CN106093410A - Listeria or the colloidal gold strip of Listeria monocytogenes and application thereof in detection food based on monoclonal antibody - Google Patents
Listeria or the colloidal gold strip of Listeria monocytogenes and application thereof in detection food based on monoclonal antibody Download PDFInfo
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Abstract
Listeria or the colloidal gold strip of Listeria monocytogenes and application thereof in detection food based on monoclonal antibody, belong to technical field of immunoassay.PVC base plate sets gradually sample pad, nitrocellulose filter and adsorptive pads；When listeria in detection food based on monoclonal antibody, described gold labeling antibody is the colloidal gold solution of antibody 2H8；When detection Listeria Monocytogenes In Food based on monoclonal antibody, gold labeling antibody is the colloidal gold solution of antibody A number.The present invention develops listeria or the colloidal gold strip of Listeria monocytogenes in detection food based on monoclonal antibody.Listeria or the high specific of Listeria monocytogenes, high accuracy, higher sensitivity, easy quickly analysis be applicable to food.
Description
Technical field
The present invention relates to listeria or the glue of Listeria monocytogenes in a kind of detection food based on monoclonal antibody
Body gold test paper strip and application thereof, belong to technical field of immunoassay.
Background technology
Listeria monocytogenes is a kind of facultative anaerobic gram-positive bacterium, belongs to listeria.It is mainly with food
Thing is Vector of infection, is one of the most fatal food-borne pathogens in global range.Listerella is the most widely distributed,
Meat, eggs, birds, milk product, vegetable etc. all have been found to be the listerial source of infection.Can cause after infecting Listerella
The symptoms such as myalgia, nauseating, diarrhoea, serious caused blood and cerebral tissue infect, and owing to this bacterium is 4 DEG C of cold preservation temperature
Under degree still can growth and breeding, contaminated chilled food has the biggest threat to human health.Therefore a lot of countries have adopted
The measure that takes is to the Listerella controlling in food, and has formulated corresponding standard.
Listeria is divided into 10 kinds, including Listeria monocytogenes (L.monocytogenes), sheep Liszt
Bacterium (L. ivanovii), Ying Nuoke Listerella (L.innocua), Weir Listerella (L.welshimeri) etc..Therein
Single Liszt of increasing plants and is divided into again 13 kinds of hypotypes, and kind is the most various, and Listeria monocytogenes is to cause that human disease's is the most former
Cause.But, as the index of food hygiene, Listerella is usually required to exclusively carry out detection.Therefore, in detection food
Listerella and Listeria monocytogenes are respectively provided with highly important meaning.Detection listeria and Listeria monocytogenes master at present
Plating method to be had, polymerase chain reaction method (PCR) and enzyme-linked immunoassay method (ELISA).
Colloidal gold strip as detection microorganism common method have simplicity, quickly, sensitiveer, specificity good, no
Need large-scale instrument, feature easy to spread.But ELISA pairing antibody might not set up colloidal gold strip, this
It is that the requirement of antagonist affinity etc. is higher due to colloidal gold strip response time the shortest (10min).Have Liszt at present
Bacterium and the research of Listeria monocytogenes collaurum test paper method, but the monoclonal antibody or many that problems faced is mainly used
The cross reaction of the detection method of clonal antibody and foundation is difficult to reach requirement, i.e. to antibacterial the most of the same race in listeria
Or in Listeria monocytogenes kind, different serotype is the most recognizable, other miscellaneous bacteria is not existed cross reaction simultaneously, this is for reality
It is particularly significant that border is applied to sample detection.And, how to realize listeria and the accuracy of Listeria monocytogenes, special
Property detection be current colloidal gold strip method remain a need for solve problem.
The present invention uses 2 strain listeria monoclonal antibody specifics and the 1 strain Listeria monocytogenes that pairing be can be combined
Specific antibody, has been successfully established listeria specificity and Listeria monocytogenes specific colloidal gold strip method,
Monitoring for listeria in food and Listeria monocytogenes provides effective quick analysis means with control.
Summary of the invention
It is an object of the invention to overcome above-mentioned weak point, set up one and there is high specific, high accuracy, more highly sensitive
Degree, colloidal gold strip method easy and simple to handle be listeria or the quick detection of Listeria monocytogenes in food.
Technical scheme:
1, the exploitation specific colloidal gold strip of listeria: based on listeria specific pairing monoclonal antibody
2G7(picks up survey antibody) and 2H8(gold labeling antibody), the P60 albumen of this pairing antibody specificity identification listeria phage surface,
Thus there is homogeneity, the possibility of specific detection listeria.Test indicate that the colloid set up by this pairing antibody
Gold test paper strip successfully have detected 12 strain Listerellas, positive rate 100%, tests bacterium no cross reaction with other simultaneously.
2, the exploitation specific colloidal gold strip of Listeria monocytogenes: first, establishes and can increase Lee by specific detection list
The pairing antibody of this special bacterium.Experiment test shows, the specific monoclonal antibody of Listeria monocytogenes A (gold labeling antibody) can be with
Specific monoclonal antibody 2G7(of listeria pick up survey antibody) formed pairing detection Listeria monocytogenes surface P60 egg
In vain, the culture supernatant of specific detection Listeria monocytogenes and in ELISA system.
On this basis, antibody is detected based on specific monoclonal antibody 2G7(of listeria) and single increasing Liszt
The specific monoclonal antibody of bacterium A (gold labeling antibody) develops colloidal gold strip.Test indicate that this colloidal gold strip
Successfully have detected the culture supernatant of 8 strain Listeria monocytogenes, simultaneously with 4 strain Listerellas and other test bacterium no cross reaction.
Concrete mechanism is that gold labeling antibody A identifies the specific polypeptide of Listeria monocytogenes on P60 albumen, can specificity with
Listeria monocytogenes secretion P60 protein binding in supernatant, and can specific recognition listeria P60 albumen on tested survey line
Antibody 2G7 capture.
Listeria or the colloidal gold strip of Listeria monocytogenes in detection food based on monoclonal antibody, including
Adsorptive pads, nitrocellulose filter, nature controlling line, detection line, sample pad and PVC base plate, be separately furnished with golden labeling antibody；
PVC base plate sets gradually sample pad, nitrocellulose filter and adsorptive pads；Nitrocellulose filter is directly arranged at PVC
On base plate；Sample pad one end is overlapped on nitrocellulose filter, and the other end is directly arranged on PVC base plate；Adsorptive pads one end sets
Being placed on PVC base plate, the other end is overlapped on nitrocellulose filter；
Nature controlling line and detection line it is sequentially provided with on described nitrocellulose filter；It is coated sheep anti-mouse igg two on described nature controlling line to resist, inspection
Detection antibody 2G7 it is coated on survey line；
When listeria in detection food based on monoclonal antibody, described gold labeling antibody is that the gold colloidal of antibody 2H8 is molten
Liquid；When detection Listeria Monocytogenes In Food based on monoclonal antibody, gold labeling antibody is the colloidal gold solution of antibody A number.
Listeria or the colloidal gold strip of Listeria monocytogenes in described detection food based on monoclonal antibody
Preparation method, step is:
(I) preparation of gold labeling antibody:
A, the synthesis of colloidal gold solution: use the golden nanometer particle of citric acid reducing process synthesis 30nm, add in clean flask
Enter the chlorauric acid solution of 300mL mass concentration 0.01%, be heated under magnetic stirring seething with excitement completely, rapidly join in solution
The citric acid three sodium solution of 4.8mL mass concentration 1%, boils 10min by solution until color becomes bright claret, will burn
Bottle is placed in room temperature cooling, and 4 DEG C of preservations；
B, the coupling of gold labeling antibody: being respectively adopted antibody 2H8 or antibody A labelled notation colloid gold particle, detailed process is: use 0.1M
The colloidal gold solution pH that 1mL step a is prepared by solution of potassium carbonate is adjusted to 7.5；Add monoclonal antibody 2H8 or antibody A number 10 μ g
And at room temperature react 2h；Subsequently, the ultra-pure water solution of the bovine serum albumin BSA that 50 μ L mass volume ratios are 10%, room are added
Temperature is lower continues reaction 2 h；4 DEG C, centrifugal colloidal gold solution under the conditions of 6000g, 20min, remove BSA and the monoclonal anti of non-coupling
Body；With being the NaN of 0.1% containing mass concentration3, 0.2% Tween, the 10mM phosphate buffer weight of 0.01M of 0.2% sucrose
Outstanding gold labeling antibody, 4 DEG C of preservations, obtain antibody 2H8 colloidal gold solution or antibody A colloidal gold solution；
(II) preparation of colloidal gold strip: be fixed on by nitrocellulose filter on PVC base plate, sticks to nitric acid fine by adsorptive pads
Sample pad near one end of nature controlling line, is sticked to the close detection of nitrocellulose filter and PVC base plate with PVC base plate by dimension element film
The other end of line；Detection antibody 2G7 is diluted to 1mg/mL, is sprayed onto at detection line with Membrane jetter；Sheep anti mouse by 0.5 mg/mL
IgG bis-is anti-to be sprayed onto at nature controlling line, dries 2h, i.e. prepares listeria or list in detection food based on monoclonal antibody for 37 DEG C
Increase listerial colloidal gold strip.
Listeria or the colloidal gold strip of Listeria monocytogenes in described detection food based on monoclonal antibody
Application, when gold labeling antibody be antibody 2H8 time, listeria in specific detection food；When gold labeling antibody is antibody A
Number time, for specific detection Listeria Monocytogenes In Food.
Described listeria includes 4 kinds of Listerellas and 8 kinds of Listeria monocytogenes.
ATCC 19111(1/2a), ATCC 19115 (4b), ATCC 19118 described 8 kinds of Listeria monocytogenes are:
(4e), CMCC 54002 (1/2c), CMCC 54003, CMCC 54004, CMCC 54007 separate strain with chilled beef；
ATCC 19119(Listeria ivanovii), ATCC 33090(Listeria described 4 kinds of Listerellas are:
Innocua, 6a), ATCC 35897(Listeria welshimeri, 6b) and ATCC 25400(Listeria grayi).
Diluted sample: P60 albumen all with 10mM PBS be diluted to 250ng/mL, 100ng/mL, 50ng/mL, 25ng/mL,
10ng/mL, 5ng/mL, 2.5ng/mL, 1ng/mL also compare with blank PBS.When using listeria ELISA test strip, will
The Listerella bacterium solution 10mM PBS cultivated is diluted to 107 CFU/mL、5×106CFU/mL、106CFU/mL、5×105CFU/
mL、105CFU/mL、5×104 CFU/mL、104CFU/mL、103CFU/mL also compares with blank PBS.Use single increasing Liszt
During bacterium ELISA test strip, by Listeria monocytogenes culture supernatant 10mM PBS gradient dilution 3 times, 9 times, 27 times and with undiluted
Blank culture fluid compare.
Listeria or the colloidal gold strip of Listeria monocytogenes in described detection food based on monoclonal antibody
Application, concretely comprise the following steps:
Take 7 μ L gold labeling antibodies and 47 μ L re-suspension liquid, i.e. containing mass concentration be 0.2% Tween, the 10 of the 0.01M of 0.2% sucrose
MM phosphate buffer, 150 μ L testing samples react 5min in microwell plate, subsequently colloidal gold strip are inserted microwell plate
In, under chromatography effect, with the object that gold labeling antibody is combined, i.e. listeria or Listeria monocytogenes P60 albumen, from sample
Product pad is answered with sheep anti-mouse igg two anti-reflective on capture antibody, with nature controlling line on detection line, thus goes out on nature controlling line and detection line
Existing redness；By naked eyes, result is carried out interpretation, nature controlling line and detection line after 10min and occur that redness is the positive simultaneously, detection
Line does not develops the color and nature controlling line occurs that redness is feminine gender.
The detection analysis principle of the inventive method is: it is special that listeria colloidal gold strip is respectively adopted listeria
Opposite sex pairing monoclonal antibody 2G7 and 2H8 are as the capture antibody on detection line and gold mark detection antibody, with sheep anti-mouse igg two
Anti-as the coated antibody on nature controlling line.The P60 albumen on gold surface, labeling antibody identification Listerella, first with the Liszt in sample
Bacterium reacts, and the antibody 2G7 in another site of P60 albumen being identified surface, Listerella to detection line under chromatography effect is caught
Obtaining, remaining antibody is by the anti-capture of sheep anti-mouse igg two on nature controlling line, thus golden nanometer particle occurs at detection line and nature controlling line
Redness, is positive by naked eyes interpretation, and the detection line color depth is proportional with Listerella content in sample.Pairing due to screening
Antibacterial in monoclonal antibody homogeneity identification listeria, therefore detection has listeria specificity.Simultaneously because its
Its bacterium does not express P60 albumen without in detection line colour developing, only there is redness at nature controlling line.
Colloidal gold test paper for listeria monocytogenes is respectively adopted listeria monoclonal antibody 2G7 of pairing and single increasing Lee
This special bacterium specificity A is as the capture antibody on detection line and golden labeling antibody, anti-as on nature controlling line using sheep anti-mouse igg two
Coated antibody.Gold labeling antibody A can be secreted into the P60 albumen of supernatant by specific recognition Listeria monocytogenes, and in chromatography effect
The antibody 2G7 descending detection line to be identified listeria P60 albumen is captured, and remaining antibody is by the sheep anti mouse on nature controlling line
The anti-capture of IgG bis-, thus the redness of golden nanometer particle occurs at p-wire and nature controlling line, it is positive by naked eyes interpretation, detects line
Shade is proportional with Listeria monocytogenes content in sample.Owing in this pairing antibody, gold labeling antibody A can specificity, all
The P60 albumen of one property identification Listeria monocytogenes secretion, therefore detection has a Listeria monocytogenes specificity, and not with other Lee
This special bacterium produces cross reaction.Simultaneously because other bacterium does not express P60 albumen without in detection line colour developing, only going out at nature controlling line
Existing redness.
Beneficial effects of the present invention: the present invention develops the glue of listeria in detection food based on monoclonal antibody
Body gold test paper strip.Based on listeria specific pairs monoclonal antibody 2G7 and 2H8 as the capture antibody on detection line and
Gold mark detection antibody, can the P60 albumen of homogeneity identification listeria phage surface, test shows that successfully have detected 12 strains surveys
The Listerella of examination, positive rate 100%, test bacterium no cross reaction with other simultaneously.The height of listeria be applicable to food
Specificity, high accuracy, higher sensitivity, easy quickly analysis.
Also developed the detection specific colloidal gold strip of Listeria Monocytogenes In Food based on monoclonal antibody.First
First, establishing can the pairing antibody of specific detection Listeria monocytogenes.Experiment test shows, based on listeria specificity
Monoclonal antibody 2G7(detection line antibody) and the specific monoclonal antibody of Listeria monocytogenes A (gold labeling antibody), foundation
Colloidal gold strip successfully have detected 8 strain Listeria monocytogenes of test, simultaneously with 4 strain Listerellas and other test bacterium without
Cross reaction.It is applicable to the high specific of Listeria Monocytogenes In Food, high accuracy, higher sensitivity, easy quickly analysis.
Biological material specimens preservation:
Monoclonal cell strain 2G7, deposit number CGMCC No. 10875；Monoclonal cell strain 2H8, deposit number CGMCC No.
10876, it is disclosed in application number 201610039900.6；
Antibody A number, deposit number: CGMCC No. 9301, it is disclosed in application number 201410260781.8.
Accompanying drawing explanation
Fig. 1-1 listeria colloidal gold strip schematic diagram.
Fig. 1-2 colloidal gold test paper for listeria monocytogenes schematic diagram.
1, adsorptive pads；2, nitrocellulose filter；3, nature controlling line；4, detection line；5, sample pad；7, PVC base plate；6, gold mark is anti-
Body.
Fig. 2-1 listeria colloidal gold strip detection P60 albumen sensitivity schematic diagram.
Fig. 2-2 colloidal gold test paper for listeria monocytogenes detection P60 albumen sensitivity schematic diagram.
Fig. 3 listeria colloidal gold strip 12 kinds of Listerella (bacterium solution) result schematic diagrams of detection.
Fig. 4-1 colloidal gold test paper for listeria monocytogenes detects 8 kinds of Listeria monocytogenes (culture supernatant) result signals
Figure.
Fig. 4-2 colloidal gold test paper for listeria monocytogenes 4 kinds of Listerella (culture supernatant) result schematic diagrams of detection.
Fig. 5-1 listeria colloidal gold strip cross reaction schematic diagram.
Fig. 5-2 colloidal gold test paper for listeria monocytogenes cross reaction schematic diagram.
Detailed description of the invention
Embodiment 1 colloidal gold strip
Listeria or the colloidal gold strip of Listeria monocytogenes in detection food based on monoclonal antibody, including water suction
Pad 1, nitrocellulose filter 2, nature controlling line 3, detection line 4, sample pad 5, PVC base plate 7, be separately furnished with golden labeling antibody 6；
PVC base plate 7 sets gradually sample pad 5, nitrocellulose filter 2 and adsorptive pads 1；Nitrocellulose filter 2 is directly arranged
On PVC base plate 7；Sample pad 5 one end is overlapped on nitrocellulose filter 2, and the other end is directly arranged on PVC base plate 7；Water suction
Padding 1 one end to be arranged on PVC base plate 7, the other end is overlapped on nitrocellulose filter 2；
Nature controlling line 3 and detection line 4 it is sequentially provided with on described nitrocellulose filter 2；It is coated sheep anti-mouse igg two on described nature controlling line 3
Anti-, coated antibody 2G7 on detection line 4；
When listeria in detection food based on monoclonal antibody, described gold labeling antibody 6 is that the gold colloidal of antibody 2H8 is molten
Liquid；When detection Listeria Monocytogenes In Food based on monoclonal antibody, gold labeling antibody 6 is the colloidal gold solution of antibody A number.
Embodiment 2
The system of the colloidal gold strip of listeria or Listeria monocytogenes in described detection food based on monoclonal antibody
Preparation Method, step is:
(1) preparation of gold labeling antibody 6:
A, the synthesis of colloidal gold solution: use the golden nanometer particle of citric acid reducing process synthesis 30nm, add in clean flask
Enter the chlorauric acid solution of 300mL mass concentration 0.01%, be heated under magnetic stirring seething with excitement completely, rapidly join in solution
The citric acid three sodium solution of 4.8mL mass concentration 1%, boils 10min by solution until color becomes bright claret, will burn
Bottle is placed in room temperature cooling, and 4 DEG C of preservations；
B, the coupling of gold labeling antibody: being respectively adopted antibody 2H8 or antibody A labelled notation colloid gold particle, detailed process is: use 0.1M
The colloidal gold solution pH that 1mL step a is prepared by solution of potassium carbonate is adjusted to 7.5；Add monoclonal antibody 2H8 or antibody A number 10 μ g
And at room temperature react 2h；Subsequently, the ultra-pure water solution of the bovine serum albumin BSA that 50 μ L mass volume ratios are 10%, room are added
Temperature is lower continues reaction 2 h；4 DEG C, centrifugal colloidal gold solution under the conditions of 6000g, 20min, remove BSA and the monoclonal anti of non-coupling
Body；With being the NaN of 0.1% containing mass concentration3, 0.2% Tween, the 10mM phosphate buffer weight of 0.01M of 0.2% sucrose
Outstanding gold labeling antibody, 4 DEG C of preservations, obtain antibody 2H8 colloidal gold solution or antibody A colloidal gold solution；
(2) preparation of colloidal gold strip: nitrocellulose filter 2 is fixed on PVC base plate 7, adsorptive pads 1 is sticked to nitric acid
Sample pad 5, near one end of nature controlling line 3, is sticked to nitrocellulose filter 2 and PVC base plate 7 by cellulose membrane 2 and PVC base plate 7
The other end near detection line 4；Detection antibody 2G7 is diluted to 1mg/mL, is sprayed onto at detection line 4 with Membrane jetter；By 0.5 mg/
The sheep anti-mouse igg two of mL is anti-to be sprayed onto at nature controlling line 3, dries 2h, i.e. prepares Lee in detection food based on monoclonal antibody for 37 DEG C
This special Pseudomonas or colloidal gold strip of Listeria monocytogenes.
When gold labeling antibody is antibody 2H8, it is possible to listeria in specific detection food；When gold labeling antibody is
During antibody A, it is possible to for specific detection Listeria Monocytogenes In Food.
Embodiment 3 wet method detection P60 albumen
Diluted sample: P60 albumen is all diluted to 250ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 10ng/ with 10mM PBS
ML, 5ng/mL, 2.5ng/mL, 1ng/mL also compare with blank PBS.It is respectively adopted listeria colloidal gold strip and list
Increase Listerella colloidal gold strip detection.Taking 7 μ L gold labeling antibodies and 47 μ L re-suspension liquid, i.e. containing mass concentration is 0.2%
Tween, the 10 mM phosphate buffers of 0.01M of 0.2% sucrose, 150 μ L testing samples react 5min in microwell plate, with
After colloidal gold strip is inserted in microwell plate, reaction 10min after interpretation.Interpretation foundation: positive: nature controlling line, detection line
There is color simultaneously.Negative sample: nature controlling line has color, and detection line is without color.Concrete testing result is as shown in Figure 2.Test result
Illustrate that listeria colloidal gold strip and Listeria monocytogenes test strips can identify Listerella and Listeria monocytogenes
P60 albumen.
Embodiment 4 wet method detection listeria or Listeria monocytogenes
When using the detection of listeria colloidal gold strip, the Listerella bacterium solution 10mM PBS of cultivation is diluted to 107
CFU/mL、5×106 CFU/mL、106 CFU/mL、5×105 CFU/mL、105 CFU/mL、5×104 CFU/mL、104 CFU/
mL、103 CFU/mL also compares with blank PBS.Detection process shows listeria colloid gold test paper with embodiment 3, test
Bar all has cross reaction with 12 strain listeria antibacterials of test, and result is the most as shown in Figure 3.
When using colloidal gold test paper for listeria monocytogenes detection, first by culture fluid 5000g, 10min of testing sample
Be centrifuged, by supernatant 10mM PBS gradient dilution 3 times, 9 times, 27 times and compare with undiluted blank culture fluid, inspection
Survey process shows the cultivation of colloidal gold test paper for listeria monocytogenes and 8 strain Listeria monocytogenes of test with embodiment 3, test
Supernatant (18h cultivation) all has cross reaction, and does not has cross reaction with 4 strain Listerellas, and detectable extension rate is at 3-27
Times, result is specifically as shown in Fig. 4-1,4-2.
Described listeria includes 4 kinds of Listerellas and 8 kinds of Listeria monocytogenes.Described 8 kinds of Listeria monocytogenes are:
ATCC 19111(1/2a), ATCC 19115 (4b), ATCC 19118 (4e), CMCC 54002 (1/2c), CMCC 54003,
CMCC 54004, CMCC 54007 separate strain with chilled beef；Described 4 kinds of Listerellas are: ATCC 19119(Listeria
Ivanovii), ATCC 33090(Listeria innocua, 6a), ATCC 35897(Listeria welshimeri,
6b) with ATCC 25400(Listeria grayi).
Embodiment 5 cross reaction is tested
Listeria or the colloidal gold strip of Listeria monocytogenes and gram positive bacteria in test food, such as golden yellow Portugal
Grape coccus and gram negative bacteria, such as escherichia coli, Escherichia coli O 157, Salmonella, Enterobacter sakazakii, jejunum campylobacter bar
Bacterium, the cross reaction of campylobacter coli.
When in test food, listeria colloidal gold strip intersects, carry out by tested antibacterial bacterium solution, test concentrations
It is 5 × 108CFU/mL.Detection process is with embodiment 3, and result shows only to respond with the 12 strain listeria antibacterials tested,
And with other test antibacterial without intersecting, the most as shown in fig. 5-1.
When test Listeria Monocytogenes In Food belongs to colloidal gold strip intersection, carry out with tested antibacterial culturing supernatant
(not diluting).There is no cross reaction.Detection process is with embodiment 3, and result shows only have instead with the 8 strain Listeria monocytogenes tested
Should, and with 4 strain Listerellas and other test antibacterial without intersecting, specifically as shown in Fig. 5-2.
Claims (6)
1. listeria or the colloidal gold strip of Listeria monocytogenes, its feature in detection food based on monoclonal antibody
It is: include adsorptive pads (1), nitrocellulose filter (2), nature controlling line (3), detection line (4), sample pad (5) and PVC base plate (7)；
Separately it is furnished with golden labeling antibody (6)；
PVC base plate (7) sets gradually sample pad (5), nitrocellulose filter (2) and adsorptive pads (1)；Nitrocellulose filter
(2) it is directly arranged on PVC base plate (7)；Sample pad (5) one end is overlapped on nitrocellulose filter (2), and the other end is directly arranged
On PVC base plate (7)；Adsorptive pads (1) one end is arranged on PVC base plate (7), and the other end is overlapped on nitrocellulose filter (2)；
Nature controlling line (3) and detection line (4) it is sequentially provided with on described nitrocellulose filter (2)；Described nature controlling line is coated goat-anti on (3)
Mus IgG bis-resists, and detection line (4) is coated detection antibody 2G7；
When listeria in detection food based on monoclonal antibody, described gold labeling antibody (6) is the gold colloidal of antibody 2H8
Solution；When detection Listeria Monocytogenes In Food based on monoclonal antibody, gold labeling antibody (6) is the gold colloidal of antibody A number
Solution.
2. listeria or the gold colloidal of Listeria monocytogenes in detection food based on monoclonal antibody described in claim 1
The preparation method of test strips, it is characterised in that step is:
(I) preparation of gold labeling antibody (6):
A, the synthesis of colloidal gold solution: use the golden nanometer particle of citric acid reducing process synthesis 30nm, add in clean flask
Enter the chlorauric acid solution of 300mL mass concentration 0.01%, be heated under magnetic stirring seething with excitement completely, rapidly join in solution
The citric acid three sodium solution of 4.8mL mass concentration 1%, boils 10min by solution until color becomes bright claret, will burn
Bottle is placed in room temperature cooling, and 4 DEG C of preservations；
B, the coupling of gold labeling antibody: being respectively adopted antibody 2H8 or antibody A labelled notation colloid gold particle, detailed process is: use 0.1M
The colloidal gold solution that 1mL step a is prepared by solution of potassium carbonate adjusts pH to 7.5；Add monoclonal antibody 2H8 or antibody A number 10 μ g
And at room temperature react 2h；Subsequently, the ultra-pure water solution of the bovine serum albumin BSA that 50 μ L mass volume ratios are 10%, room are added
Temperature is lower continues reaction 2 h；4 DEG C, centrifugal colloidal gold solution under the conditions of 6000g, 20min, remove BSA and the monoclonal anti of non-coupling
Body；With being the NaN of 0.1% containing mass concentration3, 0.2% Tween, the 10mM phosphate buffer weight of 0.01M of 0.2% sucrose
Outstanding gold labeling antibody, 4 DEG C of preservations, obtain antibody 2H8 colloidal gold solution or antibody A colloidal gold solution；
(II) preparation of colloidal gold strip: be fixed on PVC base plate on (7) by nitrocellulose filter (2), by adsorptive pads (1)
Stick to nitrocellulose filter (2) with PVC base plate (7) near one end of nature controlling line (3), sample pad (5) is sticked to nitric acid fine
Dimension element film (2) and the other end near detection line (4) of PVC base plate (7)；Detection antibody 2G7 is diluted to 1mg/mL, with spray film
Machine is sprayed onto detection line (4) place；By anti-for the sheep anti-mouse igg two of 0.5 mg/mL nature controlling line (3) place that is sprayed onto, dry 2h for 37 DEG C, the most prepared
Listeria or the colloidal gold strip of Listeria monocytogenes in detection food based on monoclonal antibody.
3. listeria or the gold colloidal of Listeria monocytogenes in detection food based on monoclonal antibody described in claim 1
The application of test strips, it is characterised in that: when gold labeling antibody is antibody 2H8, listeria in specific detection food；
When gold labeling antibody is antibody A, for specific detection Listeria Monocytogenes In Food.
Listeria or the glue of Listeria monocytogenes in detection food based on monoclonal antibody the most according to claim 3
The application of body gold test paper strip, it is characterised in that: described listeria includes 4 kinds of Listerellas and 8 kinds of Listeria monocytogenes.
Listeria and the glue of Listeria monocytogenes in detection food based on monoclonal antibody the most according to claim 4
The application of body gold test paper strip, it is characterised in that:
Described 8 kinds of Listeria monocytogenes are: ATCC 19111(1/2a), ATCC 19115 (4b), ATCC 19118 (4e),
CMCC 54002 (1/2c), CMCC 54003, CMCC 54004, CMCC 54007 separate strain with chilled beef；
ATCC 19119(Listeria ivanovii), ATCC 33090(Listeria described 4 kinds of Listerellas are:
Innocua, 6a), ATCC 35897(Listeria welshimeri, 6b) and ATCC 25400(Listeria grayi).
Listeria or the glue of Listeria monocytogenes in detection food based on monoclonal antibody the most according to claim 3
The application of body gold test paper strip, it is characterised in that concretely comprise the following steps:
Take 7 μ L gold labeling antibody (6) and 47 μ L re-suspension liquid, i.e. containing mass concentration be 0.2% Tween, the 0.01M of 0.2% sucrose
10 mM phosphate buffers, 150 μ L testing samples react 5min in microwell plate, subsequently colloidal gold strip are inserted micropore
In plate, under chromatography effect, and the gold object that is combined of labeling antibody, i.e. listeria or Listeria monocytogenes P60 albumen, from
Sample pad (5) captures antibody in detection line (4), answers with sheep anti-mouse igg two anti-reflective on nature controlling line (3), thus at nature controlling line
(3) and in detection line (4), redness occurs；By naked eyes, result is carried out interpretation, nature controlling line (3) and detection line (4) together after 10min
Time occur that redness is the positive, detection line (4) does not develops the color and nature controlling line (3) occurs that redness is feminine gender.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106771217A (en) * | 2016-12-05 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of single method for quick for increasing listeria spp |
| CN111505280A (en) * | 2020-04-17 | 2020-08-07 | 南昌大学 | Colloidal gold immunochromatographic kit for ultrasensitive detection of listeria monocytogenes |
| CN112578015A (en) * | 2020-10-20 | 2021-03-30 | 山东大学 | Listeria monocytogenes detection test strip based on hemolysin O and preparation and application thereof |
| CN112698036A (en) * | 2020-12-22 | 2021-04-23 | 上海理工大学 | Immune test paper strip for rapidly determining cross reaction of bacterial monoclonal antibody and determination method |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101609095A (en) * | 2009-07-29 | 2009-12-23 | 中国检验检疫科学研究院 | A kind of colloidal gold immunochromatographimethod method and colloid gold immune test strip of new fast quantifying and detecting Listeria monocytogenes |
| CN201548546U (en) * | 2009-06-05 | 2010-08-11 | 中国检验检疫科学研究院 | Listeria monocytogenes collaurum test paper |
| CN202710560U (en) * | 2012-06-15 | 2013-01-30 | 北京博欧实德生物技术有限公司 | Kit for fast detecting Listeria monocytogenes |
| CN103911347A (en) * | 2013-11-09 | 2014-07-09 | 中华人民共和国吉林出入境检验检疫局 | Monoclonal antibody capable of resisting Listeria monocytogenes, and application thereof |
| CN103941012A (en) * | 2014-04-29 | 2014-07-23 | 上海理工大学 | Quick detection test strip for listeria immune colloidal gold |
| CN104178458A (en) * | 2014-06-13 | 2014-12-03 | 无锡杰圣杰康生物科技有限公司 | Listeria monocytogenes monoclonal antibody hybridoma cell strain and application thereof |
| CN105203758A (en) * | 2015-09-21 | 2015-12-30 | 天津市德恒科技有限公司 | Monotone increasing listeria monocytogene detecting method based on colloidal gold test strip |
| CN105527441A (en) * | 2016-01-21 | 2016-04-27 | 江南大学 | Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on basis of monoclonal antibodies |
-
2016
- 2016-06-07 CN CN201610396890.1A patent/CN106093410A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201548546U (en) * | 2009-06-05 | 2010-08-11 | 中国检验检疫科学研究院 | Listeria monocytogenes collaurum test paper |
| CN101609095A (en) * | 2009-07-29 | 2009-12-23 | 中国检验检疫科学研究院 | A kind of colloidal gold immunochromatographimethod method and colloid gold immune test strip of new fast quantifying and detecting Listeria monocytogenes |
| CN202710560U (en) * | 2012-06-15 | 2013-01-30 | 北京博欧实德生物技术有限公司 | Kit for fast detecting Listeria monocytogenes |
| CN103911347A (en) * | 2013-11-09 | 2014-07-09 | 中华人民共和国吉林出入境检验检疫局 | Monoclonal antibody capable of resisting Listeria monocytogenes, and application thereof |
| CN103941012A (en) * | 2014-04-29 | 2014-07-23 | 上海理工大学 | Quick detection test strip for listeria immune colloidal gold |
| CN104178458A (en) * | 2014-06-13 | 2014-12-03 | 无锡杰圣杰康生物科技有限公司 | Listeria monocytogenes monoclonal antibody hybridoma cell strain and application thereof |
| CN105203758A (en) * | 2015-09-21 | 2015-12-30 | 天津市德恒科技有限公司 | Monotone increasing listeria monocytogene detecting method based on colloidal gold test strip |
| CN105527441A (en) * | 2016-01-21 | 2016-04-27 | 江南大学 | Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on basis of monoclonal antibodies |
Non-Patent Citations (2)
| Title |
|---|
| 罗立新等: "快速检测产单核李斯特菌免疫胶体金层析法的研究", 《中国卫生检验杂志》 * |
| 罗雁非等: "胶体金标记单克隆抗体测定单增李斯特菌试剂盒制备", 《中国实验诊断学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106771217A (en) * | 2016-12-05 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of single method for quick for increasing listeria spp |
| CN111505280A (en) * | 2020-04-17 | 2020-08-07 | 南昌大学 | Colloidal gold immunochromatographic kit for ultrasensitive detection of listeria monocytogenes |
| CN112578015A (en) * | 2020-10-20 | 2021-03-30 | 山东大学 | Listeria monocytogenes detection test strip based on hemolysin O and preparation and application thereof |
| CN112698036A (en) * | 2020-12-22 | 2021-04-23 | 上海理工大学 | Immune test paper strip for rapidly determining cross reaction of bacterial monoclonal antibody and determination method |
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