CN105753981A - Anti-human respiratory syncytial virus N protein antibodies and immunochromatographic kit using the same - Google Patents
Anti-human respiratory syncytial virus N protein antibodies and immunochromatographic kit using the same Download PDFInfo
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- CN105753981A CN105753981A CN201610128340.1A CN201610128340A CN105753981A CN 105753981 A CN105753981 A CN 105753981A CN 201610128340 A CN201610128340 A CN 201610128340A CN 105753981 A CN105753981 A CN 105753981A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
- G01N2333/135—Respiratory syncytial virus
Abstract
The present invention relates to anti-human respiratory syncytial virus N protein antibodies and immunochromatographic kit for detection of human respiratory syncytial virus by using the same. The anti-human respiratory syncytial virus N protein antibodies separately recognize two linear epitopes consisting of No.21-34 amino acids and No.226-239 of human respiratory syncytial virus N protein; the human respiratory syncytial virus N protein has sequence number of AAB59852.1 in GenBank; amino acid sequence of sites No.21-34 and No.226-239 of the human respiratory syncytial virus N protein are respectively SKYTIQRSTGDSID and FGIAQSSTRGGSRV. The two kinds of rabbit anti-human respiratory syncytial virus N protein antibodies have the characteristics of good specificity, high purity, high titer and low preparation cost.
Description
Technical field
The invention belongs to field of biomedicine technology, relate to anti-human Respiratory Syncytial Virus(RSV) N protein antibody and application
The immune chromatography reagent kit of this antibody test human respiratory syncytial virus.
Background technology
Respiratory Syncytial Virus(RSV) (Respiratory Syncytial Virus, RSV) is a kind of RNA virus,
Belong to Paramyxoviridae.This disease is propagated through the air spittle and close contact.It is more common in neonate and within 6 months
Baby.Incubation period 3～7 days.Infant's symptom is heavier, can have high heat, rhinitis, pharyngitis and laryngitis, after
Show as capillary bronchitis and pneumonia.Minority sick child can concurrently tympanitis, pleurisy and myocarditis etc..Adult and
After older children infects, mainly show as the infection of the upper respiratory tract.
WHO data show, the annual respiratory syncytial virus infection person in the whole world there are about 64,000,000 examples, and 160,000 examples are dead
Die.Syncytial virus pneumonia and bronchiolitis account for China's Infant Viral Pneumonia first nearly ten years, its
Symptom the most almost cannot district with parainfluenza virus pneumonia, mild Influenza Virus Pneumonia and mild adenovirus pneumonia
Not, do not do etiological examination, be difficult to make a definite diagnosis respiratory syncytial virus infection.Hence set up simplicity, quickly, can
Row, the method that can early diagnose respiratory syncytial virus infection are the most necessary.
The detection method of Respiratory Syncytial Virus(RSV) mainly has 3 classes at present: one is isolated culture, is to confirm to infect
" goldstandard ", but owing to cultivation cycle is longer, operating procedure is more, causes this method can not enter clinically
Row quick diagnosis；Two is serological method, i.e. uses ELISA, colloidal gold immunization, skeptophylaxis glimmering
Light method and indirect hemagglutination test etc., Respiratory Syncytial Virus(RSV) antibody horizontal in detection examinee's serum, can indirectly carry
Show the existence of respiratory syncytial virus infection.But, serological test can only provide a kind of retrospective diagnosis,
And sometimes for paired sera.It addition, be difficult to the opportunity that antibody occurs grasp, children and adolescents and adult
Between there is again the difference of Respiratory Syncytial Virus(RSV) specific antibody, therefore the detection quality of existing serological method is subject to
To a definite limitation；Three is the existence utilizing Protocols in Molecular Biology detection Respiratory Syncytial Virus(RSV) DNA,
That conventional is PCR (PCR), and the method is quick, sensitive, special, is that research people breathes at present
The important means of road syncytial virus infection, but owing to experimental facilities and operation are required higher by PCR, and easily go out
Existing false positive, can't be as conventional methods for clinical diagnosis in China.Therefore, human respiratory syncytial is set up sick
The fast diagnosis method of poison specific antigen is the most necessary.Due in human respiratory syncytial virus's N protein sequence
Well-conserved so that it is to become an important examination target.Therefore the anti-human respiratory tract of high specific is obtained
Syncytial virus N protein antibody is exactly a highly important job.Currently, with respect to anti-human Respiratory Syncytial Virus(RSV) N
Protein antibodies report at most for corresponding monoclonal antibody and polyclonal antibody.Maximum excellent of monoclonal antibody
Point is exactly specific high, but preparation method is loaded down with trivial details, and production cost is high, limits its application.Polyclonal antibody
Mainly it is prepared from by animals such as the N protein immunizing rabbits of gene engineering expression.Its preparation method is simple, cost
Low, but it has the defects such as the lowest, titer is low, purity is low.Therefore, the preparation height of low cost is special
Property, the anti-human Respiratory Syncytial Virus(RSV) N protein antibody of high-titer just seem particularly significant.
Summary of the invention
For these technical problems present in background technology, it is an object of the invention to provide identification human respiratory
Two kinds of two linear epitopes that syncytial virus N protein 21-34 position and 226-239 amino acids are formed
Antibody and apply the immune chromatography reagent kit of this antibody.
Anti-human Respiratory Syncytial Virus(RSV) N protein antibody, it is characterised in that: described anti-human Respiratory Syncytial Virus(RSV) N
Protein antibodies is to identify human respiratory syncytial virus's N protein 21-34 position and 226-239 amino acids institute group respectively
Two kinds of antibody of two linear epitopes become, described human respiratory syncytial virus's N protein is in GenBank sequence
Number it is AAB59852.1；14 amino acid of described human respiratory syncytial virus's N protein 21-34 position and
14 amino acid whose amino acid sequences of 226-239 position are respectively SKYTIQRSTGDSID and FGIAQSSTRGGSRV；
By 14 amino acid and the 14 amino acid whose sequences of 226-239 position of human respiratory syncytial virus's N protein 21-34 position
Row are respectively designated as N1 and N2；Described anti-human Respiratory Syncytial Virus(RSV) N protein antibody is AbN1 and AbN2.
A kind of immunochromatography reagent formed based on foregoing anti-human Respiratory Syncytial Virus(RSV) N protein antibody
Box, it is characterised in that: described kit is immune chromatography reagent kit based on quantum dot-labeled technology or based on glue
The immune chromatography reagent kit of body gold labelling technique.
As preferably, kit provided by the present invention is immune chromatography reagent kit based on quantum dot-labeled technology
Time, the preparation method of described kit is:
1) quantum dot-labeled antibody A bN1 solution:
0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide it is sequentially added in microcentrifugal tube
EDC, with MES buffer solution constant volume as 1ml, mixed solution, after 37 DEG C of reaction 5min, add 0.34mg
Antibody A bN1 prepared, lucifuge reaction 2h, add single-ended amino-polyethyleneglycols PEG2000-NH2 to end
Concentration is 1% (m/v), closes unreacted activated carboxyl site, continues lucifuge reaction 1h；Reacted sample
Product are centrifugal (molecular cut off 100k) with super filter tube, and 6500rpm is centrifuged 5min, to volume 200ul, after ultrafiltration
Sample is transferred in common EP pipe, centrifugal except reuniting, and obtains upper clear supernate and bottom precipitation, 10000rpm
Under the conditions of centrifugal 3min；It is added to upper clear supernate on splitter Superdex-200 purify, treats that upper clear supernate is natural
Flow in cylinder, then rinse with PBS, irradiate cylinder with ultraviolet light and observe the position of sample, treat that sample starts
Start when flowing out from bottom to collect, stop collecting after collecting 1ml；Sample super filter tube after purification (is retained
Molecular weight 100k) with centrifugal except reuniting, to general in being transferred to common EP pipe after 6500rpm centrifugal concentrating to 200ul
The condition that logical EP pipe is centrifuged is 10000rpm, 3min；Liquid dilution 200 is preserved with phosphate after obtaining supernatant
Times, 4 DEG C save backup；So far quantum dot-labeled antibody A bN1 solution is prepared；
In described MES buffer solution, each constituent content is respectively: 10.66g/L MES and 0.74g/L EDTA,
The pH 7.4 of described MES buffer solution；
It is to weigh 0.29g disodium hydrogen phosphate, 0.0295g biphosphate that described phosphate preserves the preparation method of liquid
Sodium, 0.2g sodium chloride, 1g bovine serum albumin(BSA) BSA and 0.1gNaN3, it is dissolved in the deionization of 90ml
In water, after adjusting pH to 7.3 with 1mol/L NaOH, it is settled to 100ml by deionized water；
2) pad is prepared
By polyester fiber film immerse step 1) obtained by quantum dot-labeled antibody A bN1 solution in 1h, take out,
25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, and so far prepare pad；
3) sample pad is prepared
Take glass fibre element film one, glass fibre element film is soaked in sample pad treatment fluid at least 3h, then
It is placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, is cut into after specification is 4cm × 2.5cm/ bar, i.e. prepares sample
Product pad, 25 DEG C seal preservation；
The preparation method of described sample pad treatment fluid is to weigh 0.29g disodium hydrogen phosphate, 0.0295g biphosphate
Sodium, 0.2g sodium chloride, 2g bovine serum albumin(BSA) BSA, 1ml Tween-20,2g sucrose and 0.5g are poly-
Vinylpyrrolidone PVP-10, is dissolved in the deionized water of 90ml, adjusts pH to 7.3 with 1mol/L NaOH
It is settled to 100ml afterwards by deionized water；
4) detection layers is prepared
Nitrocellulose filter is cut into 4cm × 4cm size；Antibody A bN2 prepared and goat anti-rabbit igg are used
Phosphate buffer adjusts to final concentration and is respectively 2.0mg/mL and 1.0mg/mL；Antibody A bN2 that will have diluted
Loading BIODOT to draw in film instrument shower nozzle, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter, forms detection
Line；Being drawn in film instrument shower nozzle by anti-rabbit IgG diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitre
As nature controlling line on acid cellulose film, nature controlling line is 0.7cm with detection distance between centers of tracks；The celluloid that will have sprayed
37 DEG C of dry 2h of film, are cut into the specification of 4cm × 4cm, and 4 DEG C of hermetically dryings preserve；So far detection layers is prepared；
The preparation method of described phosphate buffer is: weigh 0.29g disodium hydrogen phosphate, 0.0295g di(2-ethylhexyl)phosphate
Hydrogen sodium and 0.2g sodium chloride, be dissolved in the deionized water of 90ml, adjusts pH to 7.3 with 1mol/L NaOH
It is settled to 100ml afterwards by deionized water；
5) detection card is assembled
5.1) base plate is cut into 4cm × 7.3cm size, standby；
5.2) absorbent filter is cut into 4cm × 3cm size, as adsorptive pads, standby；
5.3) by step 5.1) adhered protection film on the base plate for preparing takes off, by step 4) described in
Detection layers i.e. pastes on base plate with nature controlling line and the nitrocellulose filter detecting line, and floating face；
5.4) by step 5.2) ready adsorptive pads is assembled on base plate, makes the left side and the detection layers of adsorptive pads
Right end has the overlap of 0.2cm, and the right hand edge of adsorptive pads then aligns with the right hand edge of base plate and glues and floating；Again
By step 2) described in pad be overlapped at the left hand edge of detection layers by 0.3cm, 0.3cm sticks on base plate；
5.5) by step 3) described in sample pad be then overlapped at the left hand edge of pad by one side 0.3cm,
Another side aligns with the left hand edge of base plate, sticks on base plate and floating；By the detection plate that assembles under cutting cutter
Being cut into detection card wide for 4.0mm, 4 DEG C of hermetically dryings keep in Dark Place.
As preferably, kit provided by the present invention is immune chromatography reagent kit based on colloidal gold-labeled method
Time, the preparation method of described kit is:
1) colloidal gold labeled monoclonal antibody AbN1
1.1) 30nm colloidal gold solution is prepared:
Take a good 250ml triangular flask of silication, add 99ml ultra-pure water, by 1ml 1% (m/v) HAuCl4
Solution adds in 250ml triangular flask and mixes with ultra-pure water, and oil bath is heated and stirs to boiling；To 250ml triangle
Rapidly joining 2ml 1% (m/v) trisodium citrate aqueous solution in Ping, solution continues boiling 10min, treats 250ml
Solution in triangular flask stops heating, by the coldest for the solution in 250ml triangular flask when being changed into redness by blueness
But to 25 DEG C, in 250ml triangular flask, ultra-pure water polishing is then added to 100ml；
1.2) colloidal gold labeled monoclonal antibody AbN1:
1.2.1) take a good 50ml triangular flask of silication, add 10ml step 1.1) prepared by collaurum
Solution, adds 240ul 0.2mol/L K in collaurum liquid2CO3Regulation pH to 8.5；
1.2.2) under magnetic stirrer, antibody A bN1 is added in colloidal gold solution, the denseest to antibody
Degree is 10ug/ml, is added dropwise over when adding antibody, continues stirring 45min～60min after adding；
1.2.3) addition 2.5ml 5% (m/v) bovine serum albumin(BSA) BSA to final concentration of 1% (m/v) has been reacted,
Stirring 15～30 minutes, 4 DEG C save backup；
1.2.4) loading 50ml centrifuge tube, 2500g after antibody A bN1 marked being taken out, 4 DEG C are centrifuged 5 minutes,
Obtaining lower sediment and supernatant liquor, discard lower sediment, supernatant liquor is transferred to another 50ml centrifuge tube,
12000g, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and supernatant liquor, discard supernatant liquor, lower floor sunk
The shallow lake 10ml resuspended precipitation of collaurum buffer solution, 12000g the most again, 4 DEG C are centrifuged 30 minutes, under again obtaining
Layer precipitation and supernatant liquor, lower sediment is finally resuspended with 3ml collaurum buffer solution, and 4 DEG C save backup；
In described collaurum buffer solution, each constituent content is respectively: 10mM Tris, 1% (m/v) BSA, 1%
(v/v) Tween-20,5% (m/v) sucrose and 3 ‰ (m/v) polyvinylpyrrolidone, described colloid
The pH of gold buffer solution is 10.5；
2) pad is prepared
By polyester fiber film immerse step 1) obtained by colloidal gold labeled monoclonal antibody AbN1 solution in 1h, take out,
25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, and so far prepare pad；
3) sample pad is prepared
Take glass fibre element film one, glass fibre element film is soaked at least 2h in sample pad treatment fluid, then puts
In Biohazard Safety Equipment after 37 DEG C of aeration-dryings, it is cut into after specification is 4cm × 1.5cm/ bar, i.e. prepares sample
Pad, 25 DEG C seal preservation；
The preparation method of described sample pad treatment fluid is to weigh 0.242g Tris, 1g bovine serum albumin(BSA) BSA, 1ml
Tween-20,5g sucrose and 0.3g polyvinylpyrrolidone PVP-10, be dissolved in the deionized water of 90ml,
It is settled to 100ml by deionized water after adjusting pH to 11 with 1mol/L NaOH；
4) detection layers is prepared
Nitrocellulose filter is cut into 4cm × 2cm size；Antibody A bN2 prepared and goat anti-rabbit igg are used
Phosphate buffer adjusts to final concentration and is respectively 2.0mg/mL and 1.0mg/mL；Antibody A bN2 that will have diluted
Loading BIODOT to draw in film instrument shower nozzle, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter, forms detection
Line；Being drawn in film instrument shower nozzle by anti-rabbit IgG diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitre
As nature controlling line on acid cellulose film, nature controlling line is 0.5cm with detection distance between centers of tracks；The celluloid that will have sprayed
37 DEG C of dry 18h of film, are cut into the specification of 4cm × 2cm, and 4 DEG C of hermetically dryings preserve；So far detection layers is prepared；
The preparation method of described phosphate buffer is: weigh 0.29g disodium hydrogen phosphate, 0.0295g di(2-ethylhexyl)phosphate
Hydrogen sodium and 0.2g sodium chloride, be dissolved in the deionized water of 90ml, adjusts pH to 7.3 with 1mol/L NaOH
It is settled to 100ml afterwards by deionized water；
5) detection card is assembled
5.1) base plate is cut into 4cm × 6cm size, standby；
5.2) absorbent filter is cut into 4cm × 2.5cm size, as adsorptive pads, standby；
5.3) by step 5.1) adhered protection film on the base plate for preparing takes off, by step 4) described in
Detection layers i.e. pastes on base plate with nature controlling line and the nitrocellulose filter detecting line, and floating face；
5.4) by step 5.2) ready adsorptive pads is assembled on base plate, makes the left side and the detection layers of adsorptive pads
Right end has the overlap of 0.2cm, and the right hand edge of adsorptive pads then aligns with the right hand edge of base plate and glues and floating；Again
By step 2) described in pad be overlapped at the left hand edge of detection layers by 0.2cm, 0.4cm sticks on base plate；
5.5) by step 3) described in sample pad be then overlapped at the left hand edge of pad by one side 0.2cm,
Another side aligns with the left hand edge of base plate, sticks on base plate and floating；By the detection plate that assembles under cutting cutter
Being cut into detection card wide for 4.0mm, 4 DEG C of hermetically dryings keep in Dark Place.
The invention have the advantage that
The invention provides anti-human Respiratory Syncytial Virus(RSV) N protein antibody, this anti-human Respiratory Syncytial Virus(RSV) N protein
14 amino acid of antibody recognition human respiratory syncytial virus's N protein 21-34 position and 14 amino of 226-239 position
Two linear epitopes that acid is formed, human respiratory syncytial virus's N protein at GenBank sequence number is
AAB59852.1；14 amino acid of human respiratory syncytial virus's N protein 21-34 position and the 14 of 226-239 position
Amino acid whose amino acid sequence is respectively SKYTIQRSTGDSID and FGIAQSSTRGGSRV；By human respiratory syncytial
14 amino acid of viral N proteins 21-34 position and 14 amino acid whose sequences of 226-239 position are respectively designated as N1
And N2；Anti-human Respiratory Syncytial Virus(RSV) N protein antibody is AbN1 and AbN2.Based on human respiratory syncytial virus
Above two rabbit anti-human Respiratory Syncytial Virus(RSV) N protein antibody prepared by single linear epitope has special
The feature that property is good, purity is high, titer is high, preparation cost is cheap, can be used for producing various based on Ag-Ab
The detection kit of the high-sensitivity detection human respiratory syncytial virus of the principle of reaction.Meanwhile, anti-human based on this
Respiratory Syncytial Virus(RSV) N protein antibody has also prepared two kinds of different immune chromatography reagent kits.Two kinds of differences
Immune chromatography reagent kit can quickly, the accurate human respiratory syncytial virus in detection biological sample, it all includes
Two kinds of antibody of the present invention；Two kinds of immune chromatography reagent kits are used equally to what human respiratory syncytial virus infected
Assist diagnosis, there is higher sensitivity with specific, taken into account simple, quick, stable simultaneously and manufactured
The advantages such as low cost, it is adaptable to the inspection of clinical samples, and owing to large batch of quick inspection can be carried out,
It is also suitable for epidemiology survey.Therefore, two kinds of rabbit anti-human Respiratory Syncytial Virus(RSV) N protein of the present invention
Antibody, two kinds of immune chromatography reagent kits are respectively provided with the prospect of being widely applied and practical value.
Detailed description of the invention
It is further appreciated by the present invention, preferred embodiment cited below particularly to contribute to those of ordinary skill in the art
Describe the present invention in detail.
The source of the various materials that the present invention uses or uses and the preparation of related reagent
1, sample pad treatment fluid: weigh 0.242g Tris, 1g bovine serum albumin(BSA) (BSA), 1ml Tween-20,
5g sucrose, 0.3g polyvinylpyrrolidone (PVP-10), it is dissolved in the deionized water of 90ml, uses 1mol/L
NaOH is settled to 100ml by deionized water after adjusting pH to 11.
2, phosphate preserves liquid: weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2g
Sodium chloride, 1g bovine serum albumin(BSA) BSA and 0.1gNaN3, it is dissolved in the deionized water of 90ml, with 1
Mol/L NaOH is settled to 100ml by deionized water after adjusting pH to 7.3；
3, phosphate buffer (PBS): weigh 0.29g disodium hydrogen phosphate, 0.0295g sodium dihydrogen phosphate, 0.2
G sodium chloride, is dissolved in the deionized water of 90ml, uses deionized water after adjusting pH to 7.3 with 1mol/L NaOH
It is settled to 100ml.
4, sample treatment liquid: weigh 0.0121g trishydroxymethylaminomethane, 0.17g sodium chloride, 0.025g
Lysozyme is dissolved in 90ml deionized water, is settled to 100ml by deionized water after adjusting pH to 8.0 with hydrochloric acid.
5, antibody A bN1: make by oneself for the present invention, dilutes with PBS, shakes up, and making AC in solution is 3mg/ml.
6, antibody A bN2: make by oneself for the present invention, dilutes with PBS, shakes up, and making AC in solution is 3mg/ml.
7, goat anti-rabbit igg: for Wuhan Boster Biological Technology Co., Ltd.'s product, dilute with PBS, shake up,
Making Anti-TNF-α bulk concentration in solution is 1mg/ml.
8, quantum dot: in the present invention, quantum dot used is water-soluble CdSe/ZnS that carboxylated amphipathic polymer is modified
Quantum dot, a length of 565nm of its transmitted wave, buys from Wuhan Jia Yuan technology of quantum dots development corporation, Ltd., product
Entitled carboxyl water-soluble quantum dot-565.
9, glass fibre element film: thickness is 0.4mm, water absorption is 42mg/cm2, glass fiber diameter is
0.6-3 μm, has good hydrophily, buys in Shanghai Jinbiao Bio-Tech Co., Ltd. that (model is
BT40)。
10, polyester fiber film: thickness is 0.48mm, absorption speed is 18s/4cm, has fabulous hydrophilic
Property, for the preparation of pad, buy in Shanghai Jinbiao Bio-Tech Co., Ltd. (model is DL42).
11, nitrocellulose filter: model is Millipore Corp SHF135, has liner plate, buys in Millipore
Company.
12, absorbent filter: thickness is 0.95mm, absorption speed is 60s/4cm, and water absorption is 700mg/cm2,
There is good water imbibition, as the material making adsorptive pads.Buy in Shanghai Jinbiao Bio-Tech Co., Ltd.
(model is CH37K).
13, base plate: for high whiteness PVC material, surface coating individual layer high polymer pressure sensitive adhesive SM31-40, buy
In Shanghai Jinbiao Bio-Tech Co., Ltd..
14, human respiratory syncytial virus: Long strain, purchased from American type culture collection (ATCC), compiles
Number it is ATCC VR26.
15, the microbiological specimens used in the present invention is purchased from American type culture collection (ATCC).
Below in conjunction with embodiment, technical scheme provided by the present invention is described in detail:
The preparation of 1 two kinds of rabbit anti-human Respiratory Syncytial Virus(RSV) N protein antibody of embodiment
The preparation method of two kinds of rabbit anti-human Respiratory Syncytial Virus(RSV) N protein antibody is as follows:
1) after structure biology analysis and Related Experimental Study, selected human respiratory syncytial virus's N protein
14 amino acid of (GenBank sequence number AAB59852.1) 21-34 position and 14 amino acid groups of 226-239 position
Become small peptide respectively as preparation rabbit anti-human Respiratory Syncytial Virus(RSV) N protein antibody two linear epitopes,
These two sections of amino acid sequences are respectively SKYTIQRSTGDSID and FGIAQSSTRGGSRV, this two sequence are ordered respectively
Entitled N1 and N2；
2) by step 1) described in the C end of amino acid sequence N1, after the N end of N2 adds a cysteine respectively,
Be respectively synthesized polypeptide with many automatic peptide synthesizers and purify, two polypeptide after purification respectively with carrier protein KLH
Coupling, forms N1-KLH compound protein and N2-KLH compound protein；
3) respectively by step 2) synthesized by the emulsification of two kinds of compound proteins, many in rabbit subcutaneous abdomen respectively after emulsification
Point injection, successively injection three times, every minor tick 7-10 days；
4) after third time is injected 10-12 days, collect respectively, isolated two kinds contains the anti-human respiratory syncystial of rabbit
The serum of viral N proteins antibody, ELISA detects rabbit anti-human Respiratory Syncytial Virus(RSV) N protein antibody in serum respectively
Titer, the titer of two kinds of described rabbit anti-human Respiratory Syncytial Virus(RSV) N protein antibody is all at more than 1:60000；
5) by step 2) synthesize and two polypeptide purifying respectively with the Sepharose 4B coupling of cyanogen bromide-activated,
Form two groups of polypeptide affinity columns；
6) by step 4) obtain the two kinds serum containing rabbit anti-human Respiratory Syncytial Virus(RSV) N protein antibody are respectively
Corresponding joins step 5) in two kinds of affinity columns preparing, and after 4 DEG C of overnight incubation, antibody elution,
Obtain two kinds of rabbit anti-human Respiratory Syncytial Virus(RSV) N protein antibody；Through SDS-PAGE identify its purity all 97% with
On, the antibody that both purifies is respectively designated as AbN1 and AbN2.
Step 2 in the present embodiment)-6) it is all existing mature technology, Duo Jia biotechnology company can provide
The technological service of sequencing.Being embodied as of related experiment link in above-mentioned steps in the present embodiment, is to entrust south
Jing Jinsirui bio tech ltd completes.
" antibody " of the present invention should be construed to contain any spy with required specific binding structural domain
Opposite sex binding factor.Thus, this term covers the antibody fragment of homology, derivative, humanization therewith and resists
The function coordinate of body and antibody and homologue.The example of antibody be immunoglobulin (Ig) hypotype (as IgG, IgE,
IgM, IgD and IgA) and isotype sub-classes；Can also be the fragment comprising antigen-binding domains such as Fab, scFv,
Fv, dAb, Fd and double-chain antibody.
The preparation of embodiment 2 immune chromatography reagent kit based on quantum dot-labeled technology and application
The most quantum dot-labeled antibody A bN1
0.4nmol carboxyl water-soluble quantum dot and 800nmol carbodiimide it is sequentially added in microcentrifugal tube
(EDC), with MES buffer solution (10.66g/L MES, 0.74g/L EDTA pH 7.4) constant volume for 1ml,
Ceaselessly mixed solution, after 37 DEG C of reaction 5min, adds antibody A bN1 prepared by embodiment 1 of 0.34mg,
Lucifuge reaction 2h, adds single-ended amino-polyethyleneglycols (PEG2000-NH2) to final concentration of 1% (m/v), envelope
Close unreacted activated carboxyl site, continue lucifuge reaction 1h.Reacted sample super filter tube is centrifugal (to be retained
Molecular weight 100k), 6500rpm is centrifuged 5min, to volume 200ul, sample after ultrafiltration is transferred to common EP
In pipe, centrifugal except reuniting (10000rpm, 3min).Upper clear supernate is added to splitter (Superdex-200)
Upper purifying, treats that it flows in cylinder naturally, then rinses (liquid flows down naturally) with PBS, irradiates with ultraviolet light
Cylinder observes the position of sample at any time, starts to collect when sample starts and flows out from bottom, stops after collecting 1ml
Collect.By sample after purification with super filter tube (molecular cut off 100k) with 6500rpm centrifugal concentrating to 200ul
After be transferred to common EP pipe in centrifugal (10000rpm, 3min) except reuniting.Liquid is preserved with phosphate after obtaining supernatant
Diluting 200 times, 4 DEG C save backup.So far quantum dot-labeled antibody A bN1 is prepared.
2. the preparation of pad
Polyester fiber film is immersed 1h in the quantum dot-labeled antibody A bN1 solution obtained by step 1, takes out,
25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, and so far prepare pad.
3. the preparation of sample pad
Take glass fibre element film one, it is soaked in sample pad treatment fluid at least 3h, then is placed in biological peace
In full cabinet after 37 DEG C of aeration-dryings, it is cut into after specification is 4cm × 2.5cm/ bar, i.e. prepares sample pad, 25 DEG C
Seal and preserve.
4. the preparation of detection layers
Nitrocellulose filter is cut into 4cm × 4cm size.By antibody A bN2 prepared in embodiment 1 and goat-anti
Rabbit igg phosphate buffer adjusts to final concentration and is respectively 2.0mg/mL and 1.0mg/mL.By diluted
Antibody A bN2 loads BIODOT and draws in film instrument shower nozzle, and the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter,
Form detection line；Anti-rabbit IgG diluted loading BIODOT is drawn in film instrument shower nozzle, arranges 1.0 μ l/cm's
Amount is sprayed on as nature controlling line on nitrocellulose filter, and it is 0.7cm with detection distance between centers of tracks.The nitric acid fibre that will have sprayed
Dimension element 37 DEG C of dry 2h of film, are cut into the specification of 4cm × 4cm, and 4 DEG C of hermetically dryings preserve.So far detection is prepared
Layer.
5. the assembling of detection card
Base plate is cut into 4cm × 7.3cm size, standby.
Absorbent filter is cut into 4cm × 3cm size, as adsorptive pads, standby.
Assembly working operates in Biohazard Safety Equipment, first the adhered protection film on base plate is taken off, by step 4
Described detection layers i.e. pastes on base plate with nature controlling line and the nitrocellulose filter detecting line, and the most floating
Face.Secondly, the adsorptive pads cut out in advance is assembled on base plate so that it is the left side and the right end of detection layers have 0.2
The overlap of cm, its right hand edge then aligns with the right hand edge of base plate and glues and the most floating.Again by described in step 2
Pad is overlapped at the left hand edge of detection layers by 0.3cm, and 0.3cm sticks on base plate 7.Finally by step 3
Described sample pad is then overlapped at the left hand edge of pad by one side 0.3cm, the left side of another side and base plate
Edge aligns, and sticks on base plate and the most floating.The detection plate assembled is cut into 4.0mm under cutting cutter wide
Detection card, 4 DEG C of hermetically dryings keep in Dark Place.
6. the composition of immune chromatography reagent kit based on quantum dot-labeled technology
Immune chromatography reagent kit based on quantum dot-labeled technology is by the detection card described in step 5 and sample treatment liquid
Formed.
7. the using method of immune chromatography reagent kit based on quantum dot-labeled technology
Obtain the throat swab of person to be checked according to a conventional method, be inserted into soft the moulding equipped with 500 μ l sample treatment liquid
In material pipe, squeezable plastic tube wall, after making the sample on swab fully dissolve, takes out 120 μ L and drips in detection card
Sample pad on, after 15 minutes under uv analyzer (model is WD-9403A, Liuyi Instruments Plant, Beijing produce,
Burst of ultraviolel wavelength 365nm) observe testing result.If containing human respiratory syncytial virus's antigen in throat swab,
Then quantum dot-labeled antibody A bN1 in pad is combined, by chromatography effect elder generation and nitrocellulose filter
Antibody A bN2 combine after under ultraviolet excites detection line at can form a macroscopic fluoroscopic examination
Line, the quantum dot-labeled antibody being not associated with continues to excite lower shape at ultraviolet after chromatography is combined with goat anti-rabbit igg
Become macroscopic Article 2 fluorescence nature controlling line；If without related antigen in throat swab to be checked, the most only appearance one is glimmering
Light nature controlling line.If fluorescence nature controlling line does not occurs, then this detection card lost efficacy.
8. the application effect citing of immune chromatography reagent kit based on quantum dot-labeled technology
The using method reference of the immune chromatography reagent kit based on quantum dot-labeled technology of indication in the present embodiment
Operating procedure described in step 7.
1) specific test
Prop up former by respiratory tract common causative such as human III type parainfluenza virus virus (ATCC VR-93), people's pneumonia
Body (ATCC numbering 15531), people's CPN (AR-39 strain, ATCC numbering 53592), adenovirus hominis 3
Type (GB strain, ATCC numbering VR-3), adenovirus hominis 7 type (Gomen strain, ATCC numbering VR-7), people's A type stream
Influenza Virus (H1N1, ATCC numbering VR-1743), people's influenza B virus (ATCC numbering VR-790), influenza
Haemophilus (ATCC numbering 53781), streptococcus pneumonia (ATCC numbering 700670) etc. replace human respiratory to close
Cellular virus detects, and the kit detection PBST dilution containing these microorganisms is all negative.
2) clinical trial example
Using human respiratory syncytial virus's detection " goldstandard "-cultivation as reference, take 94 example division of respiratory diseases and breathe
The kit described in oropharyngeal swab specimen step 6 of road the infected detects, and cultivation positive rate is 15.96%
(15/94), this kit is 14.89% (14/94), and the coincidence rate of 2 kinds of methods is 94.68% (89/94).
Concrete outcome is as shown in table 1.
The testing result of table 1 clinical samples
The preparation of embodiment 3 immune chromatography reagent kit based on colloidal gold-labeled method and application
1. colloidal gold labeled monoclonal antibody AbN1
A.30nm the preparation of collaurum
Take the 250ml triangular flask that a silication is good, add 99ml ultra-pure water, 1% (m/v) HAuCl4 solution is added
Wherein mixing, oil bath is heated and stirs to boiling.Rapidly join 2ml 1% (m/v) trisodium citrate wherein
The aqueous solution, solution continues boiling 10min (during this, solution is changed into redness by blueness).Stop heating,
Allow solution naturally cool to room temperature, be then added thereto to ultra-pure water polishing to 100ml.
B. colloidal gold labeled monoclonal antibody AbN1
1) take the 50ml triangular flask that a silication is good, add the colloidal gold liquid prepared by 10ml step a, Xiang Jin
Liquid adds 240ul 0.2mol/L K2CO3Regulation pH to 8.5；
2) under magnetic stirrer, antibody A bN1 is added in colloidal gold solution, final concentration of to antibody
10ug/ml, should be added dropwise over when adding antibody, continues stirring 45min～60min after adding；
3) addition 2.5ml 5% (m/v) bovine serum albumin(BSA) (BSA) extremely final concentration of 1% (m/v) has been reacted,
Stirring 15～30 minutes, 4 DEG C save backup.
4) loading 50ml centrifuge tube, 2500g after antibody A bN1 marked being taken out, 4 DEG C are centrifuged 5 minutes,
To lower sediment and supernatant liquor, discarding lower sediment, supernatant liquor is transferred to another 50ml centrifuge tube,
12000g, 4 DEG C are centrifuged 30 minutes, obtain lower sediment and supernatant liquor, discard supernatant liquor, lower floor sunk
The shallow lake 10ml resuspended precipitation of collaurum buffer solution, 12000g the most again, 4 DEG C are centrifuged 30 minutes, under again obtaining
Layer precipitation and supernatant liquor, lower sediment is finally resuspended with 3ml collaurum buffer solution, and 4 DEG C save backup；
In above-mentioned collaurum buffer solution, each constituent content is respectively: 10mM Tris, 1% (m/v) BSA, 1% (v/v)
Tween-20,5% (m/v) sucrose and 3 ‰ (m/v) polyvinylpyrrolidone, described collaurum buffers
The pH of liquid is 10.5；
2. the preparation of pad
Polyester fiber film is immersed 1h in the colloidal gold labeled monoclonal antibody AbN1 solution obtained by step 1, takes out,
25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, and so far prepare pad.
3. the preparation of sample pad
Take glass fibre element film one, it is soaked in sample pad treatment fluid at least 2h, then is placed in biological peace
In full cabinet after 37 DEG C of aeration-dryings, it is cut into after specification is 4cm × 1.5cm/ bar, i.e. prepares sample pad, 25 DEG C
Seal and preserve.
4. the preparation of detection layers
Nitrocellulose filter is cut into 4cm × 2cm size.By antibody A bN2 prepared in embodiment 1 and goat-anti
Rabbit igg phosphate buffer adjusts to final concentration and is respectively 2.0mg/mL and 1.0mg/mL.By diluted
Antibody A bN2 loads BIODOT and draws in film instrument shower nozzle, and the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter,
Form detection line；Anti-rabbit IgG diluted loading BIODOT is drawn in film instrument shower nozzle, arranges 1.0 μ l/cm's
Amount is sprayed on as nature controlling line on nitrocellulose filter, and it is 0.5cm with detection distance between centers of tracks.The nitric acid fibre that will have sprayed
Dimension element 37 DEG C of dry 18h of film, are cut into the specification of 4cm × 2cm, and 4 DEG C of hermetically dryings preserve.So far detection is prepared
Layer.
5. the assembling of detection card
Base plate is cut into 4cm × 6cm size, standby.
Absorbent filter is cut into 4cm × 2.5cm size, as adsorptive pads, standby.
Assembly working operates in Biohazard Safety Equipment, first the adhered protection film on base plate is taken off, by step 4
Described detection layers i.e. pastes on base plate with nature controlling line and the nitrocellulose filter detecting line, and the most floating
Face.Secondly, the adsorptive pads cut out in advance is assembled on base plate so that it is the left side and the right end of detection layers have 0.2
The overlap of cm, its right hand edge then aligns with the right hand edge of base plate and glues and the most floating.Again by described in step 2
Pad is overlapped at the left hand edge of detection layers 3 by 0.2cm, 0.4) cm sticks on base plate.Finally by step 3
Described sample pad is then overlapped at the left hand edge of pad by one side 0.2cm, the left side of another side and base plate
Edge aligns, and sticks on base plate and the most floating.The detection plate assembled is cut into 4.0mm under cutting cutter wide
Detection card, 4 DEG C of hermetically dryings keep in Dark Place.
6. the composition of immune chromatography reagent kit based on colloidal gold-labeled method
Immune chromatography reagent kit based on colloidal gold-labeled method is by the detection card described in step 5 and sample treatment liquid
Formed.
7. the using method of immune chromatography reagent kit based on colloidal gold-labeled method
Obtain the throat swab of person to be checked according to a conventional method, be inserted into soft the moulding equipped with 500 μ l sample treatment liquid
In material pipe, squeezable plastic tube wall, make the sample on swab fully dissolve and after 50 DEG C of water-bath 20min, take out 120
μ L drips in the sample pad of detection card, visually observes testing result after 15 minutes.If throat swab is exhaled containing people
Inhale road syncytial viral antigens, then antibody A bN1 of the colloid gold label in pad is combined, and is acted on by chromatography
After first antibody A bN2 with nitrocellulose filter on is combined detect can be formed at line macroscopic one red
Look detection line, the colloidal gold labeled monoclonal antibody being not associated with continues to form naked eyes after chromatography is combined with goat anti-rabbit igg can
The Article 2 redness nature controlling line seen；If without related antigen in throat swab to be checked, a red nature controlling line the most only occurs.
If red nature controlling line does not occurs, then this detection card lost efficacy.
8. the application effect citing of immune chromatography reagent kit based on colloidal gold-labeled method
The using method reference of the immune chromatography reagent kit based on colloidal gold-labeled method of indication in the present embodiment
Operating procedure described in step 7.
1) specific test
With respiratory tract common causative such as I type human parainfluenza viruses (ATCC VR-94), II type human parainfluenza viruses
(ATCC VR-92), type III human parainfluenza viruses virus (ATCC VR-93), people mycoplasma pneumoniae (ATCC
Numbering 15531), people's CPN (AR-39 strain, ATCC numbering 53592), adenovirus hominis 3 type (GB strain,
ATCC numbering VR-3), adenovirus hominis 7 type (Gomen strain, ATCC numbering VR-7), influenza virus A hominis (H1N1,
ATCC numbering VR-1743), people's influenza B virus (ATCC numbering VR-790), haemophilus influenzae (ATCC
Numbering 53781), streptococcus pneumonia (ATCC numbering 700670) etc. replace human respiratory syncytial virus to detect,
The kit detection PBST dilution containing these microorganisms is all negative.
2) clinical trial example
Using Respiratory Syncytial Virus(RSV) detection " goldstandard "-cultivation as reference, take 94 example division of respiratory disease respiratory tracts
The kit described in oropharyngeal swab specimen step 6 of the infected detects, and cultivation positive rate is 15.96%
(15/94), this kit is 13.83% (13/94), and the coincidence rate of 2 kinds of methods is 95.74% (90/94).
Concrete outcome is as shown in table 1.
The testing result of table 1 clinical samples
It is pointed out that and the foregoing is only presently preferred embodiments of the present invention, not in order to limit this
Invention, all any amendment, equivalents etc. made within present invention spirit and principle should be included in this
Within bright protection domain.
Claims (4)
- The most anti-human Respiratory Syncytial Virus(RSV) N protein antibody, it is characterized in that: described anti-human Respiratory Syncytial Virus(RSV) N protein antibody is the antibody of two linear epitopes identifying that human respiratory syncytial virus's N protein 21-34 position and 226-239 amino acids formed, and described human respiratory syncytial virus's N protein is AAB59852.1 at GenBank sequence number；14 amino acid of described human respiratory syncytial virus's N protein 21-34 position and 14 amino acid whose amino acid sequences of 226-239 position are respectively SKYTIQRSTGDSID and FGIAQSSTRGGSRV；14 amino acid of human respiratory syncytial virus's N protein 21-34 position and 14 amino acid whose sequences of 226-239 position are respectively designated as N1 and N2；Described anti-human Respiratory Syncytial Virus(RSV) N protein antibody is AbN1 and AbN2.
- 2. the immune chromatography reagent kit formed based on anti-human Respiratory Syncytial Virus(RSV) N protein antibody as claimed in claim 1, it is characterised in that: described kit is immune chromatography reagent kit based on quantum dot-labeled technology or immune chromatography reagent kit based on colloidal gold-labeled method.
- The immune chromatography reagent kit that anti-human Respiratory Syncytial Virus(RSV) N protein antibody the most according to claim 2 is formed, it is characterised in that: when described kit is immune chromatography reagent kit based on quantum dot-labeled technology, the preparation method of described kit is:1) quantum dot-labeled antibody A bN1 solution:0.4 it is sequentially added in microcentrifugal tube Nmol carboxyl water-soluble quantum dot and 800 nmol carbodiimide EDC, it is 1 ml with MES buffer solution constant volume, mixed solution, after 37 DEG C of reaction 5 min, adding antibody A bN1 prepared of 0.34 mg, lucifuge reacts 2 h, adds single-ended amino-polyethyleneglycols PEG2000-NH2 to final concentration of 1%m/v, close unreacted activated carboxyl site, continue lucifuge and react 1 h；Reacted sample super filter tube is centrifuged, and 6500rpm is centrifuged 5min, to volume 200ul, is transferred to by sample after ultrafiltration in common EP pipe, centrifugal except reuniting, and obtains upper clear supernate and bottom precipitation, centrifugal 3min under the conditions of 10000rpm；It is added to upper clear supernate on splitter Superdex-200 purify, treats that upper clear supernate flows in cylinder naturally, then rinse with PBS, irradiate cylinder with ultraviolet light and observe the position of sample, start to collect when sample starts and flows out from bottom, stop collecting after collecting 1 ml；By sample super filter tube after purification with centrifugal except reuniting in being transferred to common EP pipe after 6500rpm centrifugal concentrating to 200ul, the condition being centrifuged common EP pipe is 10000rpm, 3min；Preserving liquid with phosphate after obtaining supernatant and dilute 200 times, 4 DEG C save backup；So far quantum dot-labeled antibody A bN1 solution is prepared；In described MES buffer solution, each constituent content is respectively: 10.66 g/L MES and 0.74 g/L EDTA, the pH 7.4 of described MES buffer solution；It is to weigh 0.29 g disodium hydrogen phosphate, 0.0295 g sodium dihydrogen phosphate, 0.2 g sodium chloride, 1 g bovine serum albumin(BSA) BSA and 0.1 g NaN that described phosphate preserves the preparation method of liquid3, it is dissolved in the deionized water of 90 ml, after adjusting pH to 7.3 with 1 mol/L NaOH, is settled to 100 by deionized water ml；2) pad is preparedPolyester fiber film being immersed 1 h in the quantum dot-labeled antibody A bN1 solution obtained by step 1), takes out, 25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, and so far prepare pad；3) sample pad is preparedTaking glass fibre element film one, glass fibre element film soaks in sample pad treatment fluid at least 3 h, then be placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, be cut into after specification is 4cm × 2.5cm/ bar, i.e. prepare sample pad, 25 DEG C seal and preserve；The preparation method of described sample pad treatment fluid is to weigh 0.29 g disodium hydrogen phosphate, 0.0295 g sodium dihydrogen phosphate, 0.2 g sodium chloride, 2 g bovine serum albumin(BSA) BSA, 1 ml Tween-20,2 g sucrose and 0.5 g polyvinylpyrrolidone PVP-10, it is dissolved in the deionized water of 90 ml, after adjusting pH to 7.3 with 1 mol/L NaOH, is settled to 100 by deionized water ml；4) detection layers is preparedNitrocellulose filter is cut into 4cm × 4cm size；Antibody A bN2 prepared and goat anti-rabbit igg phosphate buffer are adjusted to final concentration and be respectively 2.0 mg/mL and 1.0 mg/mL；Being drawn in film instrument shower nozzle by antibody A bN2 diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter, forms detection line；Being drawn in film instrument shower nozzle by anti-rabbit IgG diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter as nature controlling line, and nature controlling line is 0.7 cm with detection distance between centers of tracks；The nitrocellulose filter 37 DEG C sprayed being dried 2 h, is cut into the specification of 4cm × 4cm, 4 DEG C of hermetically dryings preserve；So far detection layers is prepared；The preparation method of described phosphate buffer is: weighs 0.29 g disodium hydrogen phosphate, 0.0295 g sodium dihydrogen phosphate and 0.2 g sodium chloride, is dissolved in the deionized water of 90 ml, is settled to 100 by deionized water after adjusting pH to 7.3 with 1 mol/L NaOH ml；5) detection card is assembled5.1) base plate is cut into 4cm × 7.3cm size, standby；5.2) absorbent filter is cut into 4cm × 3cm size, as adsorptive pads, standby；5.3) by step 5.1) adhered protection film on the base plate for preparing takes off, and by the detection layers described in step 4), i.e. nitrocellulose filter with nature controlling line and detection line pastes on base plate, and floating face；5.4) by step 5.2) ready adsorptive pads is assembled on base plate, and making the left side end right with detection layers of adsorptive pads have, 0.2 cm's is overlapping, and the right hand edge of adsorptive pads then aligns with the right hand edge of base plate and glues and floating；Again by step 2) described in pad be overlapped at the left hand edge of detection layers by 0.3 cm, 0.3 cm sticks on base plate；5.5) by described in step 3) sample pad then by one side 0.3 Cm is overlapped at the left hand edge of pad, and another side aligns with the left hand edge of base plate, sticks on base plate and floating；The detection plate assembled is cut under cutting cutter detection card wide for 4.0 mm, and 4 DEG C of hermetically dryings keep in Dark Place.
- The immune chromatography reagent kit that anti-human Respiratory Syncytial Virus(RSV) N protein antibody the most according to claim 2 is formed, it is characterised in that: when described kit is immune chromatography reagent kit based on colloidal gold-labeled method, the preparation method of described kit is:1) colloidal gold labeled monoclonal antibody AbN11.1) 30 nm colloidal gold solutions are prepared:Take a good 250ml triangular flask of silication, add 99ml ultra-pure water, by 1 ml 1%m/v HAuCl4Solution adds in 250ml triangular flask and mixes with ultra-pure water, and oil bath is heated and stirs to boiling；2ml 1% m/v trisodium citrate aqueous solution is rapidly joined in 250ml triangular flask, solution continues boiling 10min, solution in 250ml triangular flask stops heating when being changed into redness by blueness, solution in 250ml triangular flask is naturally cooled to 25 DEG C, in 250ml triangular flask, then adds ultra-pure water polishing to 100ml；1.2) colloidal gold labeled monoclonal antibody AbN1:1.2.1) take a good 50ml triangular flask of silication, add 10 ml steps 1.1) prepared by colloidal gold solution, in collaurum liquid add 240ul 0.2 mol/L K2CO3Regulation pH to 8.5；1.2.2) under magnetic stirrer, antibody A bN1 is added in colloidal gold solution, to final concentration of 10 ug/ml of antibody, be added dropwise over when adding antibody, after adding, continue stirring 45 min～60 min；1.2.3) having reacted addition 2.5ml 5%m/v bovine serum albumin(BSA) BSA to final concentration of 1% m/v, stirred 15～30 minutes, 4 DEG C save backup；1.2.4) 50ml centrifuge tube is loaded after antibody A bN1 marked being taken out, 2500g, 4 DEG C are centrifuged 5 minutes, obtain lower sediment and supernatant liquor, discard lower sediment, supernatant liquor is transferred to another 50ml centrifuge tube, 12000g, and 4 DEG C are centrifuged 30 minutes, obtain lower sediment and supernatant liquor, discard supernatant liquor, by the lower sediment 10 ml resuspended precipitations of collaurum buffer solution, 12000g the most again, 4 DEG C are centrifuged 30 minutes, again obtaining lower sediment and supernatant liquor, lower sediment is finally resuspended with 3ml collaurum buffer solution, and 4 DEG C save backup；In described collaurum buffer solution, each constituent content is respectively: 10mM Tris, 1%m/vBSA, 1% v/v Tween-20,5% m/v sucrose and 3 ‰ m/v polyvinylpyrrolidones, and the pH of described collaurum buffer solution is 10.5；2) pad is preparedPolyester fiber film being immersed 1 h in the colloidal gold labeled monoclonal antibody AbN1 solution obtained by step 1), takes out, 25 DEG C are cut into after rear specification is 4cm × 0.6cm/ bar after drying, and 4 DEG C of sealings save backup, and so far prepare pad；3) sample pad is preparedTaking glass fibre element film one, glass fibre element film soaks in sample pad treatment fluid at least 2h, then be placed in Biohazard Safety Equipment after 37 DEG C of aeration-dryings, be cut into after specification is 4cm × 1.5cm/ bar, i.e. prepare sample pad, 25 DEG C seal and preserve；The preparation method of described sample pad treatment fluid is to weigh 0.242g Tris, 1 g bovine serum albumin(BSA) BSA, 1 ml Tween-20,5 g sucrose and 0.3g polyvinylpyrrolidone PVP-10, it is dissolved in the deionized water of 90 ml, after adjusting pH to 11 with 1 mol/L NaOH, is settled to 100 by deionized water ml；4) detection layers is preparedNitrocellulose filter is cut into 4cm × 2cm size；Antibody A bN2 prepared and goat anti-rabbit igg phosphate buffer are adjusted to final concentration and be respectively 2.0 mg/mL and 1.0 mg/mL；Being drawn in film instrument shower nozzle by antibody A bN2 diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter, forms detection line；Being drawn in film instrument shower nozzle by anti-rabbit IgG diluted loading BIODOT, the amount arranging 1.0 μ l/cm is sprayed on nitrocellulose filter as nature controlling line, and nature controlling line is 0.5cm with detection distance between centers of tracks；The nitrocellulose filter 37 DEG C sprayed being dried 18 h, is cut into the specification of 4cm × 2cm, 4 DEG C of hermetically dryings preserve；So far detection layers is prepared；The preparation method of described phosphate buffer is: weighs 0.29 g disodium hydrogen phosphate, 0.0295 g sodium dihydrogen phosphate and 0.2 g sodium chloride, is dissolved in the deionized water of 90 ml, is settled to 100 by deionized water after adjusting pH to 7.3 with 1 mol/L NaOH ml；5) detection card is assembled5.1) base plate is cut into 4cm × 6cm size, standby；5.2) absorbent filter is cut into 4cm × 2.5cm size, as adsorptive pads, standby；5.3) by step 5.1) adhered protection film on the base plate for preparing takes off, and by the detection layers described in step 4), i.e. nitrocellulose filter with nature controlling line and detection line pastes on base plate, and floating face；5.4) by step 5.2) ready adsorptive pads is assembled on base plate, and making the left side end right with detection layers of adsorptive pads have, 0.2 cm's is overlapping, and the right hand edge of adsorptive pads then aligns with the right hand edge of base plate and glues and floating；Again by step 2) described in pad be overlapped at the left hand edge of detection layers by 0.2 cm, 0.4 cm sticks on base plate；5.5) by described in step 3) sample pad then by one side 0.2 Cm is overlapped at the left hand edge of pad, and another side aligns with the left hand edge of base plate, sticks on base plate and floating；The detection plate assembled is cut under cutting cutter detection card wide for 4.0 mm, and 4 DEG C of hermetically dryings keep in Dark Place.
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