final product；The mitragynine hemisuccinate derivative, 2- morpholines
Ethanesulfonic acid, N- hydroxy thiosuccinimides mass ratio be 10:10:5.
The mass ratio of mitragynine and alchlor is 5 in step a):1.
A concentration of 10~20mg/ml of BSA solution in above-mentioned steps b).The volume of the BSA solution is 2ml.
Sample pad, gold-labelled pad, NC films, blotting paper are pasted on bottom plate successively in step 3), sample pad and gold-labelled pad it
Between, between gold-labelled pad and NC films, be overlapped 1~2mm between NC films and blotting paper, 3.8mm cuts out item after compression.
The detection kit of mitragynine and its derivative and metabolin in a kind of detection human urine, including get stuck with it is above-mentioned
Test paper.
The detection kit of mitragynine and its derivative and metabolin in above-mentioned detection human urine, Test paper is loaded on
In getting stuck to get.
Described get stuck is got stuck for plastics.
Described get stuck can be that plastics commonly used in the art in the prior art get stuck.
The Test paper of mitragynine and its derivative and metabolin in the detection human urine of the present invention has following excellent
Point：
1) safe hurtless measure：Operator only needs urine sample pad being inserted into tested personnel's urine specimen, will not cause to hinder
Mouthful, it avoids infection；
2) convenient and efficient：Urine specimen transports to gold-labelled pad by urine sample dig pass, is not necessarily to other steps, is influenced on result
Minimum realizes mitragynine and its derivative and metabolin in fast and easy detection urine；
3) efficiently and accurately, in sample mitragynine and in the gold-labelled pad of the antigenic competition combination test strip on NC films it is special
Heterogenetic antibody improves detection efficiency, ensure that sensitivity and specificity.
The preparation method of the Test paper of mitragynine and its derivative and metabolin, letter in the detection human urine of the present invention
It is single convenient, it is at low cost, it is suitble to mass production.
Specific implementation mode
Embodiment 1
The Test paper of mitragynine and its derivative and metabolin in the detection human urine of the present embodiment, including sample pad,
Gold-labelled pad, NC films, blotting paper, PVC board；The sample pad, gold-labelled pad, NC films, blotting paper are pasted in PVC board successively；It is above-mentioned
It is coated with mitragynine antibody-colloidal gold marker, rabbit igg colloid gold label object in gold-labelled pad, is coated with respectively on above-mentioned NC films
There are mitragynine-BSA haptens (as T lines), goat anti-rabbit igg (as C lines).
The preparation method of the Test paper of mitragynine and its derivative and metabolin in the detection human urine of the present embodiment,
Include the following steps：
1) mitragynine antibody-colloidal gold marker and rabbit igg colloid gold label object is used to impregnate glass fibre, Zhi Hougan
It is dry, obtain gold-labelled pad；
2) mitragynine-BSA haptens (a concentration of 0.8mg/ml) is coated with respectively on NC films as T lines, goat anti-rabbit igg
(concentration 0.5mg/ml) is used as C lines, spare after dry；
3) gold-labelled pad in sample pad, step 1), the NC films in step 2), blotting paper are pasted on successively in PVC board, sample
Between product pad and gold-labelled pad, between gold-labelled pad and NC films, be overlapped 2mm between NC films and blotting paper, 3.8mm cuts out item after compression,
To obtain the final product.
Above-mentioned steps 1) in mitragynine antibody-colloidal gold marker preparation, include the following steps：
A) prepared by colloidal gold：1L ultra-pure waters are heated to boiling, the gold chloride that 1ml mass fractions are 10% is added into boiling water
Solution rapidly joins the citric acid three sodium solution that 1.5ml mass fractions are 10%, stirs and evenly mixs；Solution boiling is kept, until liquid
Body stops heating in claret, supplies dehydration after being cooled to room temperature, obtains colloidal gold solution；
B) preparation of antibody-colloidal gold marker：The above-mentioned colloidal gold solution for taking certain volume, is slowly added to mitragynine
Antibody makes its final concentration of 4 μ g/ml, after continuing stirring 30 minutes, the BSA solution that mass fraction is 10% is added and closes 30 points
Clock is centrifuged 30 minutes with the rotating speed of 12000r/min, discards supernatant liquid, precipitation is suspended with buffer solution, and 4 DEG C save backup；It is above-mentioned
Buffer solution is composed of the following components：The borate of a concentration of 5mmol/L, the Tween 80 that mass fraction is 0.1%, buffer solution
PH is 8.0.
Above-mentioned mitragynine antibody is made by the preparation method included the following steps：Mitragynine-BSA haptens is injected
Enter mouse, largely screened by cell fusion and to monoclonal cell to get.
Above-mentioned rabbit igg colloid gold label object is made by the preparation method included the following steps：Take the colloidal gold of certain volume
Solution is slowly added to rabbit igg and is added in colloidal gold solution, makes its final concentration of 4 μ g/ml, and after continuing stirring 30 minutes, matter is added
It measures the BSA solution that score is 10% to close 30 minutes, is centrifuged 30 minutes with the rotating speed of 12000r/min, discard supernatant liquid, precipitated
It is suspended with buffer solution, 4 DEG C save backup；Above-mentioned buffer solution is composed of the following components：The borate of a concentration of 5mmol/L, quality
The pH of the Tween 80 that score is 0.1%, buffer solution are 8.0.
Above-mentioned mitragynine-BSA haptens is made by the preparation method included the following steps：
A) 100mg is emitted into spar alkali and 40mg succinic anhydrides is dissolved in dichloro methanol solution mixing, be cooled to 0 DEG C, then added
Enter 20mg alchlors, stirs and evenly mixs reaction 2 hours, gained reaction solution is slowly added dropwise in ice HCl solution, it is heavy to be collected by filtration
It forms sediment, washing, drying are to get mitragynine hemisuccinate derivative；
B) preparation of mitragynine-BSA haptens：Mitragynine hemisuccinate derivative obtained by step a) is dissolved in
Then 2-morpholine ethane sulfonic acid, N- hydroxy thiosuccinimides is added in DMF, react at room temperature 2 hours, obtain the mitragynine of activation,
The mitragynine of activation is added in the BSA solution of a concentration of 20mg/ml of 2ml, 4 DEG C of reactions are overnight, saturating with PBS solution later
It analyses to obtain the final product；The mass ratio of the mitragynine hemisuccinate derivative, 2-morpholine ethane sulfonic acid, N- hydroxy thiosuccinimides
It is 10:10:5.
The detection kit of mitragynine in the detection human urine of the present embodiment, including plastics get stuck and are tried with above-mentioned detection
Paper.During Test paper is got stuck loaded on plastics.
Embodiment 2
The Test paper of mitragynine and its derivative and metabolin in the detection human urine of the present embodiment, including sample
Pad, gold-labelled pad, NC films, blotting paper, PVC board；The sample pad, gold-labelled pad, NC films, blotting paper are pasted in PVC board successively；On
It states and is coated with mitragynine antibody-colloidal gold marker, rabbit igg colloid gold label object in gold-labelled pad, wrapped respectively on above-mentioned NC films
There are mitragynine-BSA haptens (as the areas T), goat anti-rabbit igg (as the areas C).
The preparation method of the Test paper of mitragynine and its derivative and metabolin in the detection human urine of the present embodiment,
Include the following steps：
1) mitragynine antibody-colloidal gold marker and rabbit igg colloid gold label object is used to impregnate glass fibre, Zhi Hougan
It is dry, obtain gold-labelled pad；
2) mitragynine-BSA haptens (a concentration of 0.5mg/ml) is coated with respectively on NC films as T lines, goat anti-rabbit igg
(concentration 0.5mg/ml) is used as C lines, spare after dry；
3) gold-labelled pad in sample pad, step 1), the NC films in step 2), blotting paper are pasted on successively in PVC board, sample
Between product pad and gold-labelled pad, between gold-labelled pad and NC films, be overlapped 1mm between NC films and blotting paper, 3.8mm cuts out item after compression,
To obtain the final product.
Above-mentioned steps 1) in mitragynine antibody-colloidal gold marker preparation, include the following steps：
A) prepared by colloidal gold：1L ultra-pure waters are heated to boiling, the gold chloride that 1ml mass fractions are 10% is added into boiling water
Solution rapidly joins the citric acid three sodium solution that 1.5ml mass fractions are 10%, stirs and evenly mixs；Solution boiling is kept, until liquid
Body stops heating in claret, supplies dehydration after being cooled to room temperature, obtains colloidal gold solution；
B) preparation of antibody-colloidal gold marker：The above-mentioned colloidal gold solution for taking certain volume, is slowly added to mitragynine
Antibody makes its final concentration of 10 μ g/ml, after continuing stirring 30 minutes, the BSA solution that mass fraction is 10% is added and closes 30 points
Clock is centrifuged 40 minutes with the rotating speed of 15000r/min, discards supernatant liquid, precipitation is suspended with buffer solution, and 4 DEG C save backup；It is above-mentioned
Buffer solution is composed of the following components：The borate of a concentration of 8mmol/L, the Tween 80 that mass fraction is 0.2%；Buffer solution
PH is 8.0.
Above-mentioned rabbit igg colloid gold label object is made by the preparation method included the following steps：Take the colloidal gold of certain volume
Solution is slowly added to rabbit igg and is added in colloidal gold solution, makes its final concentration of 10 μ g/ml, after continuing stirring 30 minutes, is added
The BSA solution that mass fraction is 10% is closed 30 minutes, is centrifuged 30 minutes with the rotating speed of 15000r/min, is discarded supernatant liquid, is sunk
Shallow lake is suspended with buffer solution, and 4 DEG C save backup；Above-mentioned buffer solution is composed of the following components：The borate of a concentration of 8mmol/L, matter
Measure the Tween 80 that score is 0.2%；The pH of buffer solution is 8.0.
The preparation method is the same as that of Example 1 for above-mentioned mitragynine antibody and mitragynine-BSA haptens.
The detection kit of mitragynine and its derivative and metabolin in the detection human urine of the present embodiment, including plastics
It gets stuck and above-mentioned Test paper.During Test paper is got stuck loaded on plastics.
Embodiment 3
The Test paper of mitragynine and its derivative and metabolin in the detection human urine of the present embodiment, including sample
Pad, gold-labelled pad, NC films, blotting paper, PVC board；The sample pad, gold-labelled pad, NC films, blotting paper are pasted in PVC board successively；On
It states and is coated with mitragynine antibody-colloidal gold marker, rabbit igg colloid gold label object in gold-labelled pad, wrapped respectively on above-mentioned NC films
There are mitragynine-BSA haptens (as the areas T), goat anti-rabbit igg (as the areas C).
The preparation method of the Test paper of mitragynine, includes the following steps in the detection human urine of the present embodiment：
1) mitragynine antibody-colloidal gold marker and rabbit igg colloid gold label object are used, glass fibre, Zhi Hougan are impregnated
It is dry, obtain gold-labelled pad；
2) mitragynine-BSA haptens (a concentration of 0.3mg/ml) is coated with respectively on NC films as T lines, goat anti-rabbit igg
(concentration 1.0mg/ml) is used as C lines, spare after dry；
3) gold-labelled pad in sample pad, step 1), the NC films in step 2), blotting paper are pasted on successively in PVC board, sample
Between product pad and gold-labelled pad, between gold-labelled pad and NC films, be overlapped 2mm between NC films and blotting paper, 3.8mm cuts out item after compression,
To obtain the final product.
Above-mentioned steps 1) in mitragynine antibody-colloidal gold marker preparation, include the following steps：
A) prepared by colloidal gold：1L ultra-pure waters are heated to boiling, the gold chloride that 1ml mass fractions are 10% is added into boiling water
Solution rapidly joins the citric acid three sodium solution that 1.2ml mass fractions are 10%, stirs and evenly mixs；Solution boiling is kept, until liquid
Body stops heating in claret, supplies dehydration after being cooled to room temperature, obtains colloidal gold solution；
B) preparation of antibody-colloidal gold marker：The above-mentioned colloidal gold solution for taking certain volume, is slowly added to mitragynine
Antibody makes its final concentration of 6 μ g/ml, after continuing stirring 30 minutes, the BSA solution that mass fraction is 10% is added and closes 30 points
Clock is centrifuged 30 minutes with the rotating speed of 14000r/min, discards supernatant liquid, precipitation is suspended with buffer solution, and 4 DEG C save backup；It is above-mentioned
Buffer solution is composed of the following components：The borate of a concentration of 10mmol/L, the Tween 80 that mass fraction is 0.3%；Buffer solution
PH be 8.0.
Above-mentioned rabbit igg colloid gold label object is made by the preparation method included the following steps：Take the colloidal gold of certain volume
Solution is slowly added to rabbit igg and is added in colloidal gold solution, makes its final concentration of 6 μ g/ml, and after continuing stirring 30 minutes, matter is added
It measures the BSA solution that score is 10% to close 30 minutes, is centrifuged 30 minutes with the rotating speed of 14000r/min, discard supernatant liquid, precipitated
It is suspended with buffer solution, 4 DEG C save backup；Above-mentioned buffer solution is composed of the following components：The borate of a concentration of 10mmol/L, quality
The Tween 80 that score is 0.3%；The pH of buffer solution is 8.0.
The preparation method is the same as that of Example 1 for above-mentioned mitragynine antibody and mitragynine-BSA haptens.
The detection kit of mitragynine and its derivative and metabolin in the detection human urine of the present embodiment, including plastics
It gets stuck and above-mentioned Test paper.During Test paper is got stuck loaded on plastics.
Experimental example
Detection method：Sample pad is placed in urine specimen to be measured, is placed 15-30 seconds, until the visible urine specimen of naked eyes is climbed
Upper detection zone, you can take out, start to detect after five minutes, detection threshold value (detection minimum) is 1000ng/ml.
Positive reference product configure：Take Population with Negative urine that mitragynine reference material is diluted to 500ng/ml, 1000ng/
Ml, 1500ng/ml, 3000ng/ml are detected, the results show that reference material 1000ng/ml or less is negative, 1000ng/ml with
Upper is positive.
One-step method of the present invention quickly detects the kit of mitragynine and its derivative and metabolin in human urine, can be extensive
The Kratum service condition Primary Screening Tests that doubtful drug addict is carried out for law enforcement agency, it can also be used to when joining the army
Kratum Primary Screening Tests.
It will be apparent to those skilled in the art that technical solution that can be as described above and design, make various other
Corresponding change and deformation, and all these changes and deformation should all belong to the protection domain of the claims in the present invention
Within.
Claims (9)
1. the Test paper of mitragynine and its derivative and metabolin in a kind of detection human urine, which is characterized in that including sample
Product pad, gold-labelled pad, NC films, blotting paper, bottom plate, the sample pad, gold-labelled pad, NC films and blotting paper are pasted onto on bottom plate successively,
It is coated with mitragynine antibody-colloidal gold marker and rabbit igg colloid gold label object in the gold-labelled pad, distinguishes on the NC films
It is coated with mitragynine-BSA haptens, goat anti-rabbit igg.
2. the Test paper of mitragynine and its derivative and metabolin in a kind of detection human urine as described in claim 1
Preparation method, which is characterized in that include the following steps：
1) it uses mitragynine antibody-colloidal gold marker and rabbit igg colloid gold label object to impregnate glass fibre, dries, obtain later
Gold-labelled pad；
2) mitragynine-BSA haptens, goat anti-rabbit igg are coated with respectively on NC films, it is spare after dry；
3) sample pad, gold-labelled pad, NC films, blotting paper are pasted on bottom plate successively to obtain the final product.
3. the system of the Test paper of mitragynine and its derivative and metabolin in detection human urine according to claim 2
Preparation Method, which is characterized in that the mitragynine antibody-colloidal gold marker is made by the preparation method included the following steps：
Mitragynine antibody is added in colloidal gold solution, stirring, the closing of BSA solution is added later, is then centrifuged for, discards supernatant liquid,
Precipitation with buffer solution be resuspended to get.
4. the system of the Test paper of mitragynine and its derivative and metabolin in detection human urine according to claim 3
Preparation Method, which is characterized in that the mitragynine antibody is made by the preparation method included the following steps：With mitragynine-BSA
Haptens injecting immune mouse, largely screened by cell fusion and to monoclonal cell to get.
5. the system of the Test paper of mitragynine and its derivative and metabolin in detection human urine according to claim 3
Preparation Method, which is characterized in that the buffer solution is composed of the following components：The borate of a concentration of 5-10mmol/L, mass fraction
For the Tween 80 of 0.1-0.3%；The pH of the buffer solution is 8.0.
6. the system of the Test paper of mitragynine and its derivative and metabolin in detection human urine according to claim 2
Preparation Method, which is characterized in that a concentration of 0.2~1.5mg/ml of mitragynine-BSA haptens in step 2), the goat-anti rabbit
A concentration of 0.5~1.0mg/ml of IgG.
7. the Test paper of mitragynine and its derivative and metabolin in detection human urine according to claim 2 or 4
Preparation method, which is characterized in that the mitragynine-BSA haptens is made by the preparation method included the following steps：
A) mitragynine and succinic anhydride are dissolved in dichloro methanol solution mixing, are cooled to 0 DEG C, alchlor is then added, instead
2h is answered, reaction solution is obtained；Gained reaction solution is added dropwise in cryosel acid solution, is filtered, collects precipitation, washing, drying are to get cap column
The wooden alkali hemisuccinate derivative；The mass ratio of the mitragynine and succinic anhydride is 2.5:1；
B) the mitragynine hemisuccinate derivative obtained by step a) is dissolved in DMF, 2-morpholine ethane sulfonic acid, N- hydroxyls is then added
Base thiosuccimide reacts at room temperature 2 hours, obtains the mitragynine of activation, the mitragynine of activation is added to BSA solution
In, 4 DEG C of reactions overnight, are dialysed with PBS solution later to obtain the final product；The mitragynine hemisuccinate derivative, 2- morpholine second sulphurs
Sour, N- hydroxy thiosuccinimides mass ratio is 10:10:5.
8. the system of the Test paper of mitragynine and its derivative and metabolin in detection human urine according to claim 7
Preparation Method, which is characterized in that a concentration of 10~20mg/ml of BSA solution in step b).
9. the detection kit of mitragynine and its derivative and metabolin in a kind of detection human urine, which is characterized in that including
It gets stuck and Test paper described in claim 1.
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Cited By (1)
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