CN109959795A - A kind of development and application of matrix type test strips - Google Patents
A kind of development and application of matrix type test strips Download PDFInfo
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- CN109959795A CN109959795A CN201711432666.4A CN201711432666A CN109959795A CN 109959795 A CN109959795 A CN 109959795A CN 201711432666 A CN201711432666 A CN 201711432666A CN 109959795 A CN109959795 A CN 109959795A
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- 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
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses a kind of matrix type test strips and its applications.Test strips include sample absorption pad (1), conjugate release pad (2), reaction film (3), water absorption pad (4) and bottom plate (7)；There is antigen (or antibody) matrix dot (5) for being coated with object hapten-carrier protein conjugate on the reaction film and be coated with the mostly anti-Quality Control matrix dot (6) of goat-anti rabbit, the conjugate release pad (2) is coated with antibody (or antigen)-colloidal gold composite.Test strips provided by the present invention can detect 6 or more projects simultaneously, have the characteristics that easy to operate, high sensitivity, detection speed are fast, at low cost, be suitble to the screening and on-site supervision of great amount of samples.
Description
Technical field
The present invention relates to colloidal gold immunochromatographimethod technologies, and in particular to a kind of matrix type test strips, it can quantitative detection target
Object content.
Background technique
Colloidal gold strip is in important quick detection means a kind of at this stage, because it is simple, quick, convenient at any time
It detects the characteristics of using everywhere and is applied in various types of detection work.Its main principle utilizes the spy of antigen-antibody
Opposite sex identification combines, and traditional colloidal gold strip is that the antigen (antibody) of specificity is solid in ribbon form using film instrument is drawn
It is scheduled on nitrocellulose (NC) film, it is and fixed anti-when gold labeling antibody (or antigen) is moved to through capillary action on NC film
Former (antibody) combines, and red response line is presented on film.
There is also some problems in use because of its technical reason for colloidal gold strip product at present, wherein most important
One of problem is a lack of the corresponding multiple projects of detection of single test strips.Most of test strips testing products are single item detection,
I.e. a test strips, which can only correspond to, detects a kind of target to be measured；And the colloidal gold strip product testing item of existing entry
Mesh at most also can be only formed 1 nature controlling line and 4 detection line combining forms, that is to say, that existing test strips product at most can only one
4 kinds of target to be measured of secondary detection.
Be detected the confined principal element of the number of entry be, between the two lines drawn by existing stroke of film instrument away from
From at least 4-5mm, with the increase of the number of entry, necessarily causes the width of NC film to increase and chromatograph prolonging for distance with test strips
It is long.Specifically, general general commercial NC film width is 25mm, and with the raising for requiring width, the price of NC film can also be got over
Height improves test strips cost price；On the other hand, the chromatography height of liquid also has certain limit, becomes and increases detection project
Important limiting factor, entry test strips necessarily encounter that the chromatography reaction time is too long and chromatography insufficient height is asked when reacting
Topic；In addition, lower layer's band can generate inhibition to the colour developing of upper layer band when having a plurality of band on NC film, lead to more up face
The more shallow problem of color.Three above limiting factor result in colloidal gold entry test strip further reduce the cost and
Large-scale application.
Summary of the invention
The matrix type test strips that 6 or more (containing 6) projects can be detected simultaneously the object of the present invention is to provide two kinds
Design scheme.
A kind of matrix type test strips provided by the present invention, wherein gold-labelled pad method includes that sample absorption pad (1), conjugate are released
Put pad (2), reaction film (3), water absorption pad (4) and bottom plate (7)；Have on the reaction film and is coated with object hapten-carrier
Antigen (or antibody) matrix dot (5) of protein conjugate and it is coated with the mostly anti-Quality Control matrix dot (6) of goat-anti rabbit, the conjugate
Release pad (2) is coated with antibody (or antigen)-colloidal gold composite.
The object hapten-carrier protein conjugate is to be obtained by object haptens with carrier protein couplet, institute
Stating carrier protein is bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins etc..
The object monoclonal antibody is to prepare to obtain using object hapten-carrier protein conjugate as immunogene
, it is to be secreted to obtain by object monoclonal antibody hybridoma cell strain；The goat anti-rabbit antibody is by the immunoglobulin of rabbit
Injecting immune sheep obtains.
The sample absorption pad (1), conjugate release pad (2), reaction film (3), water absorption pad (4) are successively pasted onto bottom plate
(7) on, the conjugate release pad 1/3~1/4 is capped under sample absorption pad.
The bottom plate is the material that PVC bottom plate or other hard do not absorb water；The sample absorption pad is suction strainer paper or oil strain
Paper；The conjugate release pad is mineral wool or polyester material；The water absorption pad is blotting paper；The reaction film is cellulose nitrate
Plain film or cellulose acetate film.
Freeze-drying Jin Fayu gold-labelled pad method, which is compared, does not need conjugate release pad, and additionally needs preparation and golden micropore is lyophilized, i.e.,
Including sample absorption pad (1), reaction film (3), water absorption pad (4), bottom plate (7) and golden micropore is lyophilized；Have on the reaction film
The antigen matrix dot (5) for being coated with object hapten-carrier protein conjugate and the Quality Control matrix for being coated with goat anti-rabbit antibody
Point (6) contains antibody (or antigen)-colloidal gold composite freeze-drying gold micropore.
A second object of the present invention is to provide the methods for preparing above-mentioned test strips comprising step:
1. gold-labelled pad method
1) preparation is coated with antibody (or antigen)-colloidal gold composite conjugate release pad；
2) preparation tool is sprayed with the detection dot matrix of target antigen (antibody) and is coated with the anti-of the Quality Control point of goat anti-rabbit antibody
Answer nitrocellulose filter；
And 2) 3) 1) the conjugate release pad, reaction film and sample absorption pad, water absorption pad and the bottom plate that prepare are assembled into
Test strips.
Specifically, step respectively include:
1) object immunogen immune mouse is used, by mouse boosting cell and myeloma cell by fusion, screening, obtains mesh
The strain of object monoclonal hybridoma is marked, cell strain injection mouse peritoneal is obtained into odd contradictive hydroperitoneum, purifying obtains monoclonal antibody；
2) rabbit igg immune health goat is extracted, goat anti-rabbit antibody is obtained；
3) it is reacted with trisodium citrate with gold chloride and prepares colloidal gold；
4) object monoclonal antibody prepared by step 1) is added according to a certain percentage in the colloidal gold of step 3) preparation,
Obtain antibody-colloidal gold compound；Object antigen is added in the colloidal gold of step 3) preparation according to a certain percentage, is resisted
Original-colloidal gold composite；
5) antibody (or antigen)-colloidal gold composite is sprayed in conjugate release pad, takes out, is placed in after 37 DEG C of baking 1h
It is saved backup in dry environment；
6) it will test target antigen specking on nitrocellulose filter, be coated on reaction film and constitute detection matrix area,
The how anti-point of goat-anti rabbit is constituted into nature controlling line on nitrocellulose filter；
7) sample absorption pad is used and contains 0.5% bovine serum albumin(BSA) (volume fraction), pH 7.2,0.1mol/L phosphate
Buffer impregnates 2h, dries 2h at 37 DEG C；
8) upper sample absorption pad, conjugate release pad, reaction film, water absorption pad are pasted in order on bottom plate, sample absorbs
Pad covers conjugate release pad, is finally cut into the small item of proper width (4-20mm), adds plastic casing, is vacuum-packed, 4~30 DEG C of items
It can be reserved for 12 months under part.
2. golden method is lyophilized
1) preparation is equipped with the freeze-drying gold micropore of antibody-colloidal gold compound；
2) preparation tool is sprayed with the detection dot matrix of target antigen (antibody) and is coated with the anti-of the mostly anti-Quality Control point of goat-anti rabbit
Answer nitrocellulose filter；
3) reaction film prepared, sample absorption pad, water absorption pad and bottom plate are assembled into test strips.
Specifically, step respectively include:
1) object immunogen immune mouse is used, by mouse boosting cell and myeloma cell by fusion, screening, obtains mesh
The strain of object monoclonal hybridoma is marked, cell strain injection mouse peritoneal is obtained into odd contradictive hydroperitoneum, purifying obtains monoclonal antibody；
2) rabbit igg immune health goat is extracted, goat-anti rabbit-anti antibody is obtained；
3) it is reacted with trisodium citrate with gold chloride and prepares colloidal gold；
4) colloidal gold of the object monoclonal antibody of step 1) preparation and step 3) preparation is added according to a certain percentage
Into 200 μ L milk, pH is adjusted, then micropore freeze-drying is put into freeze dryer, freeze-drying gold is made；
5) it will test target antigen specking on nitrocellulose filter, be coated on reaction film and constitute detection matrix area,
Goat anti-rabbit antibody point is constituted into Quality Control point on nitrocellulose filter；
6) upper sample absorption pad, reaction film, water absorption pad are pasted in order on bottom plate, sample absorption pad covers conjugate and releases
Pad is put, the small item of proper width (4-20mm) is finally cut into, is vacuum-packed, can be reserved for 12 months under the conditions of 4~30 DEG C.
Third object of the present invention is to provide a kind of above-mentioned test strips of application to detect the remaining method of object, it includes
Step:
(1) sample pre-treatments；
(2) it is detected with test strips；
(3) analysis detection result.
The present invention carries out the preparation of matrix test strips using normal width NC film (25mm).According to different detection mesh numbers with
And project repeat number design dot matrix scheme and path, object hapten-carrier protein conjugate is coated with by what is handled well
Antigen (or antibody) is sprayed on nitrocellulose filter.Specifically by taking one matrix test strips of the six directions as an example, top line is set as matter
Point is controlled, the following are detection project point, each project repeats point according to required examination width design 3-4.Other test strips handle work
Skill is substantially identical as existing test strips technique.
The matrix type test strips of the present patent application, 4 detection projects can only be had by breaching original test strips product first
Limitation, the detection of 6-8 project can be carried out using the film of 25mm width, if using broader 40mm specification nitrocellulose filter
It can carry out the detection of more items.Second, this technology joined multiple projects on NC film as short as possible, avoid using more
Test strips detect waste and cost problem caused by same sample.Further, since this technology is the form using colloid gold point,
So it is possible to prevente effectively from the colour developing between entry caused by crossing is interfered.Antigen needed for this project, antibody and detection
Sample is substantially reduced compared with traditional test strip product, greatly reduces the production cost and detection unit of test strips
Testing cost.
Test strips of the invention have entry integration detection great potential, can effectively improve detection efficiency and
Testing cost is one important developing direction of the following test strips product.
Detailed description of the invention
Fig. 1 is test strips the schematic diagram of the section structure.
Specific embodiment
Below with reference to coming for specific one test strips of the dairy products six directions (golden method is lyophilized), the present invention is further explained.Ying Li
Solution, these embodiments are merely to illustrate the present invention, and are not intended to limit the scope of the invention.
The preparation of the 1 matrix type dairy products six directions of embodiment, one test strips
The preparation method of the test strips mainly comprises the steps that
1) preparation is equipped with the freeze-drying gold micropore of antibody-colloidal gold compound, and it is mould to respectively include tetracycline, melamine, chlorine
Element, tylosin, quinolone and beta-lactam antibiotic a variety of antibody for small molecular substance；
2) preparation has the reaction of the test point for being coated with object coating antigen and the Quality Control point for being coated with goat anti-rabbit antibody
Film, from top to bottom respectively goat-anti rabbit test point and beta-lactam antibiotic, tetracycline, melamine, chloramphenicol, safe happy
Rhzomorph and quinolone test point；
3) 2) reaction film prepared and sample absorption pad, water absorption pad and bottom plate are assembled into test strips.
Substep narration in detail below:
1, the preparation of object monoclonal antibody
(1) animal immune
Immunogene is injected in Balb/c Mice Body, immunizing dose is 150 μ g/, its is made to generate antiserum.
(2) cell fusion and cloning
Immune Balb/c mouse boosting cell is taken, is merged in 8:1 (quantitative proportion) ratio with SP2/0 myeloma cell, is used
Indirect competitive ELISA method measures cell supernatant, screens positive hole.Cloning is carried out to positive hole using limiting dilution assay, directly
To obtaining the hybridoma cell strain of stably excreting monoclonal antibody.
(3) cell cryopreservation and recovery
Hybridoma is made 1 × 10 with frozen stock solution6The cell suspension of a/ml, saves for a long time in liquid nitrogen.When recovery
Cryopreservation tube is taken out, 37 DEG C of water-bath middling speeds is immediately placed in and melts, after centrifugation removal frozen stock solution, moves into culture culture in glassware.
(4) preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in cell culture medium, is cultivated under the conditions of 37 DEG C, with octanoic acid-
Saturated ammonium sulfate method purifies obtained culture solution, obtains monoclonal antibody, -20 DEG C of preservations.
The cell culture medium is that calf serum and sodium bicarbonate are added into RPMI1640 culture medium, and calf serum is made to exist
Final concentration of 20% (mass fraction) in cell culture medium, the final concentration of 0.2% (matter of sodium bicarbonate in cell culture medium
Measure score)；The pH of the cell culture medium is 7.4.
5, the preparation of goat-anti rabbit-anti antibody
Using sheep as immune animal, pathogen-free domestic sheep is immunized using rabbit source antibody as immunogene, obtains goat-anti rabbit-anti
Body.
6, antibody (or antigen)-colloidal gold composite preparation
(1) preparation of colloidal gold
1% gold chloride is diluted to 0.01% (mass fraction) with double distilled deionized water, 100ml is taken to be placed in conical flask,
It is heated to boiling with thermostatic electromagnetic blender, in continuous high temperature, is persistently added with stirring 1% trisodium citrate of 2.5ml, continue
Solution is at the uniform velocity heated with stirring in stopping when bright red, is restored to original volume, 4 DEG C of guarantors with deionized water after being cooled to room temperature
It deposits.The colloidal gold appearance prepared is pure, it is bright, without precipitating and floating material.
(2) preparation of antibody-colloidal gold compound
Suitable seven kinds of (tetracycline, melamine, chloramphenicol, tylosin, quinolines are separately added into 2ml colloidal gold solution
Promise ketone, beta-lactam antibiotic and rabbit igg antibody) 1 μ g/ml antibody, pre-process 10min, add 10%BSA's
PBS solution closes 10min, and 12000rpm is centrifuged 5min, discards supernatant, and is buffered with 200 μ L 0.1M phosphate buffers (P BS)
Liquid redissolves；It debugs suitable ratio and prepares micropore freeze-drying gold reagent, gold reagent is lyophilized in micropore can long-term preservation.
7, the preparation of reaction film
First by different haptens-bovine serum albumin conjugate (tetracycline, melamine, chloramphenicol, tylosin,
Quinolone and beta-lactam antibiotic) it is pre-processed, it is different according to the protein concentration of disparity items antigen (antibody),
10-20 times is diluted with phosphate buffer (Phosphate Buffer, pH 7.2)；Add a certain amount of glycerol and cow's serum egg
It is white, make final glycerol concentration 0.2%, and BSA concentration is 0.05%.In addition suitable soluble color can be added in coating buffer
Element, for indicating antigen (antibody) position.12000rpm centrifugation 5min, takes supernatant stand-by after coating antigen has been handled.
Coating process: object haptens-cow's serum conjugate is diluted to 10mg/ml with phosphate buffer, will test
For antigen coat in the test point (T point) on nitrocellulose filter, package amount is single-point 30nL；With the phosphorus of 0.01mol/L, pH7.4
Goat-anti rabbit-anti antibody is diluted to 200 μ g/ml by phthalate buffer (PBS buffer solution), is coated on nitrocellulose filter
Quality Control point (C point), package amount are single-point 30nL.Dry 2h, spare under the conditions of the reaction film being coated with is placed in 37 DEG C.
Object coating antigen is coated with respectively on nitrocellulose reaction film and constitutes detection dot matrix, by goat anti-rabbit antibody
It is coated on composition Quality Control point on reaction film, but the matrix form is not intended to limit point sample form range of the invention.
9, the preparation of sample absorption pad
Sample absorption pad is placed in containing in 0.5% bovine serum albumin(BSA) (BSA), pH7.2,0.1mol/L phosphate buffer
2h is impregnated, 37 DEG C of baking 2h are spare.
10, the assembling of test strips
The NC film of 25mm width is pasted on test strips bottom plate, and is being stained with absorbent filter and sample pad respectively up and down.
Dot matrix scheme and path are designed according to item number, is coated with the anti-of object hapten-carrier protein conjugate for treated
Former (or antibody) is sprayed on NC film, and with 1 project, each project repeats 4 points, antigen (antibody) dilution of each point for every behavior
Liquid measure is 30nL, and film and bottom plate are cut into the test strips of suitable width using cutting machine.
It can detect single a certain determinand using entry matrix type test strips, 1-6 kind determinand can also be detected simultaneously,
Do not intersect and interfere between disparity items.
The remaining detection of object in 2 sample of embodiment (by taking dairy products detect as an example)
1, the pre-treatment of sample
2, it is detected with test strips
Appropriate antibody-colloidal gold complex solution is added in 500 μ L detection architectures (milk), and is added in right amount to mark
Quasi- product are as competitor, after mixed system pre-reaction 5min, are inserted into matrix test strips, take out test strips after reacting 5min, look into
See colour developing result.
3, analysis detection result
Negative (-): C point and T point all develop the color, and purplish red color dot is presented, and indicate that target concentration is lower than detection limit in sample；
Positive (+): C point develops the color and T point is reduced without colour developing or colour developing, indicates that target concentration is equal to or higher than inspection in sample
Survey limit；
It is invalid: not occur C point, show the deterioration failure of incorrect operating process or test strips.It in the case, should be again
It is secondary to read over specification, and retested with new test strips.
3 sample detection of embodiment limit test (by taking dairy products as an example)
1. adding quantitative object standard items to be detected in the negative sample of confirmation, additive amount is as shown in table 1, according to implementation
3 scheme of example is detected, compared with negative sample, after test strips are inserted into the positive sample containing six kinds of standard items, and difference detection
Point colour developing is substantially reduced or disappears, and shows that the test strips can effectively detect the substance to be checked in sample, detection effect with
Existing test strips product maintains an equal level, and is able to satisfy daily quick detection demand；
1. dairy products detection project of table
2. being measured using company's modified colloidal gold readout instrument to matrix type test strips product colour developing situation, to each
The colour developing value quantization of point carries out quantitative analysis to colour developing result using related software, and statistical result is as shown in table 2, with negative sample
Originally it compares, the positive sample colour developing value that six kinds of standard items are added is substantially reduced compared with negative sample, realizes sxemiquantitative type test paper
Item.
The 2 matrix type dairy products six directions of table, one test strips colour developing result
Claims (6)
1. a kind of matrix type test strips, including sample absorption pad (1), conjugate release pad (2), reaction film (3), water absorption pad (4)
With bottom plate (7)；There is antigen (or antibody) matrix for being coated with object hapten-carrier protein conjugate on the reaction film
Point (5) and it is coated with the mostly anti-Quality Control matrix dot (6) of goat-anti rabbit, the conjugate release pad (2) is coated with antibody (or antigen)-
Colloidal gold composite.
2. test strips as described in claim 1, it is characterised in that the sample absorption pad (1), conjugate release pad (2), anti-
Film (3), water absorption pad (4) is answered successively to be pasted on bottom plate (7).
3. test strips as described in claim 1, it is characterised in that do not need conjugate release pad, and additionally need preparation freeze-drying
Golden micropore includes sample absorption pad (1), reaction film (3), water absorption pad (4), bottom plate (7) and golden micropore is lyophilized；The reaction
There is the antigen matrix dot (5) for being coated with object hapten-carrier protein conjugate on film and be coated with goat anti-rabbit antibody
Quality Control matrix dot (6) contains antibody (or antigen)-colloidal gold composite freeze-drying gold micropore.
4. test strips as described in claim 1, it is characterised in that the test strips are to carry out matrix test strips system using NC film
Standby, according to different detection mesh numbers and project repeat number design dot matrix scheme and path, target is coated with by what is handled well
The antigen (or antibody) of object hapten-carrier protein conjugate is sprayed on nitrocellulose filter.
5. test strips as described in claim 1, it is characterised in that the test strips can detect 2 or more items simultaneously
Mesh, top line are set as Quality Control point, and the following are detection project points.
6. a kind of method for detecting medicament residue comprising step:
1) Sample pretreatment；
2) it is detected with the described in any item test strips of claim 1-5；
3) analysis detection result.
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| CN201711432666.4A CN109959795A (en) | 2017-12-26 | 2017-12-26 | A kind of development and application of matrix type test strips |
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| CN113687070A (en) * | 2021-09-06 | 2021-11-23 | 上海帛萘娅生物科技有限公司 | High-sensitivity semi-quantitative kit for detecting novel coronavirus neutralizing antibody |
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| 何方洋 等: ""动物组织中呋喃它酮代谢物快速检测试纸条的研制"", 《食品工业科技》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111351927A (en) * | 2020-03-17 | 2020-06-30 | 陈韬 | Antibody matrix detection method (MEGA method) aiming at pathogen antigen and multi-connected detection card |
| CN113687070A (en) * | 2021-09-06 | 2021-11-23 | 上海帛萘娅生物科技有限公司 | High-sensitivity semi-quantitative kit for detecting novel coronavirus neutralizing antibody |
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