CN108845152A - A kind of test strips and its preparation method and application method detecting amphetamine - Google Patents
A kind of test strips and its preparation method and application method detecting amphetamine Download PDFInfo
- Publication number
- CN108845152A CN108845152A CN201810532305.5A CN201810532305A CN108845152A CN 108845152 A CN108845152 A CN 108845152A CN 201810532305 A CN201810532305 A CN 201810532305A CN 108845152 A CN108845152 A CN 108845152A
- Authority
- CN
- China
- Prior art keywords
- amphetamine
- sample
- test strips
- pad
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/946—CNS-stimulants, e.g. cocaine, amphetamines
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a kind of test strips and its preparation method and application for detecting amphetamine.It successively includes sample absorption pad, conjugate release pad, reaction film, water absorption pad and bottom plate that the test strips, which are constituted,；The antibody of time-resolved fluorescence microballoon label is coated in conjugate release pad；The detection line of hapten-carrier protein conjugate and the nature controlling line of sheep anti mouse secondary antibody are coated on the reaction film；The present invention realizes the rapid immunoassay of amphetamine using immunochromatography technique with time-resolved fluorescence microballoon labelled antibody.The present invention also provides a kind of methods using amphetamine in above-mentioned test strips test sample.It is an advantage of the invention that high sensitivity, can accurate quantitative analysis, detect it is quick, easy to operate, it is economical and practical, can be realized and large batch of amphetamine sample is used for quickly detecting and on-site test.
Description
Technical field
It is specifically a kind of quantitative using time-resolved fluorescence micro-ball immune chromatography technology the present invention relates to the detection of amphetamine
Detect the test strips of amphetamine and its preparation method and application method.
Background technique
Amphetamine, i.e. amphetamine are a series of synthetic drugs that Central nervous systems have significant excitation
Prototype.Because intravenous injection has additive, and it is listed in drugs.Adverse reaction caused by amphetamine, which has, is overexcited, is uneasy, losing
It sleeps, tremble, the symptoms such as nervous and agitation.Occur to the body tolerance of amphetamine it is very fast, so long-term use person is necessary
The more take the more more.Severest consequences are exactly a kind of toxicity neuropathy after taking large dosage of amphetamine, and symptom is bigoted similar to class
Type schizophrenia.The abuse of amphetamine often occurs with the abuse of barbiturate drugs and alcohol together.Amphetamine excess or repeatedly
Using can produce ill hobby, and cause the excited dysequilibrium with process of inhibition and lead to mental symptom, therefore use should be strictly
Control.
It mainly include at present high performance liquid chromatography, gas-chromatography, liquid matter or gas for the detection of amphetamine and monitoring method
Matter is used in conjunction.These chromatographic processes have good sensitivity and specificity, but complicated for operation, and flux is not high, takes a long time, instrument
Device equipment is expensive.Enzyme linked immunosorbent assay (ELISA) (ELISA) and colloidal gold immunity chromatography are current internationally recognized mainstream skills
Art, both methods have detection speed fast, cheap, simple operation and other advantages.But ELISA detection still needs to professional,
And the long period is needed to show result；Colloidal gold method can carry out qualitative analysis to amphetamine, and easy to operate, detection time is shorter,
But sensitivity is low, interference from human factor is larger.
In conclusion the detection technique of amphetamine is still immature at present, a kind of high sensitivity, easy to operate and energy are developed
It is enough to realize that the product be used for quickly detecting to batch samples and method become problem in the urgent need to address.
Summary of the invention
It is an object of the invention to overcome it is existing detection amphetamine method existing for it is complicated for operation, take a long time, and
And the characteristics of can not realizing the quick detection to batch samples, one kind easy to operate, high sensitivity, detection speed are provided
Fastly, test strips of detection amphetamine at low cost and its preparation method and application method, to realize to large batch of amphetamine sample
Product are 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 amphetamine of the invention use following technical scheme：
A kind of test strips detecting amphetamine, including sample absorption pad, conjugate release pad, reaction film, water absorption pad and bottom plate；Its feature
It is：The antibody of time-resolved fluorescence microballoon label is coated in the conjugate release pad；It is coated on the reaction film
The detection line of hapten-carrier protein conjugate and the nature controlling line of sheep anti mouse secondary antibody.
The sample absorption pad, conjugate release pad, reaction film and water absorption pad are pasted 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 that diameter is 100 nm-500 nm is micro- as the fluorescence of marker
Ball, the functional group of surface modification, the covalent coupling for albumen, 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 plate 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 amphetamine of the invention, includes the following steps：
1）Amphetamine antibody is marked with fluorescent microsphere, and is sprayed in object release pad, amphetamine is coated with preparation
Monoclonal antibody-Fluorescent microsphere marker conjugate release pad；
2）Hapten-carrier protein conjugate and sheep anti mouse secondary antibody are sprayed on reaction film respectively 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 amphetamine antibody is then added and carries out covalent coupling, after
Block buffer is added to be closed, is 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 impregnates 2 h,
2 h are dried at 37 DEG C, are placed in dry environment and are saved backup.
The method of amphetamine in a kind of test strips test sample using above-mentioned detection amphetamine of the invention, including it is as follows
Step：
（1）Test strips and sample to be tested are restored to room temperature.
（2）Immunofluorescence analysis instrument is opened, corresponding ID card is inserted into, reads standard curve.
（3）Sample to be tested 60-120 μ L is added into the sample well of test strips, after reacting 5-10min, will test card insertion and enter
Immunofluorescence analysis instrument.
（4）The fluorescent microsphere of detection line and nature controlling line is trapped under best exciting light, issues strong fluorescent bands；
（5）The fluorescence intensity of detection line and nature controlling line is read using immunochromatographiassays assays instrument, and provides T/C value, and analyzer can lead to
It crosses built-in standard curve and calculates the concentration of amphetamine in sample, and judge yin and yang attribute.
Compared with prior art, the present invention having the following advantages：
（1）The present invention is capable of the content of amphetamine in accurate quantitative analysis sample to be tested.
（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, since its Stokes is displaced 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.
Test strips of the invention 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.The method for detecting amphetamine with test strips of the present invention, easy, quick,
Accurately, applied widely, at low cost, easy popularization and use.
Further, the 1/3-1/2 of conjugate release pad be absorbed by the sample pad covering can extend testing result observation when
Between, sample absorption pad can be made to will test liquid and fully absorb and sufficiently reacted with antibody, to reduce error.
Detailed description of the invention
Fig. 1 is a kind of structural schematic diagram of the test strips of detection amphetamine of the 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, slot cover is acquired；11, slot is acquired；12, collection tube；13, collection tube lid；14, minimum collection line.
Specific embodiment
Below 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 intended 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 scope of invention.
The test strips of a kind of detection amphetamine of the 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 antibody of time-resolved fluorescence microballoon label is coated in conjugate release pad；Reaction
The detection line 6 of hapten-carrier protein conjugate and the nature controlling line 7 of sheep anti mouse secondary antibody are coated on film.Sample absorption pad combines
Object release pad, reaction film and water absorption pad are pasted on bottom plate and the partial stack along the vertical direction of middle arbitrary neighborhood two, in conjunction with
The 1/3 of object release pad is capped under sample absorption pad, and the 1/3 of conjugate release pad, which is absorbed by the sample pad covering, can extend inspection
Result observing time is surveyed, sample absorption pad can be made to will test liquid and fully absorb and sufficiently reacted with antibody, so that error is reduced,
In other embodiments, it can also be capped under sample absorption pad with the 1/2 of conjugate release pad.Time-resolved fluorescence microballoon is
Diameter is fluorescent microsphere of the rare earth ion of 100 nm-500 nm as marker, and the functional group of surface modification is used for egg
The covalent coupling of white, antibody and nucleic acid.The excitation wavelength of time-resolved fluorescence microballoon is 300-400 nm, launch wavelength 500-
700 nm.Sample absorption pad is glass fibre element film.Conjugate release pad is glass fibre or polyester material.Reaction film is nitric acid
Cellulose membrane or cellulose acetate film.Bottom plate is the material that PVC bottom plate or other hard do not absorb water.Water absorption pad is blotting paper.
The preparation method of the test strips of the detection amphetamine mainly includes the following steps that：
1）Amphetamine antibody is marked with fluorescent microsphere, and is sprayed in object release pad, it is micro- to be coated with fluorescence with preparation
The conjugate release pad of the amphetamine antibody of ball label；
2）Hapten-carrier protein conjugate and sheep anti mouse secondary antibody are sprayed on reaction film respectively as detection line（T）And matter
Control line（C）, the detection line of amphetamine hapten-carrier protein conjugate is coated with preparation and is coated with the matter of sheep anti mouse secondary antibody
Control the reaction film of 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：
The preparation of fluorescent microsphere label amphetamine antibody：It takes 15000 rpm of 1mg fluorescent microsphere to be centrifuged 10 min, collects precipitating, use
Precipitating is resuspended in 1mL coupling buffer.Then 1 is pressed:2-1:EDC and NHS is added in 20 molar ratio, is incubated at room temperature after the concussion that is vortexed
20-30 min, 15000 rpm are centrifuged 10min, collect precipitating.Coupling buffer is added, microballoon is resuspended, is added into the solution
After mixing well, reaction 2-4 h is stirred at room temperature in 40-150 μ g amphetamine antibody, and 10000 rpm are centrifuged 10 min and remove supernatant, add
Enter 1 mL Block buffer, react at room temperature 1-2h after mixing, after Block buffer centrifuge washing 3 times, with 0.02M pH7.4's
PBS（BSA containing 0.1-1% and 0.1-5% trehalose）Precipitating is resuspended, the amphetamine of the fluorescent microsphere label as prepared resists
Body, be placed in 4 DEG C it is spare.
2, the preparation of fluorescent microsphere pad：
By the conjugate release pad pad bovine serum albumin(BSA) 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 impregnates 2 h, and it is standby to dry 2 h at 37 DEG C
With.The antibody for the amphetamine that fluorescent microsphere marks is sprayed into conjugate release pad with spray film instrument, every 1 cm conjugate release
The amphetamine antibody of pad spraying 1-9 μ L fluorescent microsphere label, 37 DEG C of dry 1-2h are placed in spare in dry environment.
3, the preparation of NC film：
Amphetamine hapten-carrier protein conjugate and sheep anti-mouse antibody are coated in respectively on NC film：With 0.02M pH7.4's
Amphetamine hapten-carrier protein conjugate is adjusted to 0.5-2mg/mL by PBS, is coated on NC film and is formed detection line, coating
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, is coated on NC film
Nature controlling line is formed, 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, is placed in dry environment
In it is spare.
4, the preparation of time-resolved fluorescence microballoon immune detection card
Sample absorption pad is successively pasted on PVC bottom plate, is coated with the conjugate release of the amphetamine antibody of fluorescent microsphere label
Pad, is coated with reaction film of the amphetamine hapten-carrier protein conjugate as detection line and sheep anti mouse secondary antibody as nature controlling line,
Water absorption pad；Wherein, conjugate release pad has 1/3 region to be absorbed by the sample pad covering, the end of conjugate release pad from starting point
It is connect with the beginning of reaction film, the end of reaction film is connected with the beginning of water absorption pad, beginning and the PVC bottom plate of sample absorption pad
Beginning alignment, the end of water absorption pad is aligned with the end of PVC bottom plate；Detection line and nature controlling line and the examination on the reaction film
The perpendicular strip tape of the appearance of paper slip；Detection line is located at the side close to the end of conjugate release pad；Nature controlling line is located at remote
The side of end from conjugate release pad；The width that 4mm is cut into after being completed, becomes immuno-chromatographic test paper 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 film respectively, as shown in Figure 2.The detection card fills after assembling
Enter aluminium foil bag, desiccant sealing is added, being placed under drying at room temperature environment can be reserved for 12 months.
A method of using amphetamine in above-mentioned test strips test 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 card is inserted into, reads standard curve；
3）Into the sample well 9 of test strips be added sample to be tested 60-120 μ L, react 5-10min after, will test card insertion enter it is immune
Fluorescence analyser；
4）The fluorescent microsphere of detection line and nature controlling line is trapped under best exciting light, issues strong fluorescent bands；
5）The fluorescence intensity of detection line and nature controlling line is read using immunochromatographiassays assays instrument, and provides T/C value, and analyzer can pass through
Built-in standard curve calculates the concentration of amphetamine in sample, and judges yin and yang attribute, as shown in Figure 3.
The detection of amphetamine content in 1 hair sample of test example
1, the foundation of amphetamine test strip standard curve
Take blank hair sample, ultrasound after shredding, added in the solution after ultrasound respectively amphetamine to final concentration of 0ppb,
3.125ppb, 6.25ppb, 12.5ppb, 25ppb, 50ppb, 100ppb, 200ppb take test strips to be detected, each sample
Replication five times.After five reproducible results of measurement are averaged, demarcated on immunofluorescence analysis instrument.
2, in hair sample amphetamine 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 solution is added, 3 min of water bath sonicator；
（2）It is detected with test strips
1. opening immunofluorescence analysis instrument, it is inserted into corresponding ID card, reads standard curve.
2. face-up laying 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, it will test card and be put into immunofluorescence analysis instrument and detected
3. being trapped in the fluorescent microsphere of detection line and nature controlling line under best exciting light, strong fluorescent bands are issued；
4. reading the fluorescence intensity of detection line and nature controlling line using immunochromatographiassays assays instrument, and T/C value is provided, analyzer can pass through
Built-in standard curve calculates the concentration of amphetamine in sample, and judges yin and yang attribute.
The detection of amphetamine content in 2 blood sample of test example
In the embodiment, all preparation methods are same as Example 1, except sample absorption pad is blood filter membrane.
1, hemolytic blood sample
（1）The foundation of amphetamine test strip standard curve
Blank blood sample is taken, presses 1 with sample diluting liquid:5-1:20 ratio is diluted, to the hemolytic blood sample after dilution
Added respectively in product solution amphetamine to final concentration of 0ppb, 3.125ppb, 6.25ppb, 12.5ppb, 25ppb, 50ppb,
100ppb, 200ppb 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 amphetamine in hemolytic blood sample
1. the pre-treatment of sample
Sample is restored to 20-25 DEG C of room temperature before a detection；
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 card, reads standard curve.
B face-up lays flat detection card, and sample to be tested 60-120 μ L is added into the sample well of test strips, reacts at room temperature 5-
After 10min, it will test card and be put into immunofluorescence analysis instrument and detected
C is trapped in the fluorescent microsphere of detection line and nature controlling line under best exciting light, issues strong fluorescent bands；
D reads the fluorescence intensity of detection line and nature controlling line using immunochromatographiassays assays instrument, and provides T/C value, and analyzer can pass through
Built-in standard curve calculates the concentration of amphetamine 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 amphetamine 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 amphetamine 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 slot cover 10
It is provided with minimum collection line 14.
The acquisition of saliva sample carries out in accordance with the following steps：
1, by saliva spit into acquisition slot in until saliva amount reach minimum gathering line.
2, acquisition slot cover is covered tightly, until hearing click, the sample diluting liquid in lid is flowed into collection tube, with saliva
Sample mixes.
3, acquisition slot is backed out, covers collection tube lid, mixing of turning upside down.
4, the saliva sample after drawing dilution is detected.
The foundation of basic parameter
Detection limit：It is carried out replication 20 times with blank sample, 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 0.5ppb.The range of linearity：Take 0ppb, 3.125ppb,
6.25ppb, 12.5ppb, 25ppb, 50ppb, 100ppb, 200ppb isoconcentration value are detected, each concentration duplicate measurements five
It is secondary, measurement mean concentration and theoretical value are subjected to linear analysis, obtain linear equation y=- 0.9649x+0.6194, R2=
0.9947.（Experimental result and analysis are shown in Table 1, Fig. 4）.
1 amphetamine standard items testing result of table
Accuracy：The amphetamine mark product for being 5ppb with sample diluting liquid compound concentration, are carried out with test strips prepared in embodiment 1
Detection repeats detection three times, and testing result, which is averaged, to be calculated.The rate of recovery=detectable concentration/be actually added into concentration ×
100%, calculate sample recovery rate be 102.24%.
Precision：It takes time-resolved fluorescence prepared in three batches of embodiments 1 to quantify amphetamine test strip, detects
5ppb concentration mark product, every batch of test strips mark product Parallel testing 10 times, the results show that CV value is respectively 4.466% in three batches of batches,
0.987%, 4.501%, CV value is 3.896% between three batches of batches.
It is seen from the above data that test strips of the present invention have more compared to the method for existing detection amphetamine
It is sensitive, more rapidly, can accurate quantitative analysis the characteristics of, do not limited by operation place, and matched detecting instrument small volume and less weight, personnel
It is required that it is lower, it is suitble to promote the use of on a large scale.
Claims (10)
1. a kind of test strips for detecting amphetamine, 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；It is wrapped on the reaction film
The nature controlling line of the detection line and sheep anti mouse secondary antibody that are had hapten-carrier protein conjugate.
2. test strips according to claim 1, it is characterised in that：The sample absorption pad, conjugate release pad, reaction film
It is pasted into 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 that diameter is 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 for preparing any one of claim 1-6 test strips, which is characterized in that include the following steps：
1）Amphetamine antibody is marked with fluorescent microsphere, and is sprayed in object release pad, amphetamine is coated with preparation
Monoclonal antibody-Fluorescent microsphere marker conjugate release pad；
2）Hapten-carrier protein conjugate and sheep anti mouse secondary antibody are sprayed on reaction film respectively 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 for preparation test strips according to claim 7, it is characterised in that：In step 1）Middle taking-up fluorescent microsphere into
Row activation, then be added amphetamine antibody carry out covalent coupling, after be added Block buffer closed, after centrifuge washing
4 DEG C save backup.
9. the method for preparation test strips according to claim 7, 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 impregnates 2 h, dries 2 h at 37 DEG C, be placed in dry environment save it is standby
With.
10. a kind of method of amphetamine in test strips test sample using any one of the claim 1-6 detection amphetamine,
It 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 card is inserted into, reads standard curve；
3）Into the sample well of test strips be added sample to be tested 60-120 μ L, react 5-10min after, will test card insertion enter to be immunized it is glimmering
Light analyzer；
4）The fluorescent microsphere of detection line and nature controlling line is trapped under best exciting light, issues strong fluorescent bands；
5）The fluorescence intensity of detection line and nature controlling line is read using immunochromatographiassays assays instrument, and provides T/C value, and analyzer can pass through
Built-in standard curve calculates the concentration of amphetamine in sample, and judges yin and yang attribute.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810532305.5A CN108845152A (en) | 2018-05-29 | 2018-05-29 | A kind of test strips and its preparation method and application method detecting amphetamine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810532305.5A CN108845152A (en) | 2018-05-29 | 2018-05-29 | A kind of test strips and its preparation method and application method detecting amphetamine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108845152A true CN108845152A (en) | 2018-11-20 |
Family
ID=64209962
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810532305.5A Pending CN108845152A (en) | 2018-05-29 | 2018-05-29 | A kind of test strips and its preparation method and application method detecting amphetamine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108845152A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101556280A (en) * | 2009-05-20 | 2009-10-14 | 江西中德生物工程有限公司 | Immunochromatography detection card for detecting fluorescent microspheres of ketamine and methyl amphetamine and preparation method thereof |
| CN101788559A (en) * | 2004-04-23 | 2010-07-28 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immune chromatography test paper strip based on up-conversion luminescence technology |
| CN102890152A (en) * | 2011-07-20 | 2013-01-23 | 天津中新科炬生物制药有限公司 | Test strip and method for fast quantitative detection of drug in blood |
| CN104422765A (en) * | 2013-08-30 | 2015-03-18 | 上海八通生物科技有限公司 | Test bar and method for quantitatively detecting micromolecular compound in sample |
| CN105486859A (en) * | 2015-11-20 | 2016-04-13 | 润和生物医药科技(汕头)有限公司 | Novel improved immunochromatographic test strip, and preparation and application thereof |
| CN107478829A (en) * | 2017-09-29 | 2017-12-15 | 亳州市新健康科技有限公司 | A kind of illicit drugs inspection kit and its preparation technology based on double competition immunochromatographic methods |
-
2018
- 2018-05-29 CN CN201810532305.5A patent/CN108845152A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101788559A (en) * | 2004-04-23 | 2010-07-28 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immune chromatography test paper strip based on up-conversion luminescence technology |
| CN101556280A (en) * | 2009-05-20 | 2009-10-14 | 江西中德生物工程有限公司 | Immunochromatography detection card for detecting fluorescent microspheres of ketamine and methyl amphetamine and preparation method thereof |
| CN102890152A (en) * | 2011-07-20 | 2013-01-23 | 天津中新科炬生物制药有限公司 | Test strip and method for fast quantitative detection of drug in blood |
| CN104422765A (en) * | 2013-08-30 | 2015-03-18 | 上海八通生物科技有限公司 | Test bar and method for quantitatively detecting micromolecular compound in sample |
| CN105486859A (en) * | 2015-11-20 | 2016-04-13 | 润和生物医药科技(汕头)有限公司 | Novel improved immunochromatographic test strip, and preparation and application thereof |
| CN107478829A (en) * | 2017-09-29 | 2017-12-15 | 亳州市新健康科技有限公司 | A kind of illicit drugs inspection kit and its preparation technology based on double competition immunochromatographic methods |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108279309B (en) | Detection test strip and detection method for PLA2R antibody | |
| CN108802407A (en) | A kind of test strips and its preparation method and application method of detection morphine | |
| CA1206899A (en) | Specific binding assays utilizing analyte-cytolsin conjugates | |
| CN108845151A (en) | A kind of test strips and its preparation method and application method detecting crystal methamphetamine | |
| EP0026176B1 (en) | Double tagged immunoassay | |
| CN108398562A (en) | Cystatin C fluorescent micro-ball immune chromatography quantitative testing test paper item and test card | |
| CN101000343A (en) | Immunological test element with improved control zone | |
| CN107976541A (en) | Test card, preparation and the detection method of synchronous detection zearalenone and aflatoxin B1 | |
| JPS6488155A (en) | Immunological inspection for detecting antibody against antigen | |
| CN109765384A (en) | A kind of canine coronavirus antibody fluorescence test strip and its preparation method and application | |
| CN113640521A (en) | Test strip and kit for quantitatively determining drugs in hair, preparation method and application | |
| CN110927395A (en) | Triple test strip for detecting methamphetamine, morphine and ketamine as well as preparation method and application method thereof | |
| WO1985000663A1 (en) | Immunometric assay using polyclonal and monoclonal antibodies and a kit for use therein | |
| CN105467116A (en) | Convenient procalcitonin detection kit | |
| CN108956974A (en) | A kind of test strips and its preparation method and application method detecting ketamine | |
| CN103926401A (en) | Immunofluorescence test paper strip for rapidly and quantitatively measuring IGFBP-7 and TIMP-2 and preparation method thereof | |
| CN108802393A (en) | A kind of test strips and its preparation method and application method of detection tetrahydro-cannabinolic acid | |
| CN205193076U (en) | Biotin - rapid detection cards of avidin system | |
| CN105785041A (en) | Test strip for quantitatively detecting calprotectin, preparation method thereof and determining method for calprotectin concentration | |
| CN205229167U (en) | Quick quantitative detection AMH's test paper | |
| CN104215757B (en) | Biological fluid sample quantitative test device and its detection method | |
| US4454226A (en) | Enzymatic immunoassay | |
| CN114544564A (en) | Morphine and methamphetamine combined determination reagent based on AIE nano-microspheres and preparation method and application thereof | |
| CN109856407A (en) | A kind of canine distemper virus antibody fluorescence test strip and its preparation method and application | |
| CN108226468A (en) | A kind of test strips for detecting NT-proBNP and preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20181120 |