CN107727870A - A kind of tropomyosin quick detection immuomagnetic bead chromatographic test strip and its application - Google Patents
A kind of tropomyosin quick detection immuomagnetic bead chromatographic test strip and its application Download PDFInfo
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- CN107727870A CN107727870A CN201711240546.4A CN201711240546A CN107727870A CN 107727870 A CN107727870 A CN 107727870A CN 201711240546 A CN201711240546 A CN 201711240546A CN 107727870 A CN107727870 A CN 107727870A
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/53—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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
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Abstract
The invention belongs to detection field, and in particular to tropomyosin in the aquatic products such as Species of Crustacea shrimp crab mollusk, the sample-pretreating method and its detection kit of tropomyosin suitable for field quick detection Species of Crustacea.Upper surface including bottom plate and bottom plate is pasted with sample pad, pad, nitrocellulose filter, adsorptive pads to the other end successively from one end；The sample pad, pad, nitrocellulose filter, adsorptive pads are staggeredly pasted onto bottom plate successively；The pad for the marked by magnetic bead that the pad is modified through antigen myosin (Tm) specific murine system monoclonal antibody；The nitrocellulose filter is the pre-coated detection line for having Tm to detect antigen and the nitrocellulose filter of the control line of goat anti-mouse IgG.In the present invention, the sample-pretreating method and its quick detection kit of tropomyosin a kind of Species of Crustacea suitable for field quick detection；The tropomyosin content that can reach in naked eyes visible range in detection sample is 5 μ g/mL.
Description
Technical field
The invention belongs to detection field, and in particular to tropomyosin in the aquatic products such as Species of Crustacea shrimp crab mollusk
In vain, suitable for field quick detection Species of Crustacea tropomyosin sample-pretreating method and its detection kit.
Background technology
Aquatic products Prawn, crab and lobster are shell-fish main allergic food, and tropomyosin is wherein most important mistake
Quick original, the increasingly sophisticated and serious and traditional cooking skill of its caused allergic symptom can not degrade tropomyosin, so
Often cause food poisoning；Current detection technique with analyze holoprotein or enzymolysis after skin section amino acid sequence mass spectrum skill
Art, detection coding anaphylactogen DNA fragmentation PCR (Polymerase Chain Reaction, PCR) technology and
The immunology detection technology of diagnosis allergy originality is representative.Because operation requires that height is not particularly suited for Site Detection；So exploitation
A kind of kit suitable for Site Detection tropomyosin is necessary.
A kind of colloidal gold strip for detecting aquatic product anaphylactogen has been invented by Zhongsheng Beikong Biological Science ＆ Technology Co., Ltd.,
It is characterized in that be made up of (patent No. CN201410231103.9) carrier board, nitrocellulose membrane etc., but its sample pre-treatments is complicated
It is unfavorable for field quick detection.Magnetic immuno-chromatographic method and the inspection of quick detection tropomyosin have been invented by Shanghai Ocean University
The preparation (patent No. CN102043055.A) of paper slip is tested, 0.15 μ g/ can be reached by quantitatively detecting tropomyosin with magnetic signal instrument
ML, but immunomagnetic beads is added in sample adds the operating time in detection, will also be detected and preceding place by instrument
Reason method is numerous and diverse, time-consuming.
It need to can only be detected plus sample liquid in test strips in detection after combination, when shortening detection operation
Between；The sensitivity that naked eyes directly detect is improved, 0.5 μ g/mL can be reached.Can realize it is simple and crude in the wild under the conditions of inspection
Survey.
The content of the invention
Therefore the task of the present invention is to develop one kind to realize quick detection, and and can reaches qualitative detection and can and accomplished
The kit of quantitative detection (5 μ g/mL) purpose.
The tropomyosin quick detection immuomagnetic bead chromatographic test strip of the present invention, including the upper surface of bottom plate and bottom plate is certainly
One end is pasted with sample pad, pad, nitrocellulose filter, adsorptive pads to the other end successively；The sample pad, pad, nitre
Acid cellulose film, adsorptive pads are staggeredly pasted onto bottom plate successively；The pad is to be repaiied through anti-Tm specific murines system monoclonal antibody
The pad of the marked by magnetic bead of decorations；The nitrocellulose filter is the pre-coated detection line and goat anti-mouse that have Tm to detect antigen
The nitrocellulose filter of IgG control line.
Wherein, the preparation method of the pad of the marked by magnetic bead through the modification of anti-Tm specific murines system monoclonal antibody is
After sample pad treatment fluid and the mixed solution of pad treatment fluid that magnetic bead is dissolved in, at the uniform velocity it is added dropwise on pad；It is described
The volume ratio of magnetic bead and mixed liquor is 1：25-30；The volume ratio of the sample pad treatment fluid and pad treatment fluid is 1：2.It is excellent
Choosing, the speed being at the uniform velocity added dropwise is 60-70 μ l/cm.The magnetic Nano magnetic bead that marked antibody is uniformly added dropwise in pad
On, reduce the complex operations that anaphylactogen magnetic Nano probe is added drop-wise to sample pad by scene.At sample treatment liquid and pad
Manage sample treatment liquid and the preferred volume ratio of pad treatment fluid in the mixed solution of liquid and improve magnetic bead in this mixed liquor
Solubility is so as to improving the sensitivity of detection so that need to can only be examined plus sample liquid in test strips in detection
Survey, shorten the detection operating time；The sensitivity that naked eyes directly detect is improved, 0.5 μ g/mL can be reached.It can realize
Detection under the conditions of field is simple and crude.
Preferably, the sample pad treatment fluid is to be told containing 0.1--0.3g/ml PVP, 1--2.5% percents by volume
The borate buffer solution solution of temperature -20.
Preferably, the pad treatment fluid is containing 0.01--0.03g/ml sucrose, 0.01--0.03g/ml BSA
Borate buffer solution.
Wherein, the tropomyosin quick detection immuomagnetic bead chromatographic test strip also includes being covered in sample pad, combined
Pad, nitrocellulose filter, the transparent plastic diaphragm seal on adsorptive pads upper strata.
The application process of above-mentioned tropomyosin quick detection immuomagnetic bead chromatographic test strip, comprises the following steps：
(1) acetone extraction to colourless shrimp is added in Tris-HCl buffer solutions, mixes, 3- is heated under fluidized state
8min, rapid cooling, centrifugation, supernatant is taken to be used to detect；
(2) supernatant in step (1) is added in the sample pad of kit, it is sample that only obvious band, which occurs, in control line
Contain tropomyosin in product.
Wherein, during acetone extraction to the colourless shrimp of the step (1), the ratio of shrimp and acetone is 1g:4-6mL.
Wherein, the Tris-HCl buffer solutions of the step (1) are NaCl containing 0.4--0.6mol/L, pH 9.2.
Wherein, quality and volume ratio are 1 in the shrimp and Tris-HCl buffer solutions of the step (1)：4-6.
Wherein, the method for the mixing of the step (1) is stirring 30--50s, ultrasonic mixing 8-12min.
It is an object of the invention to overcome above-mentioned the shortcomings of the prior art, develop a kind of scene that is applied to and quickly examine
Survey the sample-pretreating method and its quick detection kit of tropomyosin in Species of Crustacea；Can be in the visible model of naked eyes
It is 5 μ g/mL to reach the tropomyosin content in detection sample in enclosing, total processing time 40min.
Brief description of the drawings
Fig. 1 is the schematic diagram of the kit of the present invention.
Fig. 2 is the testing result schematic diagram of kit produced by the present invention, wherein 1 is feminine gender, 2 be the positive.
Fig. 3 is that supernatant prepared by embodiment 2 carries out gel electrophoresis (SDS-PAGE) and Western blotting (Western-
Blooting) detect to determine the purity and content of the tropomyosin of extraction.
Embodiment
With reference to embodiment, the invention will be further described：
Sample pad treatment fluid is the boron containing 0.1g/ml PVP, the Tween-20 of 1% percent by volume in following examples
Acid buffering Solutions Solution.
Pad treatment fluid is the borate buffer solution containing 0.02g/ml sucrose, 0.01g/ml BSA.
The magnetic bead of anti-Tm specific murines system monoclonal antibody modification, Aorun Weina New Material Science and Technology Co., Ltd., Shanghai
Embodiment 1 prepares test strips
1st, pad is prepared：Pad is the combination of the marked by magnetic bead through the modification of anti-Tm specific murines system monoclonal antibody
Pad；The 60 μ l magnetic beads through antibody labeling are dissolved in 1680 μ l sample pad treatment fluid and the mixed solution of pad treatment fluid
(sample pad treatment fluid：Pad treatment fluid is 1：2), mixed liquor is uniformly added dropwise in pad by 70 μ l/cm speed afterwards
On.Vacantly uniformly to be added dropwise during dropwise addition.Dried two hours after being added dropwise.
2nd, test strips are prepared：Test strips are again by blotting paper, nitrocellulose filter, pad, sample after pad is dried
Product pad is staggeredly pasted onto bottom plate successively from top to bottom；The nitrocellulose filter for it is pre-coated have Tm detect antigen detection line and
The nitrocellulose filter of the control line of goat anti-mouse IgG.Finally cover layer protecting film on whole test strips again.
Comparative example 1 prepares test strips
1st, pad is prepared：Pad is the combination of the marked by magnetic bead through the modification of anti-Tm specific murines system monoclonal antibody
Pad；The 60 μ l magnetic beads through antibody labeling are dissolved in 1680 μ l sample pad treatment fluid and pad treatment fluid (1:2) mix molten
In liquid, then mixed liquor is uniformly added dropwise on pad by 70 μ l/cm speed, dried two hours after being added dropwise.
2nd, test strips are prepared：Test strips are again by blotting paper, nitrocellulose filter, pad, sample after pad is dried
Product pad is staggeredly pasted onto bottom plate successively from top to bottom；The nitrocellulose filter for it is pre-coated have Tm detect antigen detection line and
The nitrocellulose filter of the control line of goat anti-mouse IgG.Finally cover layer protecting film on whole test strips again.
Comparative example 2 prepares test strips
1st, pad is prepared：Pad is the combination of the marked by magnetic bead through the modification of anti-Tm specific murines system monoclonal antibody
Pad；The 60 μ l magnetic beads through antibody labeling are dissolved in 1680 μ l sample pad treatment fluid and the mixed solution of pad treatment fluid
(sample pad treatment fluid：Pad treatment fluid is 2：1), mixed liquor is uniformly added dropwise in pad by 70 μ l/cm speed afterwards
On.Vacantly uniformly to be added dropwise during dropwise addition.Dried two hours after being added dropwise.
2nd, test strips are prepared：Test strips are again by blotting paper, nitrocellulose filter, pad, sample after pad is dried
Product pad is staggeredly pasted onto bottom plate successively from top to bottom；The nitrocellulose filter for it is pre-coated have Tm detect antigen detection line and
The nitrocellulose filter of the control line of goat anti-mouse IgG.Finally cover layer protecting film on whole test strips again.
Test result：
The ELISA test strip limit that comparative example 1 and comparative example 2 are produced all can only achieve 10 μ g/ml, reduce the spirit of detection
Sensitivity.But the test limit of the test strips of the preparation of embodiment 1 can reach 5 μ g/ml.
Embodiment 2
(1) acetone and shrimp are pressed into 1g：5ml is configured, with acetone extraction to colourless shrimp, by 1:5 (w/v) ratio adds
Enter in Tris-HCl buffer solutions (NaCl containing 0.5mol/L, pH 9.2), be homogenized 30s with electric homogenizer, gained mixed liquor is carried out
10min is ultrasonically treated, heats 5min afterwards, rapid cooling, by resulting solution with mini centrifuge 10min, takes supernatant
For detecting；
Supernatant is subjected to SDS-PAGE and Western-blooting detections to determine the pure of the tropomyosin of extraction
Degree and content, as shown in accompanying drawing 3.It can be seen that the sample handled with this method, foreign protein band is few, obtained tropomyosin content
It is high.
(2) gained supernatant solution is detected sample, and 120 μ L samples are added in the sample pad of test strips, chromatographs 8min
After observe result, be as a result that only control line obvious band occurs, be positive.Fig. 2 is seen, shown in label 2.
The present embodiment total time is 40min.
Described above is presently preferred embodiments of the present invention, but the present invention should not be limited to disclosed in the embodiment
Content.So every do not depart from the lower equivalent or modification completed of spirit disclosed in this invention, the model that the present invention protects is both fallen within
Enclose.
Claims (10)
1. a kind of tropomyosin quick detection immuomagnetic bead chromatographic test strip, it is characterised in that upper including bottom plate and bottom plate
Surface is pasted with sample pad, pad, nitrocellulose filter, adsorptive pads to the other end successively from one end；The sample pad, combination
Pad, nitrocellulose filter, adsorptive pads are staggeredly pasted onto bottom plate successively；The pad is to resist through anti-Tm specific murines system monoclonal
The pad of the marked by magnetic bead of body modification；The nitrocellulose filter resists for the pre-coated detection line for having Tm to detect antigen and goat
The nitrocellulose filter of the control line of mouse IgG.
2. the tropomyosin quick detection immuomagnetic bead chromatographic test strip according to claim 1, it is characterised in that described
The preparation method of the pad of marked by magnetic bead through the modification of anti-Tm specific murines system monoclonal antibody is the sample for being dissolved in magnetic bead
After product pad the mixed solution for the treatment of fluid and pad treatment fluid, at the uniform velocity it is added dropwise on pad；The body of the magnetic bead and mixed liquor
Product is than being 1：25-30；The volume ratio of the sample pad treatment fluid and pad treatment fluid is 1：2.
3. the tropomyosin quick detection immuomagnetic bead chromatographic test strip according to claim 2, it is characterised in that described
Sample pad treatment fluid is the Tween-20 containing 0.1-0.3g/ml polyvinylpyrrolidones (PVP), 1-2.5% percents by volume
Borate buffer solution.
4. the tropomyosin quick detection immuomagnetic bead chromatographic test strip according to claim 2, it is characterised in that described
Pad treatment fluid is the borate buffer containing 0.01-0.03g/ml sucrose, 0.01-0.03g/ml bovine serum albumin(BSA)s (BSA)
Solution.
5. the tropomyosin quick detection immuomagnetic bead chromatographic test strip according to claim 1, it is characterised in that described
Tropomyosin quick detection immuomagnetic bead chromatographic test strip also includes being covered in sample pad, pad, nitrocellulose filter, suction
The transparent plastic diaphragm seal on water cushion upper strata.
6. the application process of tropomyosin quick detection immuomagnetic bead chromatographic test strip according to claim 1, it is special
Sign is, comprises the following steps：
(1) acetone extraction to colourless shrimp is added in Tris-HCl buffer solutions, mixes, 3- is heated under fluidized state
8min, rapid cooling, centrifugation, supernatant is taken to be used to detect；
(2) supernatant in step (1) is added in the sample pad of kit, it is in sample that only obvious band, which occurs, in control line
Contain tropomyosin.
7. according to the application process described in claim 6, it is characterised in that the acetone extraction of the step (1) is to colourless
During shrimp, the ratio of shrimp and acetone is 1g:4-6mL.
8. according to the application process described in claim 6, it is characterised in that the Tris-HCl buffer solutions of the step (1) are
NaCl containing 0.4-0.6mol/L, pH 9.2.
9. according to the application process described in claim 6, it is characterised in that the shrimp and Tris-HCl of the step (1) delay
Quality and volume ratio are 1 in fliud flushing：4-6.
10. according to the application process described in claim 6, it is characterised in that the method for the mixing of the step (1) is stirring
30-50s, ultrasonic mixing 8-12min.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711240546.4A CN107727870B (en) | 2017-11-30 | 2017-11-30 | Immunomagnetic bead chromatography test strip for rapid detection of tropomyosin and application thereof |
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| CN201711240546.4A CN107727870B (en) | 2017-11-30 | 2017-11-30 | Immunomagnetic bead chromatography test strip for rapid detection of tropomyosin and application thereof |
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| CN107727870B CN107727870B (en) | 2020-10-16 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110275019A (en) * | 2019-08-01 | 2019-09-24 | 郑州迈迪迅医疗科技有限公司 | A kind of novel chromatography detection kit |
| CN110596377A (en) * | 2019-08-01 | 2019-12-20 | 郑州迈迪迅医疗科技有限公司 | Dry-type immunochromatography diagnosis device based on microfluid |
| CN113341151A (en) * | 2021-03-19 | 2021-09-03 | 合肥工业大学 | Detection card for crustacean allergen and preparation method and application thereof |
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| CN101566631A (en) * | 2008-04-24 | 2009-10-28 | 北京科美东雅生物技术有限公司 | Magnetic immunochromatographic test strip for combined detection of HIV-1+2 antibody and P24 antigen and preparation method thereof |
| CN101762692A (en) * | 2009-06-24 | 2010-06-30 | 北京科美东雅生物技术有限公司 | Magnetic immunochromatographic strip for detection of tuberculosis (TB) antibody in blood and preparation method thereof |
| CN102043055A (en) * | 2010-10-27 | 2011-05-04 | 上海海洋大学 | Rapid immunomagnetic bead chromatographic method for main allergens in aquatic products |
| CN102156191A (en) * | 2011-05-18 | 2011-08-17 | 上海海洋大学 | Magnetic immune chromatography method capable of synchronously detecting Tm and Pa allergens |
| CN104101708A (en) * | 2013-04-11 | 2014-10-15 | 中国科学院化学研究所 | Up-conversion fluorescent/magnetic nanoparticles-based immune-chromatographic test paper and making method thereof |
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2017
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Patent Citations (5)
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|---|---|---|---|---|
| CN101566631A (en) * | 2008-04-24 | 2009-10-28 | 北京科美东雅生物技术有限公司 | Magnetic immunochromatographic test strip for combined detection of HIV-1+2 antibody and P24 antigen and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110275019A (en) * | 2019-08-01 | 2019-09-24 | 郑州迈迪迅医疗科技有限公司 | A kind of novel chromatography detection kit |
| CN110596377A (en) * | 2019-08-01 | 2019-12-20 | 郑州迈迪迅医疗科技有限公司 | Dry-type immunochromatography diagnosis device based on microfluid |
| CN113341151A (en) * | 2021-03-19 | 2021-09-03 | 合肥工业大学 | Detection card for crustacean allergen and preparation method and application thereof |
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| CN107727870B (en) | 2020-10-16 |
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