CN106226518A - Canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof - Google Patents
Canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof 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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Abstract
The invention provides canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof, relate to colloidal gold colloidal gold detection test paper strip technical field, the detecting step solving the existence of existing CDV detection technique is loaded down with trivial details, time-consuming, needs professional to carry out operating and detect the problem relying primarily on laboratory.This test strips includes PVC base plate, be pasted onto successively the sample pad on PVC base plate, gold-marking binding pad, with detection line and the nitrocellulose filter of nature controlling line, absorption pad: gold-marking binding pad is coated with canine distemper virus monoclonal antibody N18 of purification and the coupling label of gold colloidal, detection line on nitrocellulose filter is coated with canine distemper virus monoclonal antibody C42 of purification, and the nature controlling line on nitrocellulose filter is coated with sheep anti-mouse igg.The test strips of the present invention is simple to operate quickly, testing result understand be prone to judgements, high specificity, sensitivity height, without advantages such as instrument and equipments.
Description
Technical field
The present invention relates to colloidal gold colloidal gold detection test paper strip technical field, be specifically related to a kind of canine distemper virus colloid gold immune layer
Analysis test strip and preparation method thereof.
Background technology
Canine distemper (Canine Distemper, CD) is by canine distemper virus (Canine Distemper Virus, CDV)
A kind of high degree in contact of causing, lethal infectious diseases.Infected dogs distemper virus fur-bearing animal clinical symptoms is depressed, the body temperature of spirit
Rising, anorexia, having loose bowels, there is nervous symptoms in the later stage, shows as ataxia, quadriplegia etc..Finally, spit out white foams, scream
And it is dead.It is referred to as one of big epidemic disease of fur-bearing animal three.
Existing canine distemper disease virus detection method mainly includes serum neutralization test, immunofluorescence test, ELISA, RT-
PCR, virus purification and inclusion body inspection.But these methods are required for professional to be operated under specific apparatus, and
The most several hours to several days.Hence set up and a kind of operate simple, quick, reliable and cheap canine distemper disease clinically
Virus detection method is extremely important.
Summary of the invention
Loaded down with trivial details, time-consuming in order to solve the detecting step of existing CDV detection technique existence, need professional to operate
And relying primarily on the problem of laboratory, the present invention provides a kind of canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and system thereof
Preparation Method.
The present invention solves that the technical scheme that technical problem is used is as follows:
The canine distemper virus colloidal gold immunochromatographydetection detection test paper bar of the present invention, including PVC base plate, is pasted onto PVC successively
Sample pad on base plate, gold-marking binding pad, with detection line and the nitrocellulose filter of nature controlling line, absorption pad: gold-marking binding pad
It is coated with canine distemper virus monoclonal antibody N18 of purification and the coupling label of gold colloidal, the detection on nitrocellulose filter
Line is coated with canine distemper virus monoclonal antibody C42 of purification, and the nature controlling line on nitrocellulose filter is coated with sheep anti-mouse igg.
Present invention also offers the preparation method of above-mentioned canine distemper virus colloidal gold immunochromatographydetection detection test paper bar, including with
Lower step:
Step one: canine distemper virus monoclonal antibody N18, the preparation of C42: select the female Balb/c mice of 6～8 week old,
Only, after 7 days, difference lumbar injection two strain canine distemper virus monoclonal antibody hybridoma cell, connects lumbar injection norphytane 0.5mL/
The amount of kind is 4 × 106～6 × 106Cell/mL/ is only；Extracting ascites after mouse web portion expands respectively, 5000rpm is centrifuged
15min, takes supernatant；By ascites supernatant respectively with the ammonium sulfate precipitation 2 times of 50% saturation, after dialysis desalination, it is thus achieved that purification
Canine distemper virus monoclonal antibody N18 and canine distemper virus monoclonal antibody C42；
Step 2: the preparation of the coupling label of canine distemper virus monoclonal antibody N18 and gold colloidal: stepwise dilution method is true
The usage ratio determining canine distemper virus monoclonal antibody N18 and colloidal gold solution is 8.8 μ g～9.6 μ g:1mL, according to this consumption
Ratio is added dropwise over canine distemper virus monoclonal antibody N18, adds the bovine serum albumin of final concentration of 5%, obtains after stirring
Canine distemper virus monoclonal antibody N18 and the coupling label of gold colloidal, use low temperature supercentrifugation to this coupling label
It is purified；
Step 3: the assembling of test strips: use gold spraying instrument by the coupling of canine distemper virus monoclonal antibody N18 Yu gold colloidal
Label is sprayed on glass fibre element film, natural drying under room temperature condition, seals, it is thus achieved that gold-marking binding pad；Use and draw film instrument by dog
Distemper virus monoclonal antibody C42, sheep anti-mouse igg are drawn on film detection line on nitrocellulose filter and nature controlling line respectively；Will
The sample pad handled well, gold-marking binding pad, nitrocellulose filter are pasted onto on PVC base plate successively, cutting, assemble, seal, it is thus achieved that
Canine distemper virus monoclonal antibody colloidal gold colloidal gold detection test paper strip, room temperature preservation.
Further, in step one, two strain canine distemper virus monoclonal antibody hybridoma cells use following methods to prepare:
The canine distemper virus inoculation Balb/c mice that will purify, after booster immunization, collects its splenocyte, with preprepared myeloma
Cell merges, and cultivates with 1%HAT culture fluid；Merge about 2 weeks, use indirect ELISA method to filter out two strains positive
Hybridoma cell strain, and the positive hybridoma cell after screening is cloned, obtain two strain canine distemper virus monoclonal antibodies
Hybridoma cell strain.
Further, colloidal gold solution described in step 2 uses trisodium citrate reduction method to prepare: take 1% chlorauric acid solution
The ultra-pure water of 1ml addition 99ml is configured to the chlorauric acid solution of final concentration of 0.01%, after being heated to boiling, adds 1% Fructus Citri Limoniae
Acid trisodium 1ml also continues heating, and solution is transferred to the black-and-blue claret that eventually becomes by faint yellow, continues heating 5 after colour stable
Minute, room temperature cools down, it is thus achieved that colloidal gold solution, 4 DEG C save backup, a diameter of 30nm of colloid gold particle.
Further, in step 2, described stepwise dilution method determines that canine distemper virus monoclonal antibody N18 is molten with gold colloidal
The detailed process of the amount ratio row of liquid is as follows: the pH value regulating colloidal gold solution with the solution of potassium carbonate of 0.1mol/L is 8.4, to
11 centrifuge tubes are separately added into 1ml colloidal gold solution；I.e. content after canine distemper virus monoclonal antibody N18 stepwise dilution is divided
It is not 2 μ g, 4 μ g, 6 μ g, 8 μ g, 10 μ g, 12 μ g, 14 μ g, 16 μ g, 18 μ g, 20 μ g, joins No. 2 pipes in the order described above to 11
In number pipe, and mix with the colloidal gold solution in centrifuge tube, in No. 2 pipes to No. 11 pipes, after 5 minutes, be separately added into the chlorine of 10%
Changing sodium solution 1ml mixing, room temperature stands more than 2 hours observed results, and No. 1 pipe is blank；
To in No. 4 pipes, there is coagulation phenomenon from red to blue in No. 2 pipes, in No. 5 pipes to No. 11 pipes, keep the red of gold colloidal
Color is constant；Therefore, centrifuge tube that is No. 5 pipe that gold colloidal is red constant and canine distemper virus monoclonal antibody N18 addition is minimum
In canine distemper virus monoclonal antibody N18 content, be the minimum steady needed for stable 1ml colloidal gold solution quantitative, at this
Add 20%～30% on the basis of low stable quantity and be the canine distemper disease poison cell Dan Ke needed for stable 1ml colloidal gold solution
The actually used amount of grand antibody N18, i.e. canine distemper virus cell monoclonal antibodies N18 with the usage ratio of colloidal gold solution are
8.8 μ g～9.6 μ g:1mL.
Further, in step 2, use the detailed process that this coupling label is purified by low temperature supercentrifugation
As follows: first gold mark canine distemper virus monoclonal antibody N18 that volume is V to be centrifuged 40 points with 3000rpm/min at 4 DEG C
Clock, Aspirate supernatant, abandon precipitation: be centrifuged 40 minutes with 10000rpm/min at 4 DEG C by supernatant again, supernatant discarded, use
The PBS buffer solution solution of 0.01mol/L, pH7.4 is precipitated to the original volume i.e. volume of gold mark canine distemper virus monoclonal antibody N18
The 1/10 of V, it is thus achieved that gold mark canine distemper virus monoclonal antibody N18 of purification, 4 DEG C save backup.
Further, containing 1%BSA and 0.02% sodium azide in described PBS.
Further, the labelled amount of the coupling label of canine distemper virus monoclonal antibody N18 and gold colloidal be 8.8 μ g～
9.6μg:1mL。
Further, in step 3, the labelled amount of canine distemper virus monoclonal antibody C42 is 2 μ g/cm.
Further, in step 3, the labelled amount of sheep anti-mouse igg is 4 μ g/cm.
The invention has the beneficial effects as follows: the canine distemper virus colloidal gold immunochromatographydetection detection test paper bar of the present invention has special
Property strong, sensitivity is high, the feature such as simple and quick, simple and quick, it is easy to promote, it is adaptable to plant of basic unit is detected, have
Wide market prospect.
Enzyme linked immunological principle is combined by the present invention with colloidal gold chromatographic technology, preparation detection canine distemper virus colloid gold immune
Chromatography detecting test paper strip also intends being applied to clinic, improves the prevention ability of Aleutian disease, has quick, detection simple to operate
Result understand be prone to judgement, high specificity, sensitivity high, without advantages such as instrument and equipments, therefore, be highly suitable for scene, door
The place laboratory condition such as examining limited carries out clinical sample detection.The invention provides the preparation method of above-mentioned test strips, suitable
For commercial production.
Accompanying drawing explanation
Fig. 1 is the structural representation of the canine distemper virus colloidal gold immunochromatographydetection detection test paper bar of the present invention.
Fig. 2 is that the canine distemper virus colloidal gold immunochromatographydetection detection test paper bar result of the present invention judges schematic diagram.
In figure: 1, PVC base plate, 2, sample pad, 3, gold-marking binding pad, 4, nitrocellulose filter, 5, detection line, 6, Quality Control
Line, 7, absorption pad.
Detailed description of the invention
The canine distemper virus colloidal gold immunochromatographydetection detection test paper bar of the present invention, including PVC base plate 1, sample pad 2, Jin Biao
Pad 3, with detection the line 5 and nitrocellulose filter 4 of nature controlling line 6, absorption pad 7, sample pad 2, gold-marking binding pad 3, with
Detection line 5 and the nitrocellulose filter 4 of nature controlling line 6, absorption pad 7 are pasted onto on PVC base plate 1 successively, and gold-marking binding pad 3 is coated with
Canine distemper virus monoclonal antibody N18 of purification and the coupling label of gold colloidal, the detection line 5 on nitrocellulose filter 4 wraps
Being had canine distemper virus monoclonal antibody C42 of purification, the nature controlling line 6 on nitrocellulose filter 4 is coated with sheep anti-mouse igg.
The using method of the canine distemper virus colloidal gold immunochromatographydetection detection test paper bar of the present invention is as follows:
With the urine sample of plastic tips absorption animal to be detected in well, result within 5～10 minutes, can be shown, right
Whether i.e. can determine whether in tested mink body with canine distemper virus or by canine distemper disease than detection line 5 and the color of control line 6
Poison infects.
Result judges as shown in Figure 2:
1, positive: at detection line 5 and nature controlling line 6, a red stripes respectively to occur, it is determined that for the positive；Detection line 5 band
The depth of color and luster changes according to the height detecting canine distemper virus antigenic content in sample, and the highest colour band of antigenic content is the deepest, instead
The most shallow.
2, negative: to occur that red stripes do not occur in a red stripes, detection line 5 at nature controlling line 6, illustrate to detect in sample
Antigen without canine distemper virus exists.
3, invalid: only to have band at detection line 5 or all occur without obvious band at detection line 5 and nature controlling line 6, being considered as examination
Paper slip detection is invalid.
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail.
Embodiment 1 canine distemper virus monoclonal antibody N18, the preparation of C42
(1) the canine distemper virus inoculation Balb/c mice that will purify, after booster immunization, collects its splenocyte, with the most accurate
The myeloma cell got ready is merged, and cultivates with 1%HAT culture fluid；Merge about 2 weeks, use indirect ELISA method screening
Go out two strain positive hybridoma cell strains, and the positive hybridoma cell after screening is cloned, obtain two strain canine distemper virus
Monoclonal antibody hybridoma cell strain；
(2) selecting the female Balb/c mice of 6～8 weeks tinkling of pieces of jade, lumbar injection norphytane 0.5mL/ only, after 7 days, note respectively by abdominal cavity
Penetrating two strain canine distemper virus monoclonal antibody hybridoma cells, inoculum concentration is 4 × 106～6 × 106Cell/mL/ is only；Treat mice
After abdominal part expands, (about one week) extracts ascites respectively, and 5000rpm is centrifuged 15min, takes supernatant；Ascites supernatant is satisfied with 50% respectively
With the ammonium sulfate precipitation 2 times of degree, after dialysis desalination, it is thus achieved that canine distemper virus monoclonal antibody N18 of purification and canine distemper virus
Monoclonal antibody C42.
The preparation of embodiment 2 colloidal gold solution
Trisodium citrate reduction method is used to prepare colloidal gold solution: to take the ultra-pure water that 1% chlorauric acid solution 1ml adds 99ml
It is configured to the chlorauric acid solution of final concentration of 0.01%, after being heated to boiling, adds 1% trisodium citrate 1ml and continue heating,
Solution is transferred to the black-and-blue claret that eventually becomes by faint yellow, continues heating 5 minutes after colour stable, and room temperature cools down rear 4 DEG C of guarantors
Deposit standby, it is thus achieved that colloidal gold solution.Draw a small amount of colloid gold particle transmission competing observation of electricity, colloid gold particle size basic
Causing, be evenly distributed, colloid gold particle diameter about 40nm is qualified.
Embodiment 3 canine distemper virus monoclonal antibody N18 and the coupling label of gold colloidal (golden mark canine distemper virus Dan Ke
Grand antibody N18) preparation
(1) stepwise dilution method determines the usage ratio of canine distemper virus monoclonal antibody N18 and colloidal gold solution: use
The pH value of the solution of potassium carbonate regulation colloidal gold solution of 0.1mol/L is 8.4, takes 11 clean centrifuge tubes, numbered No. 1 pipe
To No. 11 pipes, often pipe adds 1ml colloidal gold solution；I.e. contain after the canine distemper virus monoclonal antibody N18 stepwise dilution of purification
Amount is respectively 2 μ g, 4 μ g, 6 μ g, 8 μ g, 10 μ g, 12 μ g, 14 μ g, 16 μ g, 18 μ g, 20 μ g, joins No. 2 pipes in the order described above
To No. 11 pipes, and mix with the colloidal gold solution in centrifuge tube, after 5 minutes, to No. 11 pipes, be separately added into 10% at No. 2 pipes
Sodium chloride solution 1ml mixing, room temperature stand more than 2 hours observed results；No. 1 Guan Zhongwei adds canine distemper virus monoclonal anti
Body N18 and sodium chloride solution, be set to control tube；
Canine distemper virus monoclonal antibody N18 addition is not enough to the centrifuge tube (No. 2 pipes are to No. 4 pipes) of stable colloid gold,
Coagulation phenomenon from red to blue i.e. occurs, and canine distemper virus monoclonal antibody N18 addition to meet or exceed minimum steady quantitative
Centrifuge tube (No. 5 pipes are to No. 11 pipes), then the redness keeping gold colloidal is constant.Therefore, the red constant and canine distemper disease of gold colloidal
Canine distemper virus monoclonal antibody N18 content (8 μ g) in the centrifuge tube (No. 5 pipes) that poison monoclonal antibody N18 addition is minimum,
It is the minimum steady needed for stable 1ml colloidal gold solution quantitative, on the basis of this minimum steady is quantitative, adds 10%～20%
It is the actually used amount of canine distemper virus cell monoclonal antibodies N18 needed for stable 1ml colloidal gold solution, i.e. canine distemper disease
Poison cell monoclonal antibody N18 is 8.8 μ g～9.6 μ g:1mL with the usage ratio of colloidal gold solution.
(2) pH value with 0.1mol/L solution of potassium carbonate regulation colloidal gold solution is 8.4, under magnetic stirring, according to upper
State the usage ratio (8.8 μ g～9.6 μ g:1mL) of canine distemper virus monoclonal antibody N18 and the colloidal gold solution determined to colloid
Gold solution is added dropwise over canine distemper virus monoclonal antibody N18, continues stirring 20 minutes, add the Sanguis Bovis seu Bubali of final concentration of 5%
Pure albumen, is stirred for 15 minutes, it is thus achieved that canine distemper virus monoclonal antibody N18 is golden with the coupling label of gold colloidal marks dog
Distemper virus monoclonal antibody N18,4 DEG C save backup.
(3) purification of gold mark canine distemper virus monoclonal antibody N18
Use low temperature supercentrifugation purification gold mark canine distemper virus monoclonal antibody N18, unmarked to remove in solution
Canine distemper virus monoclonal antibody N18 and the fully gold colloidal of labelling and the various polymerizations that are likely to be formed in the markers
Thing.First gold is marked canine distemper virus monoclonal antibody N18 (volume is V) at 4 DEG C, with 3000rpm/min low-speed centrifugal 40
Minute, Aspirate supernatant, abandon precipitation；Again by supernatant at 4 DEG C, it is centrifuged 40 minutes with 10000rpm/min, supernatant discarded, uses
PBS buffer solution (including 1%BSA and the 0.02% sodium azide) dissolution precipitation of 0.01mol/L, pH7.4 (refers to original volume
Gold mark canine distemper virus monoclonal antibody N18 volume V), substantially stabilized after overnight: at 4 DEG C, then with 10000rpm/min be centrifuged
40 minutes, supernatant discarded, dissolve with the PBS buffer solution (including 1%BSA and 0.02% sodium azide) of 0.01mol/L, pH7.4
It is precipitated to the 1/10 of original volume (referring to the volume V of gold mark canine distemper virus monoclonal antibody N18), it is thus achieved that the gold mark dog of purification
Distemper virus monoclonal antibody N18, gold mark canine distemper virus monoclonal antibody N18 of this purification is positioned at bottom centrifuge tube, for deeply
Red bulk precipitate, 4 DEG C save backup.
The assembling of embodiment 4 test strips
(1) use gold spraying instrument that the coupling label of canine distemper virus monoclonal antibody N18 Yu gold colloidal is sprayed on glass fibers
Dimension element film, canine distemper virus monoclonal antibody N18 is 8.8 μ g～9.6 μ g:1mL with the labelled amount of the coupling label of gold colloidal,
Natural drying under room temperature condition, seals, it is thus achieved that gold-marking binding pad 3,4 DEG C save backup；
(2) use a stroke film instrument that canine distemper virus monoclonal antibody C42, sheep anti-mouse igg are drawn film respectively in celluloid
On detection line 5 on film 4 and nature controlling line 6, canine distemper virus monoclonal antibody C42, the labelled amount of sheep anti-mouse igg are respectively 2 μ g/
Cm, 4 μ g/cm, natural drying under room temperature condition, seal, it is thus achieved that (be coated with the canine distemper virus Dan Ke of purification with detection line 5
Grand antibody C42) and the nitrocellulose filter 4,4 DEG C of nature controlling line 6 (being coated with sheep anti-mouse igg) save backup；
(3) sample pad 2 handled well above-mentioned, gold-marking binding pad 3, with detection line 5 and the celluloid of nature controlling line 6
The materials such as film 4, absorption pad 7 are pasted onto on PVC base plate successively, cutting, assemble, seal, it is thus achieved that the canine distemper virus glue of the present invention
Body gold immunochromatographydetecting detecting test strip, room temperature preservation is standby.Test strips after assembling is as shown in Figure 1.
Embodiment 5 specific test
Use the present invention test strips to canine distemper virus (CDV), Canine Parvovirus (CPV), canine coronavirus (CCV),
Canine parainfluenza virus (CPIV), Pseudorabies virus (PRV), Avian pneumo-encephalitis virus (NDV) detect.
Result shows: the mink urine sample that only canine distemper virus cell toxicant sample and canine distemper virus infect occurs substantially
Detection line 5 and control line 6, remaining sample detection is feminine gender, illustrates that the test strips of the present invention has good specificity.
Embodiment 6 sensitivity tests
Select colloidal gold strip prepared by the same batch canine distemper virus cell toxicant sample to the variable concentrations of dilution
Detecting, diluted concentration is followed successively by 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3.13 μ g/
mL.Result shows: when viral dilution liquid concentration is more than or equal to 3.13 μ g/mL, red bar all can occurs at detection line and nature controlling line
Band, it was demonstrated that the sensitivity of test strips of the present invention is stronger.
Embodiment 7 replica test
(1) repeatability detection in group:
Each by ELISA test strip canine distemper virus negative sample, the canine distemper virus positive with a batch of present invention
30 parts of samples (repeating test for three times).Result shows, the feminine gender of the ELISA test strip of the present invention, positive findings are respectively 30 examples,
This shows that the test strips of the present invention has good repeatability.
(2) repeatability detection between group:
With the ELISA test strip canine distemper virus negative sample of the present invention of 3 different batches, canine distemper virus positive sample
The each 30 parts of samples of product (repeat test for three times).Result shows, the feminine gender of each batch ELISA test strip, positive findings are respectively 30
Example, this again shows that the test strips of the present invention has good repeatability.
Claims (10)
1. canine distemper virus colloidal gold immunochromatographydetection detection test paper bar, including PVC base plate, is pasted onto the sample on PVC base plate successively
Product pad, gold-marking binding pad, with detection line and the nitrocellulose filter of nature controlling line, absorption pad, it is characterised in that: described gold mark knot
Close pad and be coated with the coupling label of canine distemper virus monoclonal antibody N18 and gold colloidal, the inspection on described nitrocellulose filter
Survey line is coated with canine distemper virus monoclonal antibody C42 of purification, and the nature controlling line on described nitrocellulose filter is coated with goat-anti
Mus IgG.
2. the method for preparation canine distemper virus colloidal gold immunochromatographimethod detection method test strips described in claim 1, its feature
It is, comprises the following steps:
Step one, canine distemper virus monoclonal antibody N18, the preparation of C42: select the female Balb/c mice of 6～8 week old, abdominal cavity
Injection norphytane 0.5mL/, difference lumbar injection two strain canine distemper virus monoclonal antibody hybridoma cell, inoculum concentration after 7 days
It is 4 × 106-6×106Cell/mL/ is only；Extracting ascites after mouse web portion expands respectively, 5000rpm is centrifuged 15min, takes
Clearly；By ascites supernatant respectively with the ammonium sulfate precipitation 2 times of 50% saturation, after dialysis desalination, it is thus achieved that the canine distemper virus of purification
Monoclonal antibody N18 and canine distemper virus monoclonal antibody C42；
The preparation of the coupling label of step 2, canine distemper virus monoclonal antibody N18 and gold colloidal: stepwise dilution method determines dog
The usage ratio of distemper virus monoclonal antibody N18 and colloidal gold solution is 8.8 μ g-9.6 μ g:1mL, according to this usage ratio by
It is added dropwise to canine distemper virus monoclonal antibody N18, adds the bovine serum albumin of final concentration of 5%, after stirring, obtain canine distemper
Viral monoclonal antibodies N18 and the coupling label of gold colloidal, use low temperature supercentrifugation to carry out pure to this coupling label
Change；
Step 3, the assembling of test strips: use gold spraying instrument by the coupling labelling of canine distemper virus monoclonal antibody N18 Yu gold colloidal
Thing is sprayed on glass fibre element film, natural drying under room temperature condition, seals, it is thus achieved that gold-marking binding pad；Use and draw film instrument by canine distemper
Viral monoclonal antibodies C42, sheep anti-mouse igg are drawn on film detection line on nitrocellulose filter and nature controlling line respectively；To process
Good sample pad, gold-marking binding pad, nitrocellulose filter are pasted onto on PVC base plate successively, cutting, assemble, seal, it is thus achieved that hundstaupe
Fever virus monoclonal antibody colloidal gold colloidal gold detection test paper strip, room temperature preservation.
Preparation method the most according to claim 2, it is characterised in that in step one, two strain canine distemper virus monoclonal antis
Body hybridoma uses following methods to prepare: the canine distemper virus inoculation Balb/c mice that will purify, and after booster immunization, collects
Its splenocyte, myeloma cell is merged with preprepared, cultivates with 1%HAT culture fluid；Merge about 2 weeks, use
Indirect ELISA method filters out two strain positive hybridoma cell strains, and clones the positive hybridoma cell after screening,
To two strain canine distemper virus monoclonal antibody hybridoma cell strains.
Preparation method the most according to claim 2, it is characterised in that described colloidal gold solution uses trisodium citrate reduction
Prepared by method: the ultra-pure water taking 1% chlorauric acid solution 1ml addition 99ml is configured to the chlorauric acid solution of final concentration of 0.01%, adds
Heat, to after boiling, adds 1% trisodium citrate 1ml and continues heating, and solution black-and-blue is eventually become claret-red by faint yellow transferring to
Color, continues heating 5 minutes after colour stable, room temperature cools down, it is thus achieved that colloidal gold solution, 4 DEG C save backup, colloid gold particle diameter
For 40nm.
Preparation method the most according to claim 2, it is characterised in that in step 2, described stepwise dilution method determines hundstaupe
The detailed process that fever virus monoclonal antibody N18 arranges with the amount ratio of colloidal gold solution is as follows: molten with the potassium carbonate of 0.1mol/L
The pH value of liquid regulation colloidal gold solution is 8.4, is separately added into 1ml colloidal gold solution in 11 centrifuge tubes；By canine distemper virus
After monoclonal antibody N18 stepwise dilution i.e. content be respectively 2 μ g, 4 μ g, 6 μ g, 8 μ g, 10 μ g, 12 μ g, 14 μ g, 16 μ g, 18 μ g,
20 μ g, join No. 2 pipes in the order described above in No. 11 pipes, and mix with the colloidal gold solution in centrifuge tube, after 5 minutes
No. 2 pipes to No. 11 pipes are separately added into 10% sodium chloride solution 1ml mixing, room temperature stands more than 2 hours observed results, No. 1
Pipe is blank；
To in No. 4 pipes, there is coagulation phenomenon from red to blue in No. 2 pipes, in No. 5 pipes to No. 11 pipes, keep the redness of gold colloidal not
Become；Therefore, in centrifuge tube that is No. 5 pipe that gold colloidal is red constant and canine distemper virus monoclonal antibody N18 addition is minimum
Canine distemper virus monoclonal antibody N18 content, is the minimum steady needed for stable 1ml colloidal gold solution quantitative, in this minimum steady
Add 10%～20% on the basis of Ding Liang to be canine distemper virus cell monoclonal needed for stable 1ml colloidal gold solution and resist
The actually used amount of body N18, i.e. canine distemper virus cell monoclonal antibodies N18 are 8.8 μ g with the usage ratio of colloidal gold solution
～9.6 μ g:1mL.
Preparation method the most according to claim 2, it is characterised in that in step 2, uses low temperature supercentrifugation to this
The detailed process that coupling label is purified is as follows: first gold mark canine distemper virus monoclonal antibody N18 that volume is V existed
Be centrifuged 40 minutes with 3000rpm/min at 4 DEG C, Aspirate supernatant, abandon precipitation: again by supernatant at 4 DEG C with 10000rpm/
Min is centrifuged 40 minutes, supernatant discarded, is precipitated to original volume i.e. gold mark hundstaupe by the PBS buffer solution solution of 0.01mol/L, pH7.4
The 1/10 of the volume V of fever virus monoclonal antibody N18, it is thus achieved that gold mark canine distemper virus monoclonal antibody N18 of purification, 4 DEG C of guarantors
Deposit standby.
Preparation method the most according to claim 6, it is characterised in that in described PBS containing 1%BSA and
0.02% sodium azide.
Preparation method the most according to claim 2, it is characterised in that in step 3, canine distemper virus monoclonal antibody N18
It is 8.8 μ g～9.6 μ g:1mL with the labelled amount of the coupling label of gold colloidal.
Preparation method the most according to claim 2, it is characterised in that in step 3, canine distemper virus monoclonal antibody C42
Labelled amount be 2 μ g/cm.
Preparation method the most according to claim 2, it is characterised in that in step 3, the labelled amount of sheep anti-mouse igg is 4 μ
g/cm。
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