CN104280555B - A kind of quick detection Peanut Allergen Ara h 2 colloidal gold strip and preparation method thereof - Google Patents
A kind of quick detection Peanut Allergen Ara h 2 colloidal gold strip and preparation method thereof Download PDFInfo
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- CN104280555B CN104280555B CN201410576447.3A CN201410576447A CN104280555B CN 104280555 B CN104280555 B CN 104280555B CN 201410576447 A CN201410576447 A CN 201410576447A CN 104280555 B CN104280555 B CN 104280555B
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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
- G01N33/561—Immunoelectrophoresis
Abstract
A kind of quick detection Peanut Allergen Ara h 2 colloidal gold strip and preparation method thereof, belongs to technical field of immunoassay.The antibody that the present invention is applied is the monoclonal antibody using the high specific that the immunization. Female BALB/c mouses of Ara h 2 of extraction purification are obtained from peanut crude protein.Due to using monoclonal antibody, stability and specificity are preferable, it is adaptable to Peanut Allergen Ara h 2 quick detection.
Description
Technical field
The present invention relates to the immune colloid gold Rapid detection test strip of peanut allergy ultimate constituent Ara h 2 a kind of and its preparation
Method, and the quick diagnosis to Peanut Allergen can be realized by the monitoring to Ara h 2, belong to immunoassay technology neck
Domain.
Background technology
Peanut allergy is to cause in food hypersenstivity death toll highest a kind of.Because food hypersenstivity trigger dead 90% all
It is caused by peanut.Peanut allergy is reacted because of its potential dangerous, chronicity and ever-increasing incidence of disease increasingly
It is taken seriously.
Peanut Allergen includes multiple proteins composition, and wherein Ara h 2 are that a kind of molecular mass is 17kDa ~ 20kDa
Allograft albumen, accounts for the 10% of peanut protein total amount.Ara h 2 are considered as main allergy ultimate constituent, more than 90%
Peanut allergy patient is to its allergy.Therefore, the quick diagnosis to Peanut Allergen can be realized by the monitoring to Ara h 2.
At present, it is many to the quantitative detecting method of anaphylactogen, such as double immunodiffusion, radioimmunology and efficient liquid phases
Method, PCR（PCR）Analytic approach, Histamine release experiments（HRT）, anaphylactogen finger-print quick determination method etc..
But the problem of all having each different, such as detection speed is slow, cost is high, need specific analytical instrument.And immunochromatography glue
Body gold test paper strip has the advantages that quick, processing sample size simple to operate is big, sensitivity is high and cheap, and need not be by special
Door instrument, is adapted to field quick detection, therefore have great importance for the Site Detection of a large amount of samples.
The content of the invention
Present invention aims at provide a kind of quick detection Peanut Allergen Ara h 2 colloidal gold strip, operation letter
Single, quick and convenient, sensitivity is high, and stability is good, with low cost, the rapid, high volume detection for Peanut Allergen in food.
Another object of the present invention is to provide a kind of preparation method of immunity colloidal gold test paper strip, including anti-Ara h 2
The preparation of specific antibody, the pairing screening of the double antibodies of Ara h 2 detection, the system of the immune colloidal gold detection test paper strips of Ara h 2
It is standby.The test strips are easy to operate, quick, accurate, and detection overall process only needs 5min, is not disturbed by environmental condition, and specificity is good,
Detection sensitivity is high, and lowest detection is limited to 1ng/mL.
The present invention is applied to hospital, and Peanut Allergen Ara in food or clinical sample can be achieved in enterprise and average family etc.
H 2 quick detection.
Technical scheme：A kind of quick detection Peanut Allergen Ara h 2 colloidal gold strip, including PVC
Bottom plate, sample pad and adsorptive pads are respectively equipped with PVC bottom plates two ends；Nitrocellulose filter detection layers are provided with the middle part of PVC bottom plates,
Gold conjugation pad is provided between nitrocellulose filter detection layers and sample pad；Described gold conjugation pad one end and sample
Pad, which is connected, to be stacked, and the other end is stacked in nitrocellulose filter detection layers.
Detection line and nature controlling line are disposed with the nitrocellulose filter detection layers.The gold conjugation pad coating
There are the anti-antibody A ra h 2-mAb-5 of Ara h 2 of gold mark mark（CGMCC No.9314）, anti-Ara h are coated with the detection line
2 antibody A ra h 2-mAb-1（CGMCC No.9315）.Sheep anti-mouse igg is coated with the nature controlling line.
The preparation method of the colloidal gold fast detecting test paper strip of the Peanut Allergen Ara h 2, step is：
（1）Extract Peanut Allergen crude protein：
Fresh shelled peanut is removed the peel, is handled through high speed disintegrator and obtains powdered, 10g is weighed, by 1:10（W/V）Immerse stone
Oily ether（60~90℃）4h is extracted under middle degreasing, 4 DEG C of magnetic agitations, 8000 r/mim centrifuge 10 min, abandon supernatant, precipitation is repeatedly
Extraction three times after be placed in fume hood petroleum ether is volatilized defatted peanut.1 is pressed immediately:10（W/V）Immerse 0.01 M pH7.4
Extract and stay overnight at 4 DEG C in PBS, 8000 r/min centrifuge 10 min, discard precipitation, supernatant is the extraction of peanut crude protein
Liquid.
（2）Extraction purification Ara h 2：
It is slowly added to saturated ammonium sulfate solid in peanut crude protein leaching liquor, stirring while adding, addition is completely dissolved
Just continue to add backward afterwards, until saturation degree is 40%, 4 DEG C of standings 1 h, 8000 r/min centrifuge 20 min, and collection precipitation is redissolved
In 0.01 M pH7.4 PBS, 40% saturation degree component is obtained.Saturated ammonium sulfate solid is continuously added in supernatant until saturation degree is
60%, obtain 60% saturation degree component by above-mentioned 40% saturation degree component preparation method.Equally continue the addition ammonium sulfate into supernatant to obtain
To 80% saturation degree component.Above three rank groups are dialysed in 0.01 M pH7.4 PBS reflected with SDS-PAGE after 24 h respectively
Fixed, the content highest components of Ara h 2 carry out gel permeation chromatography separation again.SDS-PAGE identifications, Ara h are carried out to eluting peak
2 content highest eluting peaks will be used for next step experiment.
（3）The preparation of the anti-monoclonal antibody specifics of Ara h 2：
Using the Ara h 2 of extraction purification in step (2) as immunogen immune BALB/c mouse, with direct after being repeatedly immunized
ELISA method is selected compatibility highest mouse and merged, and obtain 6 by screening has the thin of strong compatibility to Ara h 2
Born of the same parents' strain.
（4）The pairing screening of anti-Ara h2 monoclonal antibody specifics：
The cell line that step (3) is obtained is prepared and is marked respectively with horseradish peroxidase HRP after antibody purification.Mark
Double antibody sandwich ELISA pairing screening is carried out after remembering successfully.Needed for determining specific immunity colloidal gold fast detecting test paper strip
Want antibody；Ara h 2-mAb-5 are gold labeling antibody, and Ara h 2-mAb-1 are detection antibody.
（5）The preparation of the immune colloid gold Rapid detection test strips of Peanut Allergen Ara h 2：
A：The preparation of collaurum：
Using citrate reduction method trisodium citrate reducing agent, by gold chloride reduction, that 20nm-40nm collaurums are made is molten
Liquid：200mL deionized waters and 2mL 1% gold chloride are added in conical flask, boiling is heated with stirring to, rapidly joins 4mL 1%
Sodium citrate, continue heat 15-20min to be in shiny red, then in room temperature cool down, 4 DEG C are stored refrigerated.
B：The preparation of anti-Ara h2 antibody-colloidal gold labels：
The colloidal gold solution prepared in step A is taken, 4 μ L 0.1M K are added in every 1mL2CO3PH to 6 is adjusted, is dripped in stirring
Plus 150 μ L 0.2mg/mL antibody A ra h 2-mAb-5（CGMCC No.9314）, continue to stir in 30min, centrifugation, absorption
Clearly, liquid is preserved with gold labeling antibody to be resuspended.
Gold labeling antibody re-suspension liquid contains 10% sucrose, 0.05% Tween-20,1% BSA, 0.2% for pH's 8.2
PEG20000,1% PVP-K30 0.05M Tris-HCl solution.
C：The preparation of gold conjugation pad：
Three-dimensional planar point film gold spraying instrument instrument HM3055 is debugged, the antibody A ra h of anti-Ara h 2 of collaurum will have been marked
2-mAb-5 (CGMCC No.9314) is uniformly sprayed on glass fibre membrane, and spouting liquid 0.6pL/cm, 37 DEG C of drying are stayed overnight, envelope
Bag is standby.
D：The preparation of detection layers：
Three-dimensional planar point film gold spraying instrument instrument HM3055 is debugged, by the antibody A ra h 2-mAb-1 of anti-Ara h 2 diluted
(CGMCC No.9315) is uniformly sprayed on nitrocellulose filter, obtains detection line；The sheep anti-mouse igg diluted is uniformly sprayed on
On nitrocellulose filter, nature controlling line is obtained, spouting liquid is 0.6pL/cm, and 37 DEG C of drying are stayed overnight, and envelope is standby.
E：The assembling of test strips：
By sample pad, gold conjugation pad, nitrocellulose filter, adsorptive pads are pasted onto on PVC bottom plates successively by one end, i.e.,
Obtain the colloidal gold immunochromatographitest test strip for detecting Peanut Allergen Ara h 2.
Biological material specimens preservation：
One plant of monoclonal cell strain, cell line S, strain number is Ara h 2-mAb-5, has been preserved in Chinese microorganism strain
Preservation administration committee common micro-organisms center, abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China
Institute of microbiology of the academy of sciences, accession designation number is CGMCC No.9314, and preservation date is on May 28th, 2014；
One plant of monoclonal cell strain, cell line T, strain number is Ara h 2-mAb-1, has been preserved in Chinese microorganism strain
Preservation administration committee common micro-organisms center, abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China
Institute of microbiology of the academy of sciences, accession designation number is CGMCC No.9315, and preservation says the phase on May 28th, 2014.
The operation principle of test strips of the present invention：Using colloidal gold immunochromatographimethod technology, made from the specific antibodies of Ara h 2
For solid formation, detect in sample whether contain Ara h 2 using double antibody sandwich method principle.When in measuring samples contain Ara h
When 2, antigen is first combined with the antibody A ra h 2-mAb-5 of anti-Ara h 2 of colloid gold label, due to chromatography effect compound edge inspection
Survey layer to move forward, when running into the 2 antibody A ra h 2-mAb-1 of anti-Ara h in detection line, form antibody-antigene-gold mark
Antibody complex, is enriched with detection line, forms red precipitate line.
Beneficial effects of the present invention：Compared with prior art, expense is relatively low and simple to operate, it is not necessary to which instrument is set by the present invention
Standby and professional easily spreads to food enterprise, hospital and family.Detection speed is fast, and whole process only 5min is applied to high-volume sample
The field quick detection of product.And sensitivity is high, lowest detection line is 1ng/mL.
Brief description of the drawings
A kind of quick detection Peanut Allergen Ara h 2 colloidal gold strips of Fig. 1,1, PVC bottom plates, 2, sample pad, 3,
Gold conjugation pad, 4, nitrocellulose filter detection layers, 5, adsorptive pads, 6, detection line, 7, nature controlling line；
Fig. 2, a kind of nitrocellulose filter detection layers of quick detection Peanut Allergen Ara h 2 colloidal gold strip are shown
It is intended to.
Fig. 3 is the SDS-PAGE of three different saturation components of peanut crude protein extract solution of the present invention；
1,2：Low molecule amount standard protein；3,6：40% saturated ammonium sulphate component；4,7：60% saturated ammonium sulphate
Component；5,8：80% saturated ammonium sulphate component；
Fig. 4 is the gel permeation chromatography figure of 80% saturated ammonium sulphate component of peanut crude protein extract solution of the present invention；
Fig. 5 is that 80% saturated ammonium sulphate component of peanut crude protein extract solution of the present invention is washed after gel permeation chromatography
The SDS-PAGE at de- peak；1,2：80% ammonium sulfate component；3,4：80% eluting peak of ammonium sulfate component first（Peak 1）；5,
6：80% eluting peak of ammonium sulfate component second（Peak 2）；M：Low molecule standard protein；
Fig. 6 is the actually detected sample drawing of the colloidal gold strips of Ara h 2 prepared by the present invention；1st, 5 ppb, 2,2.5
Ppb, 3,2 ppb, 4,1 ppb, 5,0.5 ppb, 6,0 ppb.
Embodiment
The present invention is used as immunogene by extracting after anaphylactogen crude protein, immune mouse obtains antibody, by cell fusion
Technology, obtains specific recognition Ara h 2 monoclonal antibody.Using the monoclonal antibody, using colloidal gold immunochromatographimethod technology, system
A kind of standby quick detection Peanut Allergen Ara h 2 colloidal gold strip.
Embodiment 1
（1）Extract Peanut Allergen crude protein：
Fresh shelled peanut is removed the peel, is handled through high speed disintegrator and obtains powdered, 10g is weighed, by 1:10（W/V）Immerse stone
Oily ether（60~90℃）4h is extracted under middle degreasing, 4 DEG C of magnetic agitations, 8000 r/mim centrifuge 10 min, abandon supernatant, precipitation is repeatedly
Extraction three times after be placed in fume hood petroleum ether is volatilized defatted peanut.1 is pressed immediately:10（W/V）Immerse 0.01 M pH7.4
Extract and stay overnight at 4 DEG C in PBS, 8000 r/min centrifuge 10 min, discard precipitation, supernatant is peanut crude protein leaching liquor
（2）Extraction purification Ara h 2：
It is slowly added to saturated ammonium sulfate solid in peanut crude protein leaching liquor, stirring while adding, addition is completely dissolved
Just continue to add backward afterwards, until saturation degree is 40%, 4 DEG C of standings 1 h, 8000 r/min centrifuge 20 min, and collection precipitation is redissolved
In 0.01 M pH7.4 PBS, 40% saturation degree component is obtained.Saturated ammonium sulfate solid is continuously added in supernatant until saturation degree is
60%, obtain 60% saturation degree component by above-mentioned 40% saturation degree component preparation method.Equally continue the addition ammonium sulfate into supernatant to obtain
To 80% saturation degree component.Above three rank groups are dialysed in 0.01 M pH7.4 PBS reflected through SDS-PAGE after 24 h respectively
It is fixed, the content highests of Ara h 2 in 80% saturation degree component, to carrying out gel permeation chromatography separation.SDS-PAGE is carried out to eluting peak
The content highests of Ara h 2 in identification, second eluting peak.
（3）The pairing screening of anti-Ara h2 monoclonal antibody specifics：
By step（2）Obtained cell line is prepared and is marked respectively with horseradish peroxidase HRP after antibody purification.Mark
Double antibody sandwich ELISA pairing screening is carried out after remembering successfully.Needed for determining specific immunity colloidal gold fast detecting test paper strip
Want antibody；Ara h 2-mAb-5 are gold labeling antibody, and Ara h 2-mAb-1 are detection antibody.
（4）The preparation of the immune colloid gold Rapid detection test strips of Peanut Allergen Ara h 2：
A：The preparation of collaurum：
Using citrate reduction method trisodium citrate reducing agent, by gold chloride reduction, that 20nm-40nm collaurums are made is molten
Liquid；
B：The preparation of the anti-antibody-colloidal gold labels of Ara h 2：
The colloidal gold solution prepared in step A is taken, 4 μ L 0.1M K are added in every 1ml2CO3PH to 6 is adjusted, is dripped in stirring
Plus 150 μ L 0.2mg/mL antibody A ra h 2-mAb-5（CGMCC No.9314）, continue to stir 30min, supernatant is drawn in centrifugation,
It is resuspended with gold labeling antibody re-suspension liquid；
C：The preparation of gold conjugation pad：
Three-dimensional planar point film gold spraying instrument instrument HM3055 is debugged, the antibody A ra h of anti-Ara h 2 of collaurum will have been marked
2-mAb-5 (CGMCC No.9314) is uniformly sprayed on glass fibre membrane, and spouting liquid 0.6pL/cm, 37 DEG C of drying are stayed overnight, envelope
Bag is standby；
D：The preparation of detection layers：
Three-dimensional planar point film gold spraying instrument instrument HM3055 is debugged, by the antibody A ra h 2-mAb-1 of anti-Ara h 2 diluted
(CGMCC No.9315) is uniformly sprayed on nitrocellulose filter, obtains detection line；The sheep anti-mouse igg diluted is uniformly sprayed on
On nitrocellulose filter, nature controlling line is obtained, spouting liquid is 0.6pL/cm, and 37 DEG C of drying are stayed overnight, and envelope is standby；
E：The assembling of test strips：
By sample pad, gold conjugation pad, nitrocellulose filter, adsorptive pads are pasted onto on PVC bottom plates successively by one end, i.e.,
Obtain the colloidal gold immunochromatographitest test strip for detecting Peanut Allergen Ara h 2.
Claims (1)
1. a kind of preparation method of quick detection Peanut Allergen Ara h 2 colloidal gold strip, it is characterised in that step is：
A：The preparation of collaurum：
Gold chloride reduction is made by 20nm-40nm colloidal gold solutions using citrate reduction method trisodium citrate reducing agent；
B：The preparation of the anti-antibody-colloidal gold labels of Ara h 2：
The colloidal gold solution prepared in step A is taken, 4 μ L 0.1M K are added in every 1mL2CO3PH to 6 is adjusted, 150 are added dropwise in stirring
μ L 0.2mg/mL antibody A ra h 2-mAb-5, continue to stir 30min, centrifugation draws supernatant, with gold labeling antibody re-suspension liquid weight
It is outstanding；
Gold labeling antibody re-suspension liquid contains 10% sucrose, 0.05% Tween-20,1% BSA, 0.2% PEG20000,1% for pH's 8.2
PVP-K30 0.05M Tris-HCl solution；
C：The preparation of gold conjugation pad：
Three-dimensional planar point film gold spraying instrument instrument HM3055 is debugged, the antibody A ra h 2- of anti-Ara h 2 of collaurum will have been marked
MAb-5 is uniformly sprayed on glass fibre membrane, and spouting liquid 0.6pL/cm, 37 DEG C of drying are stayed overnight, and envelope is standby；
D：The preparation of nitrocellulose filter detection layers：
Three-dimensional planar point film gold spraying instrument instrument HM3055 is debugged, the antibody A ra h 2-mAb-1 of anti-Ara h 2 diluted is uniform
It is sprayed on nitrocellulose filter, obtains detection line；The sheep anti-mouse igg diluted is uniformly sprayed on nitrocellulose filter, obtained
Nature controlling line, spouting liquid is 0.6pL/cm, and 37 DEG C of drying are stayed overnight, and envelope is standby；
E：The assembling of test strips：
By sample pad, gold conjugation pad, nitrocellulose filter, adsorptive pads are pasted onto on PVC bottom plates successively by one end, that is, are obtained
Colloidal gold immunochromatographitest test strip for detecting Peanut Allergen Ara h 2；
One plant of monoclonal cell strain as gold labeling antibody, is cell line S, and strain number is Ara h 2-mAb-5, is specificity
The monoclonal antibody specific hybridoma cell strains of Ara h 2, have been preserved in China Committee for Culture Collection of Microorganisms common
Microorganism center, accession designation number is CGMCC No.9314；
One plant of monoclonal cell strain as detection antibody, is cell line T, and strain number is Ara h 2-mAb-1, is specificity
The monoclonal antibody specific hybridoma cell strains of Ara h 2, have been preserved in China Committee for Culture Collection of Microorganisms common
Microorganism center, accession designation number is CGMCC No.9315.
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| CN201410576447.3A CN104280555B (en) | 2014-10-24 | 2014-10-24 | A kind of quick detection Peanut Allergen Ara h 2 colloidal gold strip and preparation method thereof |
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Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5558869A (en) * | 1992-12-30 | 1996-09-24 | University Of Arkansas | Major peanut allergen ara h II |
| JP2002501748A (en) * | 1998-01-31 | 2002-01-22 | ユニバーシティ オブ アーカンソー | Methods and reagents for reducing allergic reactions |
| CN101393214A (en) * | 2008-11-17 | 2009-03-25 | 杭州浙大生物基因工程有限公司 | Multichannel ingestion type allergen rapid detection kit and method for making same |
| CN102914524A (en) * | 2011-08-05 | 2013-02-06 | 深圳出入境检验检疫局食品检验检疫技术中心 | Timed-resolved fluoroimmunoassay detection method of allergen peanut protein |
| CN103454412B (en) * | 2013-09-16 | 2015-02-18 | 南京博敏达生物科技有限公司 | Liquid phase chip for detecting allergen specific antibody and preparation method of liquid phase chip |
| CN103645328B (en) * | 2013-12-17 | 2016-01-13 | 中国计量学院 | A kind of preparation method of Major Peanut Allergens with IgE-binding Arah2 standard items |
| CN103675271B (en) * | 2013-12-23 | 2016-01-06 | 北京新华联协和药业有限责任公司 | Anaphylactia allergen colloidal gold diagnosis test strips and preparation method thereof |
| CN103777004B (en) * | 2014-02-14 | 2015-12-02 | 江南大学 | Immune colloid gold Rapid detection test strip of a kind of staphylococcus aureus enterotoxin E and preparation method thereof |
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