CN104237518B - The lateral flow immunochromatography of detection Listeria monocytogenes measures product and preparation method - Google Patents
The lateral flow immunochromatography of detection Listeria monocytogenes measures product and preparation method Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
A kind of lateral flow immunochromatography detecting Listeria monocytogenes measures product and preparation method, this product includes interconnective sample pad, adsorptive pads and has the detection line and the coated film of nature controlling line being separated from each other, described coated film is between described sample pad and described adsorptive pads, magnetic nano-particle labelling Listeria monocytogenes antibody it is loaded with in described sample pad, described detection line is coated with Listeria monocytogenes coated antibody, and described nature controlling line is coated with and the second antibody of described magnetic nano-particle labelling Listeria monocytogenes antibody specific bond；Described magnetic nano-particle labelling Listeria monocytogenes antibody is that the magnetic nano-particle of Listeria monocytogenes antibody to be marked and carboxyl modified is combined the polymer formed with covalent peptide bonds.This mensuration product is used for detecting Listeria monocytogenes, highly sensitive, high specificity, quick, easy, can realize the mensuration that objectifies.
Description
Technical field
The present invention relates to lateral flow immunochromatography and measure product and preparation method thereof, particularly detection single increasing Li Si
The lateral flow immunochromatography of special bacterium (Listeria monocytogenes) measures product and preparation method.
Background technology
It is to occur that lateral flow immunochromatography measures (LFIA, Lateralflow immunoassay) latter stage in 20th century
Novel immune detection method, there is simple and rapid feature, at multiple Viral diagnosis such as HIV, hepatitis B virus
And hormone test aspect is widely used.It is by combining immunolabelling technique and film chromatographic technique, in pole
In short time, it is not necessary to specific condition, result can be made and judge, have become as a kind of important convenient immunity
Detection method.
Listeria monocytogenes (Listeria monocytogenes) increases Liszt also known as single
Bacterium.Listerella is the antibacterial that a class is prevalent in environment, and Listeria monocytogenes is then Listerella
In unique a kind of zoonosis pathogenic bacterium, be also one of common food-borne pathogens, it can cause intestinal
The clinical symptoms such as gastritis disease, meningitis, septicemia, miscarriage, to neonate, anemia of pregnant woman, old people and immunity
The harm of the low crowd of power is particularly acute, and fatality rate is up to 20%.Listeria monocytogenes is widely present in
In various foods, and can survive under the conditions of the freezing after food processing and vacuum packaging etc., be food safety
A big hidden danger.About Listeria monocytogenes food pollution event abroad time have been reported that, including 2008
Occur to infect Listeria monocytogenes event and the malicious Fructus Melo thing occurred for 2011 in the U.S. at Canadian meat
It is dead that part has resulted in many people；Although the most not reporting the PI that Listeria monocytogenes is relevant
Event, but show according to quality testing department audit report, in multiple imported meat, the detection of Listeria monocytogenes
Rate is of a relatively high, therefore, sets up quick, the effective and detection method of reliable results, disappears for ensureing
The person's of expense health is significant.
At present, the traditional method of detection Listeria monocytogenes is by sample carries out secondary increasing bacterium,
Utilize Pseudonocardia and chromogenic culture medium to separate and identify.Pseudonocardia is such as
PALCAM, OXA, or chromogenic culture medium such as CHROMagar Liszt's culture medium, they are mainly by composition
The metabolite generation chemical reaction of certain material and antibacterial and produce color change, thus distinguish different
Bacterial strain.Selective medium is usually used in distinguishing the antibacterial of different genera, but is difficult to differentiate the different strains in belonging to；
Chromogenic culture medium has the feature of high specific, but owing to it is expensive, detects the shortcomings such as cycle length, real
Border range of application is the widest.In addition to conventional detection method, along with molecular detection technology and immunology detection
Technology day by day ripe, with nucleic acid amplification detection technique and euzymelinked immunosorbent assay (ELISA) be detected as the detection method that represents because of
There is high specificity and the detection fast feature of speed, the most gradually replace original detection method.But major part due to
Needing higher technology and condition and detection speed relatively slow, the scene limited in port quarantine makes
With.
Summary of the invention
The technical problem to be solved is to provide a kind of lateral flow immunity layer detecting Listeria monocytogenes
Analysis measures product and preparation method, and this mensuration Product checking Listeria monocytogenes is sensitive reliably, result is easy to protect
Deposit and quantitative analysis, easy and simple to handle quickly.
Lateral flow immunochromatography provided by the present invention measures product, including sample pad, adsorptive pads with have phase
The detection line separated mutually and the coated film of nature controlling line, described coated film is connected to described sample pad and described water suction
Between pad, described sample pad is loaded with magnetic nano-particle labelling Listeria monocytogenes antibody, described detection
Line is coated with Listeria monocytogenes coated antibody, and described nature controlling line is coated with and described magnetic nano-particle labelling
The second antibody of Listeria monocytogenes antibody specific bond；Described magnetic nano-particle labelling Listeria monocytogenes
Antibody is to be combined with covalent peptide bonds by the magnetic nano-particle of Listeria monocytogenes antibody to be marked and carboxyl modified
The polymer formed.
Above-mentioned lateral flow immunochromatography measures in product, and described detection line is less than institute with the distance of described sample pad
State the distance of nature controlling line and described sample pad.
Described Listeria monocytogenes coated antibody can be Listeria monocytogenes monoclonal antibody or Listeria monocytogenes
Polyclonal antibody；Described Listeria monocytogenes antibody to be marked can be Listeria monocytogenes monoclonal antibody or list
Increase Listerella polyclonal antibody.
Above-mentioned lateral flow immunochromatography measures in product, described Listeria monocytogenes antibody to be marked affine often
Number is 108M-1、106～108M-1Or 107～108M-1；Described Listeria monocytogenes coated antibody affine often
Number is 108M-1、106～108M-1Or 107～108M-1；The magnetic nano-particle of described carboxyl modified average
A diameter of 100nm, 80-200nm or 60-300nm；The diameter of the magnetic nano-particle of described carboxyl modified
The coefficient of variation (CV) can be 15%, 10%-20% or 10%-30%；The magnetic nano-particle of described carboxyl modified
Saturation magnetization can be 40emu/g.
Wherein, the affinity costant of described Listeria monocytogenes antibody to be marked concretely 108M-1, described list
Increase the affinity costant of Listerella coated antibody concretely 106～108M-1, the magnetic of described carboxyl modified is received
The average diameter of rice corpuscles can be 100nm, the diameter variation coefficient of the magnetic nano-particle of described carboxyl modified
(CV) can be 15%, the saturation magnetization of the magnetic nano-particle of described carboxyl modified can be 40emu/g.
The magnetic Fe of the magnetic nano-particle of described carboxyl modified concretely carboxyl modified3O4Nanoparticle.
In one embodiment of the invention, described Listeria monocytogenes antibody to be marked is available from Abnnova
The mouse monoclonal antibody of the anti-Listeria monocytogenes of numbered MAB0532 of company.Described Listeria monocytogenes bag
The mouse monoclonal antibody of the anti-Listeria monocytogenes of numbered MAB0529 of Abnnova company it is available from by antibody.
Described magnetic nano-particle is available from TELUS Science and Technology Ltd. of Shenzhen, and catalog number is the poly-of MP-2
The magnetic Fe that maleic acid hexadecanol ester (PMAH) is modified3O4Water-solubility nanocrystalline, described magnetic nano-particle is
The magnetic nano-particle of carboxyl modified, the magnetic nano-particle average diameter of described carboxyl modified is 100nm, directly
The footpath coefficient of variation (CV) is 15%, and the saturation magnetization of the magnetic nano-particle of described carboxyl modified is
40emu/g.It is sheep with the second antibody of described magnetic nano-particle labelling Listeria monocytogenes antibody specific bond
Dynamics.
In the present invention, described Listeria monocytogenes antibody to be marked and described Listeria monocytogenes coated antibody parent
The affinity costant to Listeria monocytogenes is each meant with constant.
Above-mentioned lateral flow immunochromatography measures in product, and described magnetic nano-particle labelling Listeria monocytogenes resists
Body is prepared according to the method comprised the steps: with institute after being activated by the magnetic nano-particle of described carboxyl modified
State Listeria monocytogenes antibody to be marked to carry out reaction with the mass ratio of 50:3 and obtain combining shape with covalent peptide bonds
The magnetic nano-particle become and the conjugate of Listeria monocytogenes antibody.
In the preparation method of described magnetic nano-particle labelling Listeria monocytogenes antibody, described carboxyl modified
Magnetic nano-particle 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide
Activate；In described activation, the magnetic nano-particle activation 1-ethyl-(3-used of described carboxyl modified
Dimethylaminopropyl) concentration of carbodiimide and N-hydroxysuccinimide is respectively 5mM and 10mM；Institute
The temperature stating activation is 37 DEG C, and the time is 0.5h.
In one embodiment of the invention, as follows the magnetic nano-particle of carboxyl modified is carried out
Activation: by the magnetic nano-particle of the above-mentioned carboxyl modified of 2.5mg and 5mmol1-ethyl-(3-dimethylamino
Base propyl group) carbodiimide (EDC) and 10mmol NHS (N-hydroxysuccinimide) MES buffer (0.1M,
PH4.7) 0.5h, the magnetic nano-particle of carboxyl modified after being activated are reacted at 37 DEG C in.
In the preparation method of described magnetic nano-particle labelling Listeria monocytogenes antibody, described by carboxyl modified
Magnetic nano-particle activation after carry out with the mass ratio of 50:3 with described Listeria monocytogenes antibody to be marked
Reaction be 37 DEG C concentration be 50mM pH be 8.5 borate buffer solution in react 3.5 hours.Described 50mM
PH be 8.5 borate buffer solution can prepare as follows: by 1.9g Na2B4O7.10H2O is dissolved in 100ml
In water, pH to 8.5 is adjusted to obtain the borate buffer solution that 50mM pH is 8.5.
The preparation method of described magnetic nano-particle labelling Listeria monocytogenes antibody also includes described magnetic
Nanoparticle obtains described magnetic nano particle after the conjugate BSA solution closing of Listeria monocytogenes antibody
The step of sub-labelling Listeria monocytogenes antibody, the concentration of described BSA solution is 5% (mass percentage concentration),
The temperature of described closing is 37 DEG C, and the time is 0.5h.
In the preparation method of described magnetic nano-particle labelling Listeria monocytogenes antibody, also include with BSA
The described magnetic nano-particle labelling Listeria monocytogenes antibody 0.02M pH that solution obtains after closing is 7.4
PBS carry out the step washing and suspend.
Described lateral flow immunochromatography measures product and may also include backboard and/or protecting film, can be with reagent paper, card etc.
Form exists.
Above-mentioned lateral flow immunochromatography measures product can be prepared by the following method.
The method preparing above-mentioned lateral flow immunochromatography mensuration product includes:
I, prepare above-mentioned lateral flow immunochromatography respectively and measure sample pad and described coated film described in product；
II, described sample pad step I obtained, described coated film and adsorptive pads are connected with each other, and obtain institute
State coated film lateral flow immunochromatography between described sample pad and described adsorptive pads and measure product.
Wherein, the manufacture method of described coated film includes: thought by described Listeria monocytogenes coated antibody
The concentration of 2mg/ml is coated in nitrocellulose filter and obtains described detection line, is surveyed by described lateral flow immunochromatography
Second antibody described in fixed output quota product is coated on described nitrocellulose filter and described detection with the concentration of 1mg/ml
The region that line is separated from each other, obtains described nature controlling line, has the described nitre of described detection line and described nature controlling line
Acid cellulose film is described coated film.
In the manufacture method of above-mentioned coated film, described detection line and being coated of described nature controlling line are all to use
BioJet Quanti3000 shower nozzle in the XYZ3050 spray membranous system of BioDot increases single described in 2mg/ml
The extremely described nitrocellulose filter of second antibody solution spraying described in Listerella coated antibody solution and 1mg/ml,
Complete being coated of described detection line and described nature controlling line.Wherein, Listeria monocytogenes described in 2mg/ml is coated anti-
The solvent of second antibody solution described in liquid solution and 1mg/ml can be all pH be 7.4 0.02M PBS buffering
Liquid.Described pH be 7.4 0.02M PBS can prepare as follows: weigh 2.3g Na2HPO4、
0.524g NaH2PO4.H2O, 8.77g NaCl is dissolved in water, is settled to 1L with water, adjusts pH to 7.4, obtains
PH is the 0.02M PBS of 7.4.
Wherein, the manufacture method of described sample pad includes: described magnetic nano-particle labelling list is increased Liszt
Bacteria antibody is coated in glass fibre element film with the concentration of 0.5mg/ml and obtains described sample pad.
In the manufacture method of described sample pad, described glass fibre element film is being coated described magnetic nano-particle mark
Before note Listeria monocytogenes antibody, carry out following pretreatment: described glass fibre element film is processed buffering at film
Liquid soaks 1 hour at 37 DEG C, obtains the glass fibre element film through pretreatment；Described film processes buffer
Preparation as follows: Triton X-100, BSA, sucrose are dissolved in the 0.02M PBS that above-mentioned pH is 7.4
Buffer makes the weight/mass percentage composition of Triton X-100 be 0.2%, the weight/mass percentage composition of BSA is 1%, sugarcane
The weight/mass percentage composition of sugar is 1%, adjusts pH to 7.4, obtains film and processes buffer.Described by described magnetic
It is by institute that nanometer particle to mark Listeria monocytogenes antibody is coated in glass fibre element film with the concentration of 0.5mg/ml
State magnetic nano-particle labelling Listeria monocytogenes antibody described film process buffer and be made into described magnetic Nano
Particle label Listeria monocytogenes antibody content is the liquid of 0.5mg/ml, by described liquid spray in by described
Through the glass fibre element film of pretreatment, obtain described sample pad after drying.Wherein, described spraying can be adopted
The AirJet Quanti3000 shower nozzle sprayed in membranous system with the XYZ3050 of BioDot is carried out.
The lateral flow immunochromatography of the present invention measures product can be used for detecting in foodstuff samples whether contain single increasing Lee
This special bacterium.
The lateral flow immunochromatography of the present invention measures the immunochromatography detection skill of product and magnetic compound particles labelling
Art is correlated with, and is to use magnetic compound particles as marker material, carries out a class side of fast immune chromatographic mensuration
Method, this Technology Integration phases such as magnetic Nano material chemosynthesis, labelling technique, flow measurement immunochromatography technique
The research in field, pass.The lateral flow immunochromatography of the present invention measures the principle that product chromatographs based on sidestream immune,
After adding testing sample, the Listeria monocytogenes in sample resists with magnetic nano-particle labelling Listeria monocytogenes
Body combine after chromatography to detection line (T line) place, with the Listeria monocytogenes coated antibody shape of spraying at T line
Coated antibody-antigen-magnetic marker antibody immune complex, unnecessary magnetic nano-particle labelling list is become to increase Li Si
The magnetic mark immune complex that special bacteria antibody is then formed with anti-Mus IgG at nature controlling line (C line) place.Use magnetic reagent paper
Interpretoscope measures the magnetic strength intensity of magnetic microsphere at T line, by with the threshold ratio set to and determine that it is positive
Or negative findings, C line measurement result is then as the Quality Control internal standard of this assay method.
Beneficial effects of the present invention:
The lateral flow immunochromatography of the present invention measures product and is capable of the quick of Listeria monocytogenes and Gao Ling
Sensitive detection, has the advantage that highly sensitive, high specificity, quick, easy, can realize the survey that objectifies
Fixed.The lateral flow immunochromatography of the present invention measures product and the sensitivity of Listeria monocytogenes is reached 104CFU/mL。
The present invention can effectively meet the health quarantine needs being related to Listeria monocytogenes, for the prison of relevant epidemic situation
Control and take precautions against significant.
Accompanying drawing explanation
Fig. 1 is that the lateral flow immunochromatography of detection Listeria monocytogenes measures product structure schematic diagram；
Fig. 2 is the water-solubility nanocrystalline Electronic Speculum figure that poly hexadecanol ester (PMAH) is modified；
Fig. 3 is that lateral flow immunochromatography measures detection paper value and concentration standard curve figure.
Detailed description of the invention
Below in conjunction with accompanying drawing, embodiments of the invention are elaborated.State under it is emphasized that
Bright that be merely exemplary rather than in order to limit the scope of the present invention and application thereof.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Magnetic nano-particle used in following embodiment is available from TELUS Science and Technology Ltd. of Shenzhen, produces
Product catalog number (Cat.No.) is the magnetic Fe that poly hexadecanol ester (PMAH) of MP-2 is modified3O4Water-solubility nanocrystalline (figure
2), its core is Fe3O4Nano-particle, solid content is 30mg/ml, for the magnetic nano-particle of carboxyl modified,
The magnetic nano-particle average diameter of this carboxyl modified is 100nm, and diameter variation coefficient (CV) is 15%；Should
The saturation magnetization of the magnetic nano-particle of carboxyl modified is 40emu/g.
Listeria monocytogenes antibody to be marked used in following embodiment is available from the numbering of Abnnova company
Mouse monoclonal antibody for the anti-Listeria monocytogenes of MAB0532.
Listeria monocytogenes coated antibody in following embodiment is available from the numbered of Abnnova company
The mouse monoclonal antibody of the anti-Listeria monocytogenes of MAB0529.
For making the glass fibre element film of sample pad purchased from Whatman company, commodity mesh in following embodiment
Record number is Standard17.
For making the nitrocellulose filter of coated film purchased from Whatman company, commodity mesh in following embodiment
Record number isFP。
PH in following embodiment be 7.4 0.02M PBS prepare as follows: weigh 2.3g
Na2HPO4、0.524g NaH2PO4.H2O, 8.77g NaCl is dissolved in pure water, is settled to 1L with pure water, adjusts pH
To 7.4, obtain the 0.02M PBS that pH is 7.4.
Film in following embodiment processes buffer and prepares as follows: by Triton X-100, BSA,
Sucrose is dissolved in the 0.02M PBS that above-mentioned pH is 7.4 makes the weight/mass percentage composition of Triton X-100
Be 0.2%, the weight/mass percentage composition of BSA be 1%, the weight/mass percentage composition of sucrose be 1%, adjust pH to 7.4,
Obtain film and process buffer.
The borate buffer solution that 50mM pH is 8.5 in following embodiment is prepared as follows: weigh 1.9g
Na2B4O7.10H2O is dissolved in 100ml pure water, adjusts pH to 8.5 to obtain the borate buffer solution that 50mM pH is 8.5.
As it is shown in figure 1, according to embodiments of the invention, lateral flow immunochromatography measure product include sample pad 2,
Adsorptive pads 5 and there is the detection line 3 and the coated film 6 of nature controlling line 4, sample pad 2, adsorptive pads being separated from each other
5 and coated film 6 be both mounted on a backboard 7.Wherein, described coated film 6 is connected to described sample pad 2 He
Between described adsorptive pads 5, described detection line 3 and the distance of described sample pad 2 less than described nature controlling line 4 with
The distance of described sample pad 2.Magnetic nano-particle labelling Listeria monocytogenes it is loaded with in described sample pad 2
Antibody, described detection line 3 is coated with Listeria monocytogenes coated antibody, and described nature controlling line 4 is coated with and institute
State the second antibody of magnetic nano-particle labelling Listeria monocytogenes antibody specific bond；Described magnetic nano particle
Sub-labelling Listeria monocytogenes antibody is by Listeria monocytogenes antibody to be marked and the magnetic Nano of carboxyl modified
Particle combines the polymer formed with covalent peptide bonds.
In more preferably embodiment, the thickness of product is in the scope (i.e. 4.9-5.1mm) of 5.0 ± 0.1mm.?
More preferably in embodiment, the length of adsorptive pads 5 exists in scope, the length of coated film 6 of 25.0 ± 0.1mm
The scope of 35.0 ± 0.1mm, the length of sample pad 2 is in the scope of 25.0 ± 0.1mm, detection line 3 and matter
The distance of control line 4 is in the scope of 5.0 ± 0.1mm.It has been investigated that, the dimensional structure design of above-mentioned optimization,
Mensuration product can be made to obtain preferable detection efficiency on the basis of the high accuracy of satisfied detection, and have
Effect saves the material of product.
According to embodiments of the invention, lateral flow immunochromatography measures the preparation method of product and comprises the following steps:
(1) preparation of the magnetic nano-particle label probe of carboxyl modified
Use the magnetic nano-particle of the carboxyl modified being suitable for, after activating the carboxyl on its surface, use chemistry even
Listeria monocytogenes antibody orientation is connected to the magnetic nano particle sub-surface of this carboxyl modified by the mode of connection.
(2) being coated of antigen/antibody at test section T line and C line
Use spray film instrument, at the T line of test section, spray Listeria monocytogenes coated antibody, at C line
Spraying dynamics.
(3) being coated of label probe at sample pad
Use spraying apparatus, in the anti-Listeria monocytogenes of sample pad specific location deposited magnetic microsphere labelling
Antibody (magnetic nano-particle labelling Listeria monocytogenes antibody).
(4) assembled formation of Sptting plate
According to structure chart (see Fig. 1) requirement of Sptting plate, paste as test in the middle of plastic support backboard 7
Celluloid (NC) film in district, the T line end in NC film pastes sample pad 2, C line end stickup adsorptive pads 5.
Paste transparent protective film in the above.Use reagent paper cutting machine, monoblock Sptting plate is cut as certain broadband
Paper slip, packs with the special aluminium foil bag equipped with desiccant.
(5) formation of Ag-Ab magnetic mark immune complex
Adding testing sample 1 at the well of the Sptting plate of above-mentioned assembled formation, the list in sample 1 increases Li Si
Listeria monocytogenes antibody (the magnetic nano-particle labelling Listeria monocytogenes antibody) knot of special bacterium and magnetic marker
After conjunction, chromatography arrives the Listeria monocytogenes coated antibody of spraying at T line, forms coated antibody-antigen at T line
-magnetic marker antibody immune complex, unnecessary magnetic mark Listeria monocytogenes antibody (magnetic nano-particle labelling list
Increase Listerella antibody) the magnetic mark immune complex that then formed with anti-Mus IgG at C line.
(6) magnetic mark immune complex magnetic field intensity detection
The magnetic field intensity of magnetic microsphere at T line is measured, by the threshold ratio with setting with magnetic reagent paper interpretoscope
To and determine its positive or negative result, C line measurement result is then as the Quality Control internal standard of this assay method.
The magnetic field intensity of described magnetic mark immune complex, refers to the knot under being detained respectively at T line and C line
The numerical value that the magnetic resonance detector MAR of the quantity U.S. Quantum Dot closing magnetic bead is obtained after measuring.
By the immunoreactive condition of double-antibody sandwich, research finds, can through the different clinical serum sample of a large amount of mensuration
Determine the mensuration average of variant normal sample, determine that T line is examined in this, as marginal value (cutoff)
Test sample positive or negative result originally.C line measurement result is then as the Quality Control internal standard of this assay method.
Embodiment 1, the lateral flow immunochromatography of detection Listeria monocytogenes measure the preparation of reagent paper
(1) preparation of magnetic nano-particle labelling Listeria monocytogenes antibody
With saturation magnetization as 40emu/g, average diameter as 100nm, diameter variation coefficient (CV) be 15%
The magnetic Fe modified of poly hexadecanol ester (PMAH)3O4Water-solubility nanocrystalline is (purchased from Shenzhen Taylor
This Science and Technology Ltd., catalog number is MP-2) as the magnetic nano-particle of carboxyl modified, with to list
Increasing Listerella affinity costant is 108M-1The mouse monoclonal antibody of anti-Listeria monocytogenes (purchased from Abnnova
Company, numbered MAB0532) as Listeria monocytogenes antibody to be marked, prepare magnetic by the following method
Nanometer particle to mark Listeria monocytogenes antibody:
Magnetic nano-particle MES buffer (0.1M, pH4.7) of the above-mentioned carboxyl modified taking 2.5mg is washed
After washing and use the magnet stand separation and concentration of 0.4T, resuspended with 1ml MES buffer (0.1M, pH4.7), add
Enter 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) to final concentration of 5mM, add NHS
(N-hydroxysuccinimide), to final concentration of 10mM, at 37 DEG C, reacts carboxylic after being activated half an hour
The magnetic nano-particle that base is modified.
Wash the magnetic nano-particle of carboxyl modified after this activation with the borate buffer solution of 50mM pH8.5, take
The magnetic Nano of carboxyl modified after the above-mentioned Listeria monocytogenes antibody to be marked of 0.15mg and the above-mentioned activation of 2.5mg
Mix particles fully mixes in the borate buffer solution of 50mM pH8.5.React 3.5 hours at 37 DEG C, allow anti-
Body and magnetic particle form stable covalent peptide bonds combination and obtain magnetic nano-particle and Listeria monocytogenes antibody
Conjugate.After reaction terminates, add the BSA solution of final concentration of 5% (weight/mass percentage composition) to magnetic
Nanoparticle is closed with residual activity carboxyl site on the conjugate of Listeria monocytogenes antibody, reacts
Carry out at 37 DEG C 0.5 hour.After completing, wash with the 0.02M PBS of pH7.4, resuspended obtain
25mg/ml magnetic nano-particle labelling Listeria monocytogenes antibody liquid, 4 DEG C of preservations are stand-by.
(2) lateral flow immunochromatography of detection Listeria monocytogenes measures the preparation of reagent paper
With to Listeria monocytogenes affinity costant for 108M-1The anti-list of numbered MAB0529 of Abnnova company
Increase listerial mouse monoclonal antibody, as Listeria monocytogenes coated antibody, with sheep anti-mouse igg antibody
Prepare as the second antibody with described magnetic nano-particle labelling Listeria monocytogenes antibody specific bond and be coated
Film, concrete grammar is as follows:
Using the 0.02M PBS of pH7.4, by sheep anti-mouse igg antibody, (excellent biotechnology is won in Changsha
Company limited, ABGAM-0500) it is formulated as concentration 1mg/ml solution, by Listeria monocytogenes coated antibody
The concentration of (abcam company, ab36055) is formulated as concentration 2mg/ml solution, selects the XYZ3050 of BioDot
Sheep anti-mouse igg antibody is sprayed onto nitrocellulose filter (NC by the BioJet Quanti3000 shower nozzle in spray membranous system
Film) nature controlling line (Control Line, C line) position, Listeria monocytogenes coated antibody is sprayed onto detection
Line (Test Line, T line) position, carries out dehumidifier 4 in the drying plant that relative humidity is less than 10% little
Dried for standby time after, obtains having detection line and the coated film of nature controlling line.
Processing buffer with above-mentioned film and soak all-glass paper 1 hour, the temperature of immersion is 37 DEG C, in equally
Dehumidifier condition carry out dehumidifier 4 hours after, with above-mentioned film process buffer dilution step (one) magnetic Nano
Particle label Listeria monocytogenes antibody liquid to magnetic nano-particle labelling Listeria monocytogenes antibody content is
After 0.5mg/ml, use the AirJet Quanti3000 shower nozzle in the XYZ3050 spray membranous system of BioDot
It is sprayed on the above-mentioned glass fibre element film processed preparation and is formed sample pad, in same dehumidifier condition
It is dried.In 100,000 grades of cleanings and dry workshop, above-mentioned dried having is detected line and nature controlling line
Coated film, above-mentioned sample pad, absorbent paper (as adsorptive pads), backboard and protecting film carry out as shown in Figure 1
After collocation assembles, use the width that Paperboard cutting is 5mm/ bar that the CM4000 cutting system of BioDot will post
Degree, the lateral flow immunochromatography obtaining detecting Listeria monocytogenes measures reagent paper, loads detection intermediate plate stand-by.
The structural representation of this reagent paper is as shown in Figure 1.
Embodiment 2, the lateral flow immunochromatography of detection Listeria monocytogenes measure the sensitivity technique of reagent paper
Liszt is increased using the detection list that the Listeria monocytogenes of formalin-inactivated measures embodiment 1 as testing sample
The lateral flow immunochromatography of bacterium measures the sensitivity of reagent paper.
Listeria monocytogenes type strain (NICPBP54002) is taken out from-20 DEG C of refrigerators, with LEB culture medium
After activation, after 0.3% formalin-inactivated 12h, become series concentration (10 with normal saline9、108、107、106、
105、104、103、102, 10CFU/mL) diluent, be separately added into the detection list obtained by embodiment 1
Increase listerial lateral flow immunochromatography to measure in reagent paper, and use magnetic resonance detector MAR (Magna
BioSciences, 8094-101-01&8094-101-02) read RMU (relative magnetic field strength).Detection
Step: first detected sample is recovered before detection room temperature (25 DEG C), takes detected sample 50 μ l with accurate pipettor
The lateral flow immunochromatography being vertically slowly dropped into the detection formalin-inactivated Listeria monocytogenes that embodiment 1 obtains measures
The sample pad of reagent paper, then instills the 0.02M PBS that the above-mentioned pH of 100ul is 7.4, after 20 minutes
Test with MAR.In this formalin-inactivated Listeria monocytogenes, the definition of CFU/mL is every milliliter of bacterium colony content.
Its testing result is as shown in table 1 below.Can show that from testing result the detection list of embodiment 1 increases Li Si
It is 10 that the lateral flow immunochromatography of special bacterium measures the sensitivity of reagent paper4(take T/C value 0.015 is CFU/mL
CUT-OFF, is positive findings more than or equal to 0.015, is negative findings less than 0.015；T/C value is Test-RMU/
CtlRMU)。
Formalin-inactivated Listeria monocytogenes magnetic detection paper value is with concentration curve as shown in Figure 3.
The magnetic detection paper value of table 1 formalin-inactivated Listeria monocytogenes variable concentrations diluent
Detection sensitivity is 104Individual/mL, i.e. 500 bacterium/detections, the range of linearity is 104-108, when concentration is high to 109
Time, detected value declines, Hooke effect occurs.
Embodiment 3, the lateral flow immunochromatography of detection Listeria monocytogenes measure the specific detection of reagent paper
Select sheep Listerella (L.iuanuii), Ying Nuoke Listerella (L.innocua), this Listerella of Weir
(L.welshimeri), Xi Er Listerella (L.seeligeri), Listera grayi (L.grayi), escherichia coli
O157 (Escherichia coliO157), Salmonella detect sample as cross reaction, embodiment 1 obtain
To detection Listeria monocytogenes lateral flow immunochromatography measure reagent paper detect, except with Mianyang Li Si
Special bacterium, Xi Er Listerella have beyond cross reaction, with other detection samples without intersecting.Concrete grammar and result
As follows: by physiological saline solution, it is separately added into and is detected the lateral of Listeria monocytogenes by what embodiment 1 obtained
In stream immunochromatographic measurement reagent paper, and magnetic resonance detector MAR is used to read RMU.
Table 2 specific detection result
| TestName | CtlRMU | CtlFit | Test-RMU | Test-Fit | T/C |
| Salmonella | 7447.7 | 0.9858572 | 59 | 0.9829324 | 0.007922 |
| Escherichia coli | 7733.3 | 0.9796289 | 74.2 | 0.9888651 | 0.009595 |
| Sheep Listerella | 1748.1 | 0.986193 | 2274 | 0.9881458 | 1.300841 |
| Ying Luoke Listerella | 6828.2 | 0.9875784 | 0 | 0.2110946 | 0 |
| Listera grayi | 6507.1 | 0.9852532 | 47.8 | 0.8404327 | 0.007346 |
| Xi Er Listerella | 3850.5 | 0.9870803 | 2166.7 | 0.9891912 | 0.562706 |
| This Listerella of Weir | 8100.2 | 0.9792305 | 104.7 | 0.8817242 | 0.012926 |
The experiment proves that, by magnetic compound particles, Listeria monocytogenes and Listeria monocytogenes
The research of antibody molecule characteristic, by selecting the magnetic compound particles being suitable for be oriented with specific antibody
Covalent chemical coupling, it is thus achieved that functional magnetic mark probe, and by optimizing double-antibody sandwich immunoreation
Various conditions, the lateral flow immunochromatography preparing the present invention measures product, it is achieved that increase Li Si to single
Special bacterium quickly and Sensitive Determination, have the advantage that highly sensitive, high specificity, quick, easy,
The mensuration that objectifies can be realized.The lateral flow immunochromatography of the present invention measures sensitive to Listeria monocytogenes of product
Degree reaches 104CFU/mL.The present invention can effectively meet the health quarantine needs being related to Listeria monocytogenes, right
In the monitoring of relevant epidemic situation with take precautions against significant.
Above content is that to combine concrete preferred implementation made for the present invention the most specifically
Bright, it is impossible to assert the present invention be embodied as be confined to these explanations.For technology belonging to the present invention
For the those of ordinary skill in field, without departing from the inventive concept of the premise, it is also possible to if making
Dry simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (10)
1. the lateral flow immunochromatography detecting Listeria monocytogenes measures product, it is characterised in that include
Detection line that sample pad, adsorptive pads and having is separated from each other and the coated film of nature controlling line, described coated film connects
Between described sample pad and described adsorptive pads, described sample pad is loaded with magnetic nano-particle labelling list and increases
Listerella antibody, described detection line is coated with Listeria monocytogenes coated antibody, and described nature controlling line is coated with
Second antibody with described magnetic nano-particle labelling Listeria monocytogenes antibody specific bond；Described magnetic is received
Rice corpuscles labelling Listeria monocytogenes antibody is by Listeria monocytogenes antibody to be marked and the magnetic of carboxyl modified
Nanoparticle combines the polymer formed with covalent peptide bonds；Described Listeria monocytogenes antibody to be marked is available from
The mouse monoclonal antibody of the anti-Listeria monocytogenes of numbered MAB0532 of Abnova company, described single increasing Li Si
Special bacterium coated antibody is available from the Mus monoclonal of the anti-Listeria monocytogenes of numbered MAB0529 of Abnova company
Antibody.
Lateral flow immunochromatography the most according to claim 1 measures product, it is characterised in that described carboxylic
The average diameter of the magnetic nano-particle that base is modified is 60-300nm；The magnetic nano-particle of described carboxyl modified
Diameter variation coefficient be 10%-30%；The saturation magnetization of the magnetic nano-particle of described carboxyl modified is
40emu/g。
Lateral flow immunochromatography the most according to claim 1 and 2 measures product, it is characterised in that institute
State magnetic nano-particle labelling Listeria monocytogenes antibody to prepare according to the method comprised the steps: by described
With described Listeria monocytogenes antibody to be marked with the matter of 50:3 after the magnetic nano-particle activation of carboxyl modified
Amount obtains combining the magnetic nano-particle formed and Listeria monocytogenes antibody with covalent peptide bonds than carrying out reacting
Conjugate.
Lateral flow immunochromatography the most according to claim 3 measures product, it is characterised in that described magnetic
In the preparation method of property nanometer particle to mark Listeria monocytogenes antibody, the magnetic nano particle of described carboxyl modified
Son 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide activate；
In described activation, magnetic nano-particle activation 1-ethyl-(the 3-dimethylamino third used of described carboxyl modified
Base) concentration of carbodiimide and N-hydroxysuccinimide is respectively 5mM and 10mM；The temperature of described activation
Being 37 DEG C, the time is 0.5h.
Lateral flow immunochromatography the most according to claim 3 measures product, it is characterised in that described magnetic
In the preparation method of property nanometer particle to mark Listeria monocytogenes antibody, described by the magnetic Nano of carboxyl modified
Carrying out reaction with described Listeria monocytogenes antibody to be marked with the mass ratio of 50:3 after particle activation is 37
DEG C concentration be 50mM pH be 8.5 borate buffer solution in react 3.5 hours.
Lateral flow immunochromatography the most according to claim 3 measures product, it is characterised in that described magnetic
Property nanometer particle to mark Listeria monocytogenes antibody preparation method in also include by described magnetic nano-particle with
The conjugate BSA solution of Listeria monocytogenes antibody obtains described magnetic nano-particle labelling list and increases after closing
The step of Listerella antibody, the mass percentage concentration of described BSA solution is 5%, and the temperature of described closing is
37 DEG C, the time is 0.5h.
7. measuring product according to described lateral flow immunochromatography arbitrary in claim 1-2,4-6, it is special
Levying and be, the distance of described detection line and described sample pad is less than the distance of described nature controlling line with described sample pad.
8. prepare the method that in claim 1-6, arbitrary described lateral flow immunochromatography measures product,
It is characterized in that, including:
I, prepare arbitrary described lateral flow immunochromatography in claim 1-6 respectively to measure described in product
Sample pad and described coated film；
II, described sample pad step I obtained, described coated film and adsorptive pads are connected with each other, and obtain institute
State coated film lateral flow immunochromatography between described sample pad and described adsorptive pads and measure product.
Method the most according to claim 8, it is characterised in that the manufacture method of described coated film includes:
Described Listeria monocytogenes coated antibody is thought, and the concentration of 2mg/ml is coated in nitrocellulose filter and obtains described
Detection line, resists described in described lateral flow immunochromatography mensuration product arbitrary in claim 1-6 second
Body is coated on described nitrocellulose filter the region being separated from each other with described detection line with the concentration of 1mg/ml,
Obtaining described nature controlling line, the described nitrocellulose filter with described detection line and described nature controlling line is described
Coated film.
Method the most according to claim 8 or claim 9, it is characterised in that the making side of described sample pad
Method includes: by described magnetic nano-particle labelling Listeria monocytogenes antibody arbitrary in claim 1-6 with
The concentration of 0.5mg/ml is coated in glass fibre element film and obtains described sample pad.
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| US6727073B1 (en) * | 1999-11-19 | 2004-04-27 | Binax, Inc. | Method for detecting enteric disease |
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