CN108802393A - A kind of test strips and its preparation method and application method of detection tetrahydro-cannabinolic acid - Google Patents
A kind of test strips and its preparation method and application method of detection tetrahydro-cannabinolic acid Download PDFInfo
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Abstract
The invention discloses a kind of test strips of detection tetrahydro-cannabinolic acid and its preparation method and application methods.It includes sample absorption pad, conjugate release pad, reaction film, water absorption pad and bottom plate successively that the test strips, which are constituted,；The antibody of time-resolved fluorescence microballoon label is coated in conjugate release pad；The nature controlling line of the detection line and sheep anti mouse secondary antibody of hapten-carrier protein conjugate is coated on the reaction film；The present invention realizes the rapid immunoassay of tetrahydro-cannabinolic acid with time-resolved fluorescence microballoon labelled antibody using immunochromatography technique.The present invention also provides a kind of methods using tetrahydro-cannabinolic acid in above-mentioned ELISA test strip sample.It is an advantage of the invention that high sensitivity, can accurate quantitative analysis, detection is quick, easy to operate, economical and practical, can realize and is used for quickly detecting to large batch of tetrahydro-cannabinolic acid sample and Site Detection.
Description
Technical field
It is specifically a kind of to utilize time-resolved fluorescence micro-ball immune chromatography skill the present invention relates to the detection of tetrahydro-cannabinolic acid
Art quantitatively detects the test strips of tetrahydro-cannabinolic acid and its preparation method and application method.
Background technology
Hemp is present in marihuana, there is the effects that cough-relieving, analgesia, sleeping.It can aspirate, take orally, chew, after sucking
Sense in high spirits can be generated and cause habituation.Drug addict will produce that will appear illusion, vain hope and class inclined as sucked large dosage of hemp
State is held, with disturbance in thinking, the symptoms such as dual personality occurs in alienation.Taking for a long time may cause insomnia, appetite
Decline edgy, irritability, is vomitted, is trembled, poet's comprehension and failure of memory, and immunity degradation is easy to obtain various diseases
Disease, it is in poor health to make, it becomes thin, but will not generally lead to death.The main component of hemp is tetrahydro-cannabinolic acid（THC）,
It is the strongest psychoactive compositions of Central nervous systemic effect.
Include at present mainly high performance liquid chromatography, gas-chromatography, liquid for the detection of tetrahydro-cannabinolic acid and monitoring method
Matter or makings are used in conjunction.These chromatographic processes have good sensitivity and specificity, but complicated for operation, and flux is not high, take
Longer, instrument and equipment is expensive.Enzyme linked immunosorbent assay (ELISA) (ELISA) and colloidal gold immunity chromatography are internationally recognized at present
Mainstream technology, both methods have detection speed fast, cheap, simple operation and other advantages.But ELISA detections still need to profession
Personnel, and the long period is needed to show result；Colloidal gold method can carry out tetrahydro-cannabinolic acid qualitative analysis, easy to operate, inspection
The survey time is shorter, but sensitivity is low, interference from human factor is larger.
In conclusion the detection technique of tetrahydro-cannabinolic acid is still immature at present, a kind of high sensitivity, easy to operate is developed
And it can realize that the product be used for quickly detecting to batch samples and method become problem in the urgent need to address.
Invention content
It is an object of the invention to overcome it is existing detection tetrahydro-cannabinolic acid method existing for it is complicated for operation, take compared with
The characteristics of growing, and can not realizing the quick detection to batch samples provides one kind easy to operate, high sensitivity, detection
Speed is fast, test strips of detection tetrahydro-cannabinolic acid at low cost and its preparation method and application method, to realize to high-volume
Tetrahydro-cannabinolic acid sample be used for quickly detecting and on-site supervision.
In order to achieve the object of the present invention, a kind of test strips of detection tetrahydro-cannabinolic acid of the invention use following technology
Scheme：It is a kind of detection tetrahydro-cannabinolic acid test strips, including sample absorption pad, conjugate release pad, reaction film, water absorption pad and
Bottom plate；It is characterized in that：The antibody of time-resolved fluorescence microballoon label is coated in the conjugate release pad；The reaction
The nature controlling line of the detection line and sheep anti mouse secondary antibody of hapten-carrier protein conjugate is coated on film.
The sample absorption pad, conjugate release pad, reaction film and water absorption pad are pasted onto on bottom plate and middle arbitrary neighborhood
Two partial stack, the 1/3-1/2 of conjugate release pad are capped under sample absorption pad along the vertical direction.
The time-resolved fluorescence microballoon is that the rare earth ion of a diameter of 100 nm-500 nm is micro- as the fluorescence of marker
Ball, the functional group of surface modification, for albumen, the covalent coupling of antibody and nucleic acid.
The excitation wavelength of the time-resolved fluorescence microballoon is 300-400 nm, and launch wavelength is 500-700 nm.
The sample absorption pad is glass fibre element film.
The conjugate release pad is glass fibre or polyester material.
The reaction film is nitrocellulose filter or cellulose acetate film.
The bottom plate is the material that PVC bottom plates or other hard do not absorb water.
The water absorption pad is blotting paper.
A kind of method of test strips preparing above-mentioned detection tetrahydro-cannabinolic acid of the present invention, includes the following steps：
1）Tetrahydro-cannabinolic acid antibody is marked with fluorescent microsphere, and is sprayed in object release pad, is coated with preparing
The conjugate release pad of tetrahydro-cannabinolic acid monoclonal antibody-Fluorescent microsphere marker；
2）Hapten-carrier protein conjugate and sheep anti mouse secondary antibody are sprayed at respectively on reaction film and are used as detection line（T）And matter
Control line（C）；
3）By 1）With 2）Conjugate release pad, reaction film and sample absorption pad, water absorption pad and the bottom plate prepared is assembled into test paper
Item.
In step 1）Middle taking-up fluorescent microsphere is activated, and tetrahydro-cannabinolic acid antibody is then added and carries out covalent coupling,
After be added Block buffer closed, saved backup for 4 DEG C after centrifuge washing.
In step 1）In conjugate release pad bovine serum albumin(BSA) containing 0.2-1%（Mass fraction）, pH 7.4
0.02-0.05 mol/L Tris-HCl（The trehalose and 0.02-0.1% Tween-20 of the % containing 0.1-5）Buffer solution impregnates 2 h,
2 h are dried at 37 DEG C, are placed in dry environment and are saved backup.
Tetrahydro-cannabinolic acid in a kind of ELISA test strip sample using above-mentioned detection tetrahydro-cannabinolic acid of the present invention
Method includes the following steps：
（1）Test strips and sample to be tested are restored to room temperature.
（2）Immunofluorescence analysis instrument is opened, corresponding ID cards is inserted into, reads standard curve.
（3）Sample to be tested 60-120 μ L are added into the sample well of test strips, after reacting 5-10min, detection card insertion is entered
Immunofluorescence analysis instrument.
（4）The fluorescent microsphere of detection line and nature controlling line is trapped under best exciting light, sends out strong fluorescent bands；
（5）The fluorescence intensity of detection line and nature controlling line is read using immunochromatographiassays assays instrument, and provides T/C values, and analyzer can lead to
It crosses built-in standard curve and calculates the concentration of tetrahydro-cannabinolic acid in sample, and judge yin and yang attribute.
Compared with prior art, the present invention having the following advantages：
（1）The present invention can be in accurate quantitative analysis sample to be tested tetrahydro-cannabinolic acid content.
（2）The present invention uses the reaction system of independent nature controlling line and detection line, does not interfere with each other and influences, and uses T/C
The mode of value is demarcated, and ensure that the accuracy of test result.
（3）The present invention uses time-resolved fluorescence microballoon, due to its Stokes displacement big (> 150nm) and fluorescence lifetime ratio
High 5~6 orders of magnitude of background substance fluorescence lifetime can effectively eliminate the interference of various non-specific fluorescences, improve detection
Sensitivity.
The present invention test strips have high sensitivity, high specificity, at low cost, easy to operate, detection time is short, is not examined
The advantages of measurement equipment limits, storage is simple, long shelf-life.With the method for ELISA test strip tetrahydro-cannabinolic acid of the present invention, easy,
Quickly, accurate, applied widely, at low cost, easily promote the use of.
Further, the 1/3-1/2 of conjugate release pad be absorbed by the sample pad covering can extend testing result observation when
Between, it can make sample absorption pad that will detect liquid and fully absorb and fully reacted with antibody, to reduce error.
Description of the drawings
Fig. 1 is a kind of structural schematic diagram of the test strips of detection tetrahydro-cannabinolic acid of the present invention；
Fig. 2 is the structural schematic diagram of detection card；
Fig. 3 is test strips sample detection result schematic diagram；
Fig. 4 is the standard curve of test strips of the present invention；
Fig. 5 is saliva collecting device structural schematic diagram；
1, sample absorption pad；2, conjugate release pad；3, reaction film；4, water absorption pad；5, bottom plate；6, detection line；7, nature controlling line；8,
Detect window；9, sample well；10, capping is acquired；11, slot is acquired；12, collection tube；13, collection tube lid；14, minimum collection line.
Specific implementation mode
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this
Invention, and be not used to limit the scope of the invention.In addition, the range that those skilled in the art limits in the appended claims
Interior to carry out various changes or modification to the present invention, these are changed or modification should equally fall into the protection domain of invention.
The test strips of a kind of detection tetrahydro-cannabinolic acid of the present invention, as shown in Figure 1, including sample absorption pad 1, conjugate
Release pad 2, reaction film 3, water absorption pad 4 and bottom plate 5；The anti-of time-resolved fluorescence microballoon label is coated in conjugate release pad
Body；The detection line 6 of hapten-carrier protein conjugate and the nature controlling line 7 of sheep anti mouse secondary antibody are coated on reaction film.Sample absorbs
Pad, conjugate release pad, reaction film and water absorption pad are pasted onto on bottom plate and middle arbitrary neighborhood two is partly folded along the vertical direction
Add, the 1/3 of conjugate release pad is capped under sample absorption pad, and the 1/3 of conjugate release pad is absorbed by the sample pad covering can
To extend testing result observing time, it can make sample absorption pad that will detect liquid and fully absorb and fully reacted with antibody, to
Reduce error, in other embodiments, can also conjugate release pad 1/2 be capped under sample absorption pad.Time resolution
Fluorescent microsphere is fluorescent microsphere of the rare earth ion of a diameter of 100 nm-500 nm as marker, and surface modification is functional
Group, for albumen, the covalent coupling of antibody and nucleic acid.The excitation wavelength of time-resolved fluorescence microballoon is 300-400 nm, hair
The a length of 500-700 nm of ejected wave.Sample absorption pad is glass fibre element film.Conjugate release pad is glass fibre or polyester material.
Reaction film is nitrocellulose filter or cellulose acetate film.Bottom plate is the material that PVC bottom plates or other hard do not absorb water.Water absorption pad
For blotting paper.
The preparation method of the test strips of the detection tetrahydro-cannabinolic acid mainly includes the following steps that：
1）Tetrahydro-cannabinolic acid antibody is marked with fluorescent microsphere, and is sprayed in object release pad, is coated with preparing
The conjugate release pad of the tetrahydro-cannabinolic acid antibody of fluorescent microsphere label；
2）Hapten-carrier protein conjugate and sheep anti mouse secondary antibody are sprayed at respectively on reaction film and are used as detection line（T）And matter
Control line（C）, the detection line of tetrahydro-cannabinolic acid hapten-carrier protein conjugate is coated with preparation and is coated with sheep anti mouse two
The reaction film of anti-nature controlling line；
3）By 1）With 2）Conjugate release pad, reaction film and sample absorption pad, water absorption pad and the bottom plate prepared is assembled into test paper
Item.
Substep narration in detail below：
1, the preparation of Fluorescent microsphere marker：
Fluorescent microsphere marks the preparation of tetrahydro-cannabinolic acid antibody：It takes 15000 rpm of 1mg fluorescent microspheres to centrifuge 10 min, collects
Precipitation is resuspended with 1mL coupling buffers and is precipitated.Then 1 is pressed:2-1:EDC and NHS is added in 20 molar ratio, and be vortexed concussion rear chamber
Temperature is incubated 20-30 min, and 15000 rpm centrifuge 10min, collect precipitation.Coupling buffer is added, microballoon is resuspended, into the solution
40-150 μ g tetrahydro-cannabinolic acid antibody is added, after mixing well, reaction 2-4 h, 10000 rpm centrifugation 10 is stirred at room temperature
Min removes supernatant, and 1 mL Block buffers are added, 1-2h is reacted at room temperature after mixing, after Block buffer centrifuge washing 3 times, uses
The PBS of 0.02M pH7.4（BSA containing 0.1-1% and 0.1-5% trehaloses）Precipitation is resuspended, the fluorescent microsphere label as prepared
Tetrahydro-cannabinolic acid antibody, be positioned over 4 DEG C it is spare.
2, the preparation of fluorescent microsphere pad：
By the conjugate release pad pad bovine serum albumin(BSA)s of % containing 0.1-0.5（Mass fraction）, pH 7.4 0.01-0.05M
Tris-HCl（The trehalose and 0.01-1 % Tween-20 of the % containing 0.1-5）Buffer solution impregnates 2 h, and it is standby to dry 2 h at 37 DEG C
With.The antibody for the tetrahydro-cannabinolic acid that fluorescent microsphere marks is sprayed into conjugate release pad with spray film instrument, every 1 cm is combined
Object release pad sprays the tetrahydro-cannabinolic acid antibody of 1-9 μ L fluorescent microspheres label, and 37 DEG C of dry 1-2h are placed in standby in dry environment
With.
3, the preparation of NC films：
Tetrahydro-cannabinolic acid hapten-carrier protein conjugate and sheep anti-mouse antibody are coated in respectively on NC films：Use 0.02M
Tetrahydro-cannabinolic acid hapten-carrier protein conjugate is adjusted to 0.5-2mg/mL by the PBS of pH7.4, is coated on shape on NC films
At detection line, package amount is 5-10 μ L/cm；Sheep anti mouse secondary antibody is adjusted to 0.1-0.5mg/mL with the PBS of 0.01M pH7.4,
It is coated on NC films and forms nature controlling line, package amount is 5-10 μ L/cm.The reaction film being coated with is placed in 37 DEG C of dry 1-2 h,
It is placed in spare in dry environment.
4, the preparation of time-resolved fluorescence microballoon immune detection card
It pastes sample absorption pad successively on PVC bottom plates, is coated with the combination of the tetrahydro-cannabinolic acid antibody of fluorescent microsphere label
Object release pad is coated with tetrahydro-cannabinolic acid hapten-carrier protein conjugate as detection line and sheep anti mouse secondary antibody as matter
Control the reaction film of line, water absorption pad；Wherein, conjugate release pad has 1/3 region to be absorbed by the sample pad covering, conjugate from initiating terminal
The end of release pad and the beginning of reaction film connect, and the end of reaction film is connected with the beginning of water absorption pad, the beginning of sample absorption pad
End is aligned with the beginning of PVC bottom plates, and the end of water absorption pad is aligned with the end of PVC bottom plates；Detection line on the reaction film and
The nature controlling line strip tape perpendicular with the appearance of the test strips；Detection line is located at one close to the end of conjugate release pad
Side；Nature controlling line is located remotely from the side of the end of conjugate release pad；The width that 4mm is cut into after being completed, becomes and exempts from
Epidemic disease chromatograph test strip.
Immuno-chromatographic test paper strip is fixed on plastic bottom card, test strips surface is compressed with face card, and face is stuck in corresponding test paper
Sample well 9 and detection window 8 are reserved in the part of sample absorption pad and NC films respectively, as shown in Figure 2.The detection card fills after assembling
Enter aluminium foil bag, drier sealing is added, is positioned under drying at room temperature environment and can save 12 months.
A method of using tetrahydro-cannabinolic acid in above-mentioned ELISA test strip sample, this approach includes the following steps：
1）Test strips and sample to be tested are restored to room temperature；
2）Immunofluorescence analysis instrument is opened, corresponding ID cards is inserted into, reads standard curve；
3）Sample to be tested 60-120 μ L are added into the sample well 9 of test strips, after reacting 5-10min, detection card insertion are entered immune
Fluorescence analyser；
4）The fluorescent microsphere of detection line and nature controlling line is trapped under best exciting light, sends out strong fluorescent bands；
5）The fluorescence intensity of detection line and nature controlling line is read using immunochromatographiassays assays instrument, and provides T/C values, and analyzer can pass through
Built-in standard curve calculates the concentration of tetrahydro-cannabinolic acid in sample, and judges yin and yang attribute, as shown in Figure 3.
The detection of tetrahydrocannabinol acid content in 1 hair sample of test example
1, the foundation of tetrahydro-cannabinolic acid test strip standard curve
Blank hair sample is taken, ultrasound after shredding adds tetrahydro-cannabinolic acid in the solution after ultrasound to final concentration of respectively
0ppb, 5ppb, 10pb, 20ppb, 50ppb, 100ppb, 200ppb, 500ppb take test strips to be detected, and each sample repeats
It measures five times.After five reproducible results of measurement are averaged, demarcated on immunofluorescence analysis instrument.
2, in hair sample tetrahydro-cannabinolic acid detection
（1）The pre-treatment of sample
1. sample is restored to 20-25 DEG C of room temperature before detection；
2. weighing 20-50 mg sample of hair to shred into polystyrene centrifuge tube；
3. 1 mL sample extracting solutions are added, 3 min of water bath sonicator；
（2）It is detected with test strips
1. opening immunofluorescence analysis instrument, corresponding ID cards are inserted into, standard curve is read.
2. face-up keeping flat detection card, sample to be tested 60-120 μ L, room temperature reaction are added into the sample well of test strips
After 5-10min, detection card is put into immunofluorescence analysis instrument and is detected
3. being trapped in the fluorescent microsphere of detection line and nature controlling line under best exciting light, strong fluorescent bands are sent out；
4. reading the fluorescence intensity of detection line and nature controlling line using immunochromatographiassays assays instrument, and T/C values are provided, analyzer can pass through
Built-in standard curve calculates the concentration of tetrahydro-cannabinolic acid in sample, and judges yin and yang attribute.
The detection of tetrahydrocannabinol acid content in 2 blood sample of test example
In the embodiment, all preparation methods are same as Example 1, except sample absorption pad is hemofiltration film.
1, hemolytic blood sample
（1）The foundation of tetrahydro-cannabinolic acid test strip standard curve
Blank blood sample is taken, 1 is pressed with sample diluting liquid:5-1:20 ratio is diluted, to the hemolytic blood sample after dilution
Added respectively in product solution tetrahydro-cannabinolic acid to final concentration of 0ppb, 5ppb, 10pb, 20ppb, 50ppb, 100ppb,
200ppb, 500ppb take test strips to be detected, each sample replication five times.Five reproducible results of measurement are made even
After mean value, demarcated on immunofluorescence analysis instrument.
（2）The detection of tetrahydro-cannabinolic acid in hemolytic blood sample
1. the pre-treatment of sample
Sample is restored to 20-25 DEG C of room temperature before a detections；
B presses 1 with sample diluting liquid:5-1:20 ratio is diluted hemolytic blood sample.
2. being detected with test strips
A opens immunofluorescence analysis instrument, is inserted into corresponding ID cards, reads standard curve.
B face-up keeps flat detection card, and sample to be tested 60-120 μ L are added into the sample well of test strips, reacts at room temperature 5-
After 10min, detection card is put into immunofluorescence analysis instrument and is detected
C is trapped in the fluorescent microsphere of detection line and nature controlling line under best exciting light, sends out strong fluorescent bands；
D reads the fluorescence intensity of detection line and nature controlling line using immunochromatographiassays assays instrument, and provides T/C values, and analyzer can pass through
Built-in standard curve calculates the concentration of tetrahydro-cannabinolic acid in sample, and judges yin and yang attribute.
2, fresh blood samples
The embodiment is identical as above-mentioned hemolytic blood Samples EXAMPLE, but can be direct without dilution before fresh blood samples test
Sample-adding is detected.
The detection of tetrahydrocannabinol acid content in 3 urine sample of test example
In the embodiment, all preparation methods of test strips and sample detection methods are same as Example 1.
The detection of tetrahydrocannabinol acid content in 4 saliva sample of test example
In the embodiment, all preparation methods of test strips and sample detection methods are same as Example 1, and the acquisition of saliva can use
Saliva collecting device acquires slot 11, collection tube 12, collection tube lid 13, on collection tube 12 as shown in figure 5, including acquisition capping 10
It is provided with minimum collection line 14.
The acquisition of saliva sample carries out in accordance with the following steps：
1, until saliva amount reaches minimum gathering line in saliva being spat into acquisition slot.
2, acquisition capping is covered tightly, until hearing click, the sample diluting liquid in lid flows into collection tube, with saliva
Sample mixes.
3, acquisition slot is backed out, covers collection tube lid, turn upside down mixing.
4, the saliva sample after dilution is drawn to be detected.
The foundation of basic parameter
Detection limit：Replication is carried out with blank sample 20 times, calculates the mean value M and standard deviation SD of 20 results, it is equal with blank
Value plus twice of standard deviation（M+2SD）The detection of method for reporting limits, result 5ppb.The range of linearity：Take 0ppb, 5ppb, 10pb,
20ppb, 50ppb, 100ppb, 200ppb, 500ppb isoconcentration value are detected, and each concentration duplicate measurements five times will measure
Mean concentration carries out linear analysis with theoretical value, obtains linear equation y=- 9212x+2.5273, R2=0.9923.（Experiment knot
Fruit and analysis are shown in Table 1, Fig. 4）.
1 tetrahydro-cannabinolic acid standard items testing result of table
Accuracy：The tetrahydrocannabinol acidity scale product for being 30ppb with sample diluting liquid compound concentration, with prepared examination in embodiment 1
Paper slip is detected, and repeats detection three times, testing result, which is averaged, to be calculated.The rate of recovery=detectable concentration/is actually added into dense
Degree × 100%, calculate sample recovery rate be 104.25%.
Precision：Time-resolved fluorescence prepared in three batches of embodiments 1 is taken to quantify tetrahydro-cannabinolic acid test strip,
Detect 40ppb concentration mark product, every batch of test strips mark product Parallel testing 10 times, the results show that CV values are respectively in three batches of batches
2.572%, 1.914%, 2.673%, CV values are 3.510% between three batches of batches.
It is seen from the above data that test strips of the present invention, the method for comparing existing detection tetrahydro-cannabinolic acid,
Have the characteristics that it is sensitiveer, more rapidly, can accurate quantitative analysis, do not limited by operation place, and mating detecting instrument is small and exquisite light
Just, personnel requirement is lower, is suitble to promote the use of on a large scale.
Claims (10)
1. a kind of test strips of detection tetrahydro-cannabinolic acid, including sample absorption pad, conjugate release pad, reaction film, water absorption pad
And bottom plate；It is characterized in that：The antibody of time-resolved fluorescence microballoon label is coated in the conjugate release pad；The reaction
The nature controlling line of the detection line and sheep anti mouse secondary antibody of hapten-carrier protein conjugate is coated on film.
2. test strips according to claim 1, it is characterised in that：The sample absorption pad, conjugate release pad, reaction film
It is pasted onto with water absorption pad on bottom plate and the partial stack along the vertical direction of middle arbitrary neighborhood two, the 1/3-1/ of conjugate release pad
2 are capped under sample absorption pad.
3. test strips according to claim 1, it is characterised in that：The time-resolved fluorescence microballoon is a diameter of 100
Fluorescent microsphere of the rare earth ion of nm-500 nm as marker, the functional group of surface modification, for albumen, antibody and
The covalent coupling of nucleic acid.
4. test strips according to claim 3, it is characterised in that：The excitation wavelength of the time-resolved fluorescence microballoon is
300-400 nm, launch wavelength are 500-700 nm.
5. test strips according to claim 1, it is characterised in that：The conjugate release pad is glass fibre or polyester material
Material.
6. test strips according to claim 1, it is characterised in that：The reaction film is nitrocellulose filter or acetate fiber
Plain film.
7. a kind of method preparing any one of claim 1-6 test strips, which is characterized in that include the following steps：
1）Tetrahydro-cannabinolic acid antibody is marked with fluorescent microsphere, and is sprayed in object release pad, is coated with preparing
The conjugate release pad of tetrahydro-cannabinolic acid monoclonal antibody-Fluorescent microsphere marker；
2）Hapten-carrier protein conjugate and sheep anti mouse secondary antibody are sprayed at respectively on reaction film and are used as detection line（T）And matter
Control line（C）；
3）By 1）With 2）Conjugate release pad, reaction film and sample absorption pad, water absorption pad and the bottom plate prepared is assembled into test paper
Item.
8. the method according to claim 7 for preparing test strips, it is characterised in that：In step 1）Middle taking-up fluorescent microsphere into
Row activation, then be added tetrahydro-cannabinolic acid antibody carry out covalent coupling, after be added Block buffer closed, centrifuge
It is saved backup for 4 DEG C after washing.
9. the method according to claim 7 for preparing test strips, it is characterised in that：In step 1）In conjugate release pad
With bovine serum albumin(BSA) containing 0.2-1%（Mass fraction）, pH 7.4 0.02-0.05 mol/L Tris-HCl（The %'s containing 0.1-5
Trehalose and 0.02-0.1% Tween-20）Buffer solution impregnates 2 h, dries 2 h at 37 DEG C, be placed in dry environment preserve it is standby
With.
10. tetrahydrochysene is big in a kind of ELISA test strip sample using any one of the claim 1-6 detection tetrahydro-cannabinolic acids
The method of numb phenolic acid, which is characterized in that this approach includes the following steps：
1）Test strips and sample to be tested are restored to room temperature；
2）Immunofluorescence analysis instrument is opened, corresponding ID cards is inserted into, reads standard curve；
3）Sample to be tested 60-120 μ L are added into the sample well of test strips, after reacting 5-10min, detection card insertion are entered immune glimmering
Light analyzer；
4）The fluorescent microsphere of detection line and nature controlling line is trapped under best exciting light, sends out strong fluorescent bands；
5）The fluorescence intensity of detection line and nature controlling line is read using immunochromatographiassays assays instrument, and provides T/C values, and analyzer can pass through
Built-in standard curve calculates the concentration of tetrahydro-cannabinolic acid in sample, and judges yin and yang attribute.
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| CN113049827A (en) * | 2021-03-12 | 2021-06-29 | 郑州大学 | Time-resolved fluorescence immunochromatography reagent strip, preparation method and hemp concentration quantitative detection method |
| CN113340757A (en) * | 2021-05-24 | 2021-09-03 | 金华海关综合技术服务中心 | Method and device for rapidly detecting tetrahydrocannabinol in cosmetics |
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Application publication date: 20181113 |