CN108918896A - A kind of FITC test strips and its preparation method and application method detecting Benzodiazepine - Google Patents
A kind of FITC test strips and its preparation method and application method detecting Benzodiazepine Download PDFInfo
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Abstract
The invention discloses a kind of FITC test strips for detecting Benzodiazepine and its preparation method and application methods.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 FITC 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 Benzodiazepine using immunochromatography technique with FITC labelled antibody.The present invention also provides a kind of methods using Benzodiazepine 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 Benzodiazepine sample is used for quickly detecting and on-site test.
Description
Technical field
It is specifically a kind of to utilize FITC immunochromatography technique quantitative detection benzene phenodiazine the present invention relates to the detection of Benzodiazepine
Tall and erect FITC test strips and its preparation method and application method.
Background technique
Benzodiazepine, 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 Benzodiazepine, which has, is overexcited, no
Pacify, have a sleepless night, trembling, the symptoms such as nervous and agitation.Occur to the body tolerance of Benzodiazepine it is very fast, so taking for a long time
Person the more must take the more more.Severest consequences are exactly a kind of toxicity neuropathy, symptom class after taking large dosage of Benzodiazepine
Like class paranoid schizophrenia.The abuse of Benzodiazepine often occurs with the abuse of barbiturate drugs and alcohol together.Benzene phenodiazine
Zhuo Chaoliang or Reusability can produce ill hobby, and cause excited and process of inhibition dysequilibrium and lead to mental symptom,
Therefore use should be controlled strictly.
At present for Benzodiazepine detection and monitoring method mainly include high performance liquid chromatography, gas-chromatography, liquid matter or
Makings is used in conjunction.These chromatographic processes have good sensitivity and specificity, but complicated for operation, and flux is not high, take a long time,
Instrument and equipment is expensive.Enzyme linked immunosorbent assay (ELISA) (ELISA) and colloidal gold immunity chromatography are current internationally recognized mainstreams
Technology, both methods have detection speed fast, cheap, simple operation and other advantages.But ELISA detection still needs to professional people
Member, and the long period is needed to show result；Colloidal gold method can carry out qualitative analysis, easy to operate, detection time to Benzodiazepine
It is shorter, but sensitivity is low, interference from human factor is larger.
In conclusion the detection technique of Benzodiazepine is still immature at present, develop a kind of high sensitivity, it is easy to operate and
Can be realized the product being used for quickly detecting to batch samples and method becomes problem in the urgent need to address.
Summary of the invention
It is an object of the invention to overcome it is existing detection Benzodiazepine method existing for it is complicated for operation, take a long time,
And the characteristics of can not realizing the quick detection to batch samples, provides one kind easy to operate, high sensitivity, detection speed
Fastly, FITC test strips of detection Benzodiazepine at low cost and its preparation method and application method, to realize to large batch of benzene
Phenodiazine Zhuo sample is used for quickly detecting and on-site supervision.
In order to achieve the object of the present invention, a kind of FITC test strips of detection Benzodiazepine of the invention use following technology
Scheme：It is a kind of detect Benzodiazepine FITC 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 FITC label is coated in the conjugate release pad；Half is coated on the reaction film
The detection line of antigen-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 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 FITC test strips preparing above-mentioned detection Benzodiazepine of the invention, includes the following steps：
1）Benzodiazepine antibody is marked with FITC, and is sprayed in object release pad, Benzodiazepine is coated with preparation
The conjugate release pad of monoclonal antibody-FITC marker；
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 configuration FITC solution, then be added Benzodiazepine antibody carry out covalent coupling, after be added envelope
It closes buffer to be closed, be saved backup for 4 DEG C after centrifuge washing.
In step 1）It is middle that the antibody of the FITC Benzodiazepine marked is sprayed into conjugate release pad with spray film instrument, every 1
Cm conjugate release pad sprays the Benzodiazepine antibody of 1-9 μ L FITC label, and 37 DEG C of dry 1-2h are placed in standby in dry environment
With.
Benzodiazepine in a kind of FITC test strips test sample of the above-mentioned detection Benzodiazepine of application method of the 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 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 FITC 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 Benzodiazepine 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 Benzodiazepine 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.
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.It is easy, fast with the method for test strips of the present invention detection Benzodiazepine
Fast, accurate, 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 FITC test strips of detection Benzodiazepine 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 FITC test strips of a kind of detection Benzodiazepine 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 FITC label is coated in conjugate release pad；It is wrapped on reaction film
There are the detection line 6 of hapten-carrier protein conjugate and the nature controlling line 7 of sheep anti mouse secondary antibody.Sample absorption pad, conjugate 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, conjugate release
The 1/3 of 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 testing result
Observing time can make sample absorption pad will test liquid and fully absorb and sufficiently react with antibody, so that error is reduced, at other
In embodiment, it can also be capped under sample absorption pad with the 1/2 of conjugate release pad.It is various that FITC is that one kind is usually used in marking
The fluorescent dye of albumen, is mainly used in Immunofluorescence test.FITC can be in conjunction with various antibody proteins, and the antibody in conjunction with after is not
The specificity with antigen binding is lost, and the yellow-green fluorescence that it is issued under ultraviolet wavelength can be examined via optical instrument
It surveys, realizes quantitative detection.Sample absorption pad is glass fibre element film.Conjugate release pad is glass fibre or polyester material.Instead
Answering film is nitrocellulose filter 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 FITC test strips of the detection Benzodiazepine mainly includes the following steps that：
1）Benzodiazepine antibody is marked with FITC, and is sprayed in object release pad, FITC label is coated with preparation
Benzodiazepine antibody 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）, the detection line of Benzodiazepine hapten-carrier protein conjugate is coated with preparation and is coated with sheep anti mouse secondary antibody
The reaction film of 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 FITC marker：
The preparation of FITC label Benzodiazepine antibody：The carbonate that Benzodiazepine monoclonal antibody to be marked is dissolved in 1M is delayed
Fliud flushing（pH9.0）In, antibody final concentration is not less than 2mg/mL.The FITC solution for preparing 1mg/mL is protected from light with DMSO（DMSO solution
It needs ready-to-use）.It is protected from light, the FITC solution of 50-100 μ L is slowly added into 1mL antibody-solutions, in the process that FITC is added
In mix gently antibody-solutions.Mixed solution is protected from light, 4 DEG C be stirred to react 8h.Then the NH of 5M is added4Cl, 4 DEG C of stirrings
2h.Then PBS is placed reaction liquid into（pH7.4）Middle dialysis, until dialyzate is refrigerant.The benzene two of the FITC label as prepared
The antibody of nitrogen Zhuo, be placed in 4 DEG C it is spare.
2, the preparation of FITC pad：
The antibody of the FITC Benzodiazepine marked is sprayed into conjugate release pad with spray film instrument, every 1 cm conjugate release pad
The Benzodiazepine antibody of 1-9 μ L FITC label is sprayed, 37 DEG C of dry 1-2h are placed in spare in dry environment.
3, the preparation of NC film：
Benzodiazepine hapten-carrier protein conjugate and sheep anti-mouse antibody are coated in respectively on NC film：With 0.02M pH7.4
PBS Benzodiazepine hapten-carrier protein conjugate is adjusted to 0.5-2mg/mL, be coated on NC film and form 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, is coated on NC
Nature controlling line is formed on film, 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 drying
It is spare in environment.
4, the preparation of FITC immune detection card
Sample absorption pad is successively pasted on PVC bottom plate, is coated with the conjugate release pad of the Benzodiazepine antibody of FITC label,
It is coated with reaction film of the Benzodiazepine 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
Well and detection window are reserved respectively in the part of sample absorption pad and NC film.The detection card is packed into aluminium foil bag after assembling, and adds
Enter desiccant sealing, being placed under drying at room temperature environment can be reserved for 12 months.
A kind of method of Benzodiazepine in the above-mentioned test strips test sample of application method, 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 FITC 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 Benzodiazepine in sample, and judges yin and yang attribute, as shown in Figure 3.
The detection of Benzodiazepine content in 1 hair sample of test example
1, the foundation of Benzodiazepine test strip standard curve
Take blank hair sample, ultrasound after shredding, added in the solution after ultrasound respectively Benzodiazepine to final concentration of 0ppb,
0.4ppb, 0.8ppb, 1.6ppb, 3.2ppb, 6.4ppb, 12.8ppb, 25.6ppb, 51.2ppb, 102.4bbp take test strips
It is detected, each sample replication five times.After five reproducible results of measurement are averaged, in immunofluorescence analysis instrument
On demarcated.
2, in hair sample Benzodiazepine 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 FITC 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 Benzodiazepine in sample, and judges yin and yang attribute.
The detection of Benzodiazepine 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 Benzodiazepine 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 Benzodiazepine to final concentration of 0ppb, 0.4ppb, 0.8ppb, 1.6ppb, 3.2ppb, 6.4ppb,
12.8ppb, 25.6ppb, 51.2ppb, 102.4bbp take test strips to be detected, each sample replication five times.It will measurement
Five reproducible results be averaged after, demarcated on immunofluorescence analysis instrument.
（2）The detection of Benzodiazepine 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 FITC 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 Benzodiazepine 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 Benzodiazepine 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 Benzodiazepine 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 0.2ppb.The range of linearity：Take 0ppb, 0.4ppb,
0.8ppb, 1.6ppb, 3.2ppb, 6.4ppb, 12.8ppb, 25.6ppb, 51.2ppb, 102.4bbp isoconcentration value are detected,
Each concentration duplicate measurements five times, will measurement mean concentration and theoretical value carry out linear analysis, obtain linear equation y=-
2.0321x+1.6886 R2=0.9912.（Experimental result and analysis are shown in Table 1, Fig. 4）.
1 Benzodiazepine standard items testing result of table
Accuracy：The Benzodiazepine mark product for being 10ppb with sample diluting liquid compound concentration, are detected with prepared test strips,
Repeat detection three times, testing result, which is averaged, to be calculated.The rate of recovery=detectable concentration/is actually added into concentration × 100%, calculates
Obtaining sample recovery rate is 107.1%.
Precision：It takes time-resolved fluorescence prepared in three batches of embodiments 1 to quantify Benzodiazepine test strip, detects
20ppb concentration mark product, every batch of test strips mark product Parallel testing 10 times, the results show that CV value is respectively in three batches of batches
10.630%, 14.556%, 12.511%, CV value is 12.738% between three batches of batches.
It is seen from the above data that test strips of the present invention have compared to the method for existing detection Benzodiazepine
It is sensitiveer, more rapidly, can accurate quantitative analysis the characteristics of, do not limited by operation place, and matched detecting instrument small volume and less weight, people
Member requires lower, suitable popularization and use on a large scale.
Claims (8)
1. a kind of FITC test strips for detecting Benzodiazepine, including sample absorption pad, conjugate release pad, reaction film, water absorption pad
And bottom plate；It is characterized in that：The antibody of FITC label is coated in the conjugate release pad；Half is coated on the reaction film
The detection line of antigen-carrier protein conjugate and the nature controlling line of sheep anti mouse secondary antibody.
2. FITC test strips according to claim 1, it is characterised in that：It is the sample absorption pad, conjugate release pad, anti-
Film and water absorption pad is answered to be pasted on bottom plate and the partial stack along the vertical direction of middle arbitrary neighborhood two, the 1/ of conjugate release pad
It is capped under sample absorption pad.
3. FITC test strips according to claim 1, it is characterised in that：The conjugate release pad is glass fibre or gathers
Ester material.
4. FITC test strips according to claim 1, it is characterised in that：The reaction film is nitrocellulose filter or acetic acid
Cellulose membrane.
5. a kind of method for preparing any one of claim 1-4 FITC test strips, which is characterized in that include the following steps：
1）Benzodiazepine antibody is marked with FITC, and is sprayed in object release pad, Benzodiazepine is coated with preparation
The conjugate release pad of monoclonal antibody-FITC marker；
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.
6. the method according to claim 5 for preparing FITC test strips, it is characterised in that：In step 1）It is molten to configure FITC
Liquid, then be added Benzodiazepine antibody carry out covalent coupling, after be added Block buffer closed, 4 after centrifuge washing
It DEG C saves backup.
7. the method according to claim 5 for preparing FITC test strips, it is characterised in that：In step 1）It is middle to use spray film instrument
The antibody of the Benzodiazepine of FITC label is sprayed into conjugate release pad, and every 1 cm conjugate release pad sprays 1-9 μ L
The Benzodiazepine antibody of FITC label, 37 DEG C of dry 1-2h are placed in spare in dry environment.
8. Benzodiazepine in a kind of FITC test strips test sample using any one of the claim 1-4 detection Benzodiazepine
Method, 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 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 FITC 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 Benzodiazepine in sample, and judges yin and yang attribute.
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Application publication date: 20181130 |