CN105572375B - Mink Parvovirus Enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip and preparation method thereof - Google Patents
Mink Parvovirus Enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip and preparation method thereof Download PDFInfo
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Abstract
Mink Parvovirus Enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip and preparation method thereof is related to the detection technique field of fur-bearing animal viral infectious cause of disease.The colloidal gold colloidal gold detection test paper strip of the present invention includes：Sample pad, gold-labelled pad, NC films and absorption pad spray VP2 albumen in the detection line of the NC films, sheep anti mouse secondary antibody are sprayed on the nature controlling line of the NC films.The preparation method of the Mink Parvovirus Enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip of the present invention, mainly includes the following steps that：Prokaryotic expression Mink Parvovirus (MEV) VP2 and VP1 albumen and its purifying；The preparation of Mink Parvovirus (MEV) monoclonal antibody；The preparation of colloidal gold solution；The preparation of gold labeling antibody solution；The preparation of gold-labelled pad；The pretreatment of NC films；Assembling.The present invention contributes to Fur Animal Feeding field to purify Mink Parvovirus Enteritis, facilitates the operation of base animal doctor personnel.
Description
Technical field
The present invention relates to the detection technique fields of fur-bearing animal viral infectious cause of disease, and in particular to a kind of mink is tiny
Viral enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip and preparation method thereof.
Background technology
Mink viral enteritis incidence caused by mink enteritis virus (Mink enteritis virus, MEV) and dead
Rate height is died, is had brought tremendous economic losses to mink breeding industry.However this disease can only be exempted from without special therapy using vaccine
Epidemic disease is prevented, so the diagnosis to mink viral enteritis is particularly important.Be mainly to the diagnosis of MEV at present HA-HI and
Traditional PCR method, HA is cumbersome, time and effort consuming, and needs to prepare the red blood cell of pig；And PCR is to develop in recent years
The molecular biological testing come is mainly used for the detection of viral nucleic acid, but PCR method needs the instrument and equipment of profession,
And operating personnel's needs could be in special experiment in-house operation by special training.
Since Faulk in 1971 and Taylor foundes colloidal gold-labeled method, colloidal gold become after fluorescent marker,
Enzyme marks and another novel immunolabelling technique after radioimmunoassay label.Colloidal gold technique have it is convenient and efficient,
Special sensitivity, stability is strong, do not need to special installation and reagent, result judge the advantages that intuitive, just because of these characteristics make it
There is very big practicability in heavy workload, the better simply animal test quarantine real work of facility, meet base animal doctor
Detection and big face animal detection demand, so be widely used in animal doctor's infectious disease, especially viral infectious,
At present this technology has been applied in the detection of many animals virus.
Invention content
Of the existing technology in order to solve the problems, such as, the present invention provides a kind of glue of quick detection mink enteritis virus antigen
Body gold test paper strip and preparation method thereof.
The present invention is as follows for technical scheme applied to solve the technical problem：
The Mink Parvovirus Enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip of the present invention, including：Sample pad, Jin Biao
Pad, NC films and absorption pad, spray Mink Parvovirus VP2 albumen in the detection line of the NC films, on the nature controlling line of the NC films
Spray sheep anti mouse secondary antibody；The preparation method of the gold-labelled pad is：Using 20nm colloidal gold solutions label Mink Parvovirus Dan Ke
Grand antibody obtains gold labeling antibody solution, its point sample amount according to 6 μ l/cm is sprayed on glass film and obtains gold-labelled pad；The water
Ermine parvovirus monoclonal antibody is prepared using Mink Parvovirus VP1 albumen, the Mink Parvovirus VP1 albumen
SEQ ID NO in amino acid sequence such as sequence table:Shown in 2.
Further, the Mink Parvovirus VP2 protein concentrations are 0.85mg/ml, and the sheep anti mouse secondary antibody is a concentration of
1mg/ml。
The present invention also provides the preparations of above-mentioned Mink Parvovirus Enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip
Method includes the following steps：
Step 1: MEVB plants of VP2 genes of PCR amplification Mink Parvovirus and VP1 portion genes, directed cloning to protokaryon table
It up to vector construction recombinant plasmid, and converts into host strain BL21, through obtaining Mink Parvovirus VP2 after purification after IPTG inductions
Albumen and Mink Parvovirus VP1 albumen；
Step 2: the Mink Parvovirus VP1 albumen using purifying prepares Mink Parvovirus monoclonal antibody；
Step 3: 20nm colloidal gold solutions are prepared using citric acid reduction method；
Step 4: gold labeling antibody solution is obtained using 20nm colloidal gold solutions label Mink Parvovirus monoclonal antibody；
Step 5: glass film is after pretreatment, gold labeling antibody solution is sprayed on glass film according to the point sample amount of 6 μ l/cm,
Obtain gold-labelled pad；
Step 6: the Mink Parvovirus VP2 albumen of a concentration of 0.85mg/ml after purification is sprayed on the detection of NC films
On line, the sheep anti mouse secondary antibody of a concentration of 1mg/ml is sprayed on the nature controlling line of NC films；
Step 7: NC films, gold-labelled pad, sample pad and absorption pad are assembled into colloidal gold colloidal gold detection test paper strip.
Preferably, in step 1, the prokaryotic expression carrier is PET-30a.
Preferably, in step 1, the purification process of Mink Parvovirus VP2 albumen and Mink Parvovirus VP1 albumen is：
It is purified using His labels, purpose egg is can obtain through His Bind Purification Kit Purification Kits
In vain.
Preferably, it in step 2, prepares Mink Parvovirus monoclonal using the Mink Parvovirus VP1 albumen of purifying and resists
The specific method of body is：
(1) by the Mink Parvovirus VP1 protein immunization BABL/c mouse of purifying, first immunisation is by Mink Parvovirus
VP1 albumen using intraperitoneal injection mode with after Freund's complete adjuvant in equal volume emulsification, being immunized, once being exempted from interval of two weeks
Epidemic disease is immunized 4 times altogether, wherein the 2nd time, the 3rd time is emulsified using incomplete Freund's adjuvant, the 4th is that the reinforcement of 3 days before merging is exempted from
Epidemic disease；
(2) mice serum antibody titer, Mink Parvovirus antibody effect are measured by indirect ELISA method after booster immunization
Valency reaches 105It is merged above, it is sterile to take mouse spleen, it is merged after grinding centrifugation with myeloma cell SP2/0, using indirect
ELISA method is screened and limiting dilution assay is subcloned, and finishing screen selects secretion water resistant ermine parvovirus antibody
14 plants of hybridoma cell strain chooses the strong cell strain of affinity using the method for preparing antibody in vivo, prepares Mink Parvovirus
Monoclonal antibody.
Preferably, in step 3, use citric acid reduction method prepare the specific methods of 20nm colloidal gold solutions for：
After 1% chlorauric acid solution 1mL is taken to add in the abundant mixings of deionized water 99mL, it is heated to boiling；When in stirring
1% citric acid three sodium solution 1.5mL, mixing are added in, and it is made to keep fluidized state, solution colour is then turned successively by light yellow
Become light/dark balance, black, darkviolet, finally stabilize to claret, continue to boil 5min, be supplemented to after cooling with deionized water
100mL obtains 20nm colloidal gold solutions, and 4 DEG C are kept in dark place.
Preferably, in step 4, the specific preparation method of gold labeling antibody solution is：
(1) the colloidal gold solution 1mL of 20nm is taken, with the K of 0.1mol/L2CO3Solution is adjusted to best pH value 8.0, and stirring is mixed
It is even；
(2) 12 μ g of Mink Parvovirus (MEV) monoclonal antibody after purification are added in, mixing is stored at room temperature 30min；
(3) it adds in PEG-20000 and BSA solution to stir and evenly mix, stands 30min；
(4) labeled good colloidal gold probe 1500r/min is centrifuged into 30min, removal precipitation；Supernatant 12000r/min
30min is centrifuged, abandons supernatant；The colloidal gold probe of precipitation is suspended and centrifuged with BSA and sodium azide solution, finally uses precipitation
The BSA of 1mL and the mixture of sodium azide solution are dissolved, and obtain gold labeling antibody solution, 4 DEG C are kept in dark place.
Preferably, in step 5, the preprocessing process of glass film is：
Glass fibre element film is cut into the strip of 5mm × 30cm, with 0.01mol/L, PH be 8.2 TB solution treatments,
It is placed in 37 DEG C of drying.
Preferably, it is using Biodot XYZ3060 instruments that VP2 albumen after purification and sheep anti mouse two is anti-zoned in step 6
Line is on NC films；Stroked parameters are：2 μ l/cm, C line of T lines, 1 μ l/cm, T lines and C lines interval 5mm；Biodot XYZ3060 instruments
Parameter is：T linear velocity 50mm/s, acceleration 1000mm/s2, Z axis decline 55mm, drop 40nl, pitch 0.4, on time
0.4；C linear velocity 50mm/s, acceleration 1000mm/s2, Z axis decline 55mm, drop 40nl, pitch 0.4, on time
0.4。
The beneficial effects of the invention are as follows：
The foundation of this colloidal gold strip will be helpful to Fur Animal Feeding field purification mink viral enteritis, facilitate base
The operation of animal doctor personnel.
The present invention can be not only used for the diagnosis of mink viral enteritis, and can be used for examining for canine parvovirus disease
It is disconnected, the clinical diagnosis to canine parvovirus enteritis such as pet clinic can be widely used in.
The present invention is particularly suitable for base animal doctor and farm for quickly detecting the parvovirus antigen in mink excrement
The execute-in-place of technical staff.
Description of the drawings
Fig. 1 is the pictorial diagram of the Mink Parvovirus Enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip of the present invention.Figure
In：S is well, and T is detection line, and C is nature controlling line.
Fig. 2 is the Mink Parvovirus Enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip result judgement signal of the present invention
Figure.Fig. 2 a are positive judgement result (one line of C lines), and Fig. 2 b are negative judgement result (C lines, T lines two lines).
Fig. 3 is specific test result figure.In figure：From left to right it is followed successively by CPV BJ14-24, MEVB, AMDV-G, CAV-
2C and CDV3 and water control.
Fig. 4 is sensitivity tests result figure.In figure：It is from left to right followed successively by water control and MEVB strain virus is imitated according to blood clotting
It (is respectively 1 that valency, which carries out 2 times to be serially diluted,:2,1:4,1:8,1:16,1:32,1:64,1:128,1:256).
Specific embodiment
The Mink Parvovirus Enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip of the present invention, constituent include：
What the detection line (T lines) on sample pad, gold-labelled pad, NC films and absorption pad, wherein NC films sprayed is VP2 albumen, and nature controlling line (C
Line) what is sprayed is sheep anti mouse secondary antibody, entire test strips, which fit together, to be wrapped in plastic clip, as shown in Figure 1.
Result judgement：As shown in Figure 2 a, nature controlling line (C lines) shows dark bands, and detection line (T lines) does not show dark bars
Band is judged to the positive.As shown in Figure 2 b, nature controlling line (C lines) and detection line (T lines) show dark bands, are judged to feminine gender.Nature controlling line
(C lines) and detection line (T lines) do not show that dark bands either nature controlling line (C lines) does not show dark bands and detection line (T lines)
Show dark bands, it is invalid to be judged to.
The preparation method of the Mink Parvovirus Enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip of the present invention, it is main to wrap
Include following steps：
Step 1: prokaryotic expression Mink Parvovirus (MEV) VP2 albumen and its purifying
Step 2: prokaryotic expression Mink Parvovirus (MEV) VP1 Partial Proteins and its purifying
Step 3: the preparation of Mink Parvovirus (MEV) monoclonal antibody
Step 4: the preparation of colloidal gold solution
Step 5: the preparation of gold labeling antibody solution
Step 6: the preparation of gold-labelled pad
Step 7: the pretreatment (scribing line of C, T line) of NC films
Step 8: the assembling of test strips.
1 prokaryotic expression Mink Parvovirus (MEV) VP2 albumen of embodiment and its purifying
The VP2 albumen of detection line (T lines) is by obtained from Prokaryotic expression, purification.
Detailed process is：(1) MEVB plants of VP2 genes of Mink Parvovirus are expanded using round pcr, amplification condition is：95
DEG C, 5min；94 DEG C, 30s, 55 DEG C, 1min, 72 DEG C, 2min；72 DEG C, 8min.By the genetic fragment directed cloning of acquisition to protokaryon
Expression vector PET-30a, structure Mink Parvovirus (MEV) VP2 gene prokaryotic recombinant plasmids PET-30a-VP2.
(2) Mink Parvovirus (MEV) VP2 gene prokaryotic recombinant plasmids PET-30a-VP2 of above-mentioned acquisition is turned
Change into host strain BL21, VP2 albumen is expressed in the form of inclusion body after IPTG inductions, carries out SDS-PAGE and Western
Blotting is analysis shows the VP2 albumen can be expressed correctly.
(3) the result shows that：The full length gene is 1755bp, and objective gene sequence is completely correct, can encode 584 amino
Acid carries out it using the His labels on Mink Parvovirus (MEV) VP2 gene prokaryotic recombinant plasmids PET-30a-VP2
Purifying can obtain destination protein by His Bind Purification Kit Purification Kits, which is
The amino acid sequence of Mink Parvovirus (MEV) VP2 albumen is as shown in the sequence 1 in sequence table.
2 prokaryotic expression Mink Parvovirus (MEV) VP1 Partial Proteins of embodiment and its purifying
Mink Parvovirus (MEV) VP1 albumen is obtained by expressing VP1 portion genes, expression and purification method ginseng
According to embodiment 1.
Detailed process is：(1) MEVB plants of VP1 portion genes of Mink Parvovirus, amplification condition are expanded using round pcr
For：95 DEG C, 5min；94 DEG C, 30s, 55 DEG C, 45s, 72 DEG C, 90s；72 DEG C, 8min.By the genetic fragment directed cloning of acquisition extremely
Prokaryotic expression carrier PET-30a, structure Mink Parvovirus (MEV) VP1 gene prokaryotic recombinant plasmids PET-30a-VP1.
(2) Mink Parvovirus (MEV) VP1 gene prokaryotic recombinant plasmids PET-30a-VP1 of above-mentioned acquisition is turned
Change into host strain BL21, VP1 albumen is expressed in the form of inclusion body after IPTG inductions, carries out SDS-PAGE and Western
Blotting is analysis shows the VP1 albumen can be expressed correctly.
(3) the result shows that：The a length of 900bp of the gene, objective gene sequence is completely correct, can encode 300 amino acid,
It is carried out using the His labels on Mink Parvovirus (MEV) VP1 gene prokaryotic recombinant plasmids PET-30a-VP1 pure
Change, can obtain destination protein by His Bind Purification Kit Purification Kits, the destination protein, that is, water
The amino acid sequence of ermine parvovirus (MEV) VP1 albumen is as shown in the sequence 2 in sequence table.
The preparation of 3 Mink Parvovirus of embodiment (MEV) monoclonal antibody
(1) by the Mink Parvovirus VP1 protein immunization BABL/c mouse of above-mentioned Prokaryotic expression, purification, first immunisation is by water
Ermine parvovirus VP1 albumen by the way of intraperitoneal injection with after Freund's complete adjuvant in equal volume emulsification, being immunized, at interval of two weeks
Primary immunization is carried out, is immunized 4 times altogether, wherein the 2nd time, the 3rd time is emulsified using incomplete Freund's adjuvant, the 4th is 3 days before fusion
Booster immunization.
(2) mice serum antibody titer, Mink Parvovirus (MEV) are measured by indirect ELISA method after booster immunization
Antibody titer reaches 105It is merged above, it is sterile to take mouse spleen, it merges, adopts with myeloma cell SP2/0 after grinding centrifugation
It is screened with indirect ELISA method and limiting dilution assay is subcloned, finishing screen selects the anti-Mink Parvovirus of secretion
(MEV) 14 plants of the hybridoma cell strain of antibody chooses the strong cell strain of affinity using the method for preparing antibody in vivo, prepares water
Ermine parvovirus (MEV) monoclonal antibody.
The preparation of 4 colloidal gold solution of embodiment
20nm colloidal gold solutions are prepared using citric acid reduction method.
Detailed process is：1% chlorauric acid solution 1mL is taken to add in the abundant mixings of deionized water 99mL to be placed on electric furnace,
It is heated to boiling；1% citric acid three sodium solution 1.5mL, rapid mixing are rapidly added in being vigorously stirred, and it is made to keep boiling
State is risen, solution colour then gradually stabilizes to claret by light yellow light/dark balance, black, the darkviolet of being changed into successively, continues to boil
5min is boiled, is supplemented to 100mL with deionized water after cooling, obtains 20nm colloidal gold solutions, 4 DEG C are kept in dark place.
The preparation of 5 gold labeling antibody solution of embodiment
(1) the colloidal gold solution 1mL of 20nm is taken, with the K of 0.1mol/L2CO3Solution is adjusted to best pH value 8.0, and stirring is mixed
It is even.
(2) 12 μ g of Mink Parvovirus (MEV) monoclonal antibody after purification are added in, mixing is stored at room temperature 30min.
(3) it adds in PEG-20000 and BSA solution to stir and evenly mix, stands 30min.
(4) labeled good colloidal gold probe 1500r/min is centrifuged into 30min, removal precipitation；Supernatant 12000r/min
30min is centrifuged, abandons supernatant；The colloidal gold probe of precipitation is suspended and centrifuged with BSA and sodium azide solution, finally uses precipitation
The BSA of 1mL and the mixture of sodium azide solution are dissolved, and obtain gold labeling antibody solution, 4 DEG C are kept in dark place.
The preparation of 6 gold-labelled pad of embodiment
One whole glass fibre element film is cut into the strip of 5mm × 30cm, with the TB solution that 0.01mol/L, PH are 8.2
Fully processing is placed in 37 DEG C of drying, according to above-mentioned size (5mm × 30cm) 6 μ l gold labeling antibody solution of spraying per cm.
Its instrument parameter is as follows：Speed 50mm/s, acceleration 1000mm/s2, Z axis decline 54.5mm.
The pretreatment (scribing line of C, T line) of embodiment 7NC films
Using Biodot XYZ3060 instruments, the VP2 albumen (0.85mg/ml) of detection line T wire tags after purification, nature controlling line
The sheep anti mouse secondary antibody (1mg/ml) of C wire tag commercializations.
VP2 albumen after purification and antibody are lined on NC films, scribing line basic parameter is：2 μ l/cm, C line of T lines, 1 μ
L/cm, T line and C lines interval 5mm.
XYZ3060 instrument parameters are：T linear velocity 50mm/s, acceleration 1000mm/s2, Z axis decline 55mm, drop are
40nl, pitch 0.4, on time 0.4；C linear velocity 50mm/s, acceleration 1000mm/s2, Z axis decline 55mm, drop are
40nl, pitch 0.4, on time 0.4.
The assembling of 8 test strips of embodiment
NC films, gold-labelled pad, sample pad and absorption pad are affixed on successively on PVC backboards, 4mm × 60mm is cut into cutting machine
Size test strips are put in the plastics card slot being sized for, and are assembled into the colloidal gold colloidal gold detection test paper strip of detection MEV antigens.
9 specific test of embodiment
With colloidal gold colloidal gold detection test paper strip detection mink enteritis virus (MEVB plants), the Aleutian Mink Disease Parvovirus of the present invention
(AMDV-G plants), fox encephalitis virus (CAV-2C plants), canine distemper virus (CDV3 plants), canine parvovirus (CPV BJ14-24
Strain) and water control.
The results are shown in Figure 3, as a result shows：Mink enteritis virus and canine parvovirus are positive as a result, and mink Ah staying
Shen virus, fox encephalitis virus and canine distemper virus and water compare the result that is negative.Thus the colloidal gold of the present invention is proved
Test strip specificity preferably, can be used not only for the diagnosis of mink enteritis virus, can be also used for the detection of canine parvovirus,
With wide application market.
10 sensitivity tests of embodiment
Test strips sensitivity tests is carried out by the use of MEVB strain virus as standard strain, by MEVB strain virus according to hemagglutinative titer
It carries out 2 times to be serially diluted, respectively 1:2,1:4,1:8,1:16,1:32,1:64,1:128,1:256, colloidal gold of the invention inspection
The detection sensitivity for testing paper slip is 1:64.The results are shown in Figure 4.
11 storage life of embodiment is tested
The colloidal gold colloidal gold detection test paper strip of the invention to 3 batches carries out storage life experiment, as a result, it has been found that the colloidal gold inspection of the present invention
The time that paper slip preserves 6 months at 4 DEG C and 25 DEG C respectively is tested, sensibility and specificity does not change significantly, therefore, this
The colloidal gold colloidal gold detection test paper strip storage life of invention is 6 months.
12 clinical practice of embodiment is tested
Clinical 146 parts of mink fecal specimens are detected, and examined with PCR using the colloidal gold colloidal gold detection test paper strip of the present invention
Survey method carries out contrasting detection, as a result, it has been found that the colloidal gold colloidal gold detection test paper strip Positive rate of the present invention is detected for 60.3%, PCR
Positive rate is 58.9%, and the coincidence rate of two methods is 90.4%.Detailed results are shown in Table 1.
The colloidal gold colloidal gold detection test paper strip of 1 present invention of table is compared with PCR detection method result
Claims (1)
1. the preparation method of Mink Parvovirus Enteritis pathogen antigen colloidal gold colloidal gold detection test paper strip, which is characterized in that including
Following steps：
Step 1: MEVB plants of VP2 genes of PCR amplification Mink Parvovirus and VP1 portion genes, directed cloning to prokaryotic expression carries
Body construction recombination plasmid, and convert into host strain BL21, through obtaining Mink Parvovirus VP2 albumen after purification after IPTG inductions
With Mink Parvovirus VP1 albumen；
The prokaryotic expression carrier is PET-30a；
The purification process of Mink Parvovirus VP2 albumen and Mink Parvovirus VP1 albumen is：It is purified using His labels,
Destination protein is can obtain through His Bind Purification Kit Purification Kits；
Step 2: the Mink Parvovirus VP1 albumen using purifying prepares Mink Parvovirus monoclonal antibody, specific method
For：
(1) by the Mink Parvovirus VP1 protein immunization BABL/c mouse of purifying, first immunisation is by Mink Parvovirus VP1 eggs
It after being emulsified in equal volume with Freund's complete adjuvant in vain, is immunized using intraperitoneal injection mode, carried out primary immunization at interval of two weeks, exempt from altogether
Epidemic disease 4 times, wherein the 2nd time, the 3rd time is emulsified using incomplete Freund's adjuvant, the 4th is the booster immunization of 3 days before fusion；
(2) mice serum antibody titer is measured by indirect ELISA method after booster immunization, Mink Parvovirus antibody titer reaches
To 105It is merged above, it is sterile to take mouse spleen, it is merged after grinding centrifugation with myeloma cell SP2/0, using indirect
ELISA method is screened and limiting dilution assay is subcloned, and finishing screen selects secretion water resistant ermine parvovirus antibody
14 plants of hybridoma cell strain chooses the strong cell strain of affinity using the method for preparing antibody in vivo, prepares Mink Parvovirus
Monoclonal antibody；
Step 3: preparing 20nm colloidal gold solutions using citric acid reduction method, specific method is：
After 1% chlorauric acid solution 1mL is taken to add in the abundant mixings of deionized water 99mL, it is heated to boiling；It is added in when in stirring
1% citric acid three sodium solution 1.5mL, mixing, and it is made to keep fluidized state, solution colour is then changed into successively by light yellow
Light/dark balance, black, darkviolet, finally stabilize to claret, continue to boil 5min, are supplemented to after cooling with deionized water
100mL obtains 20nm colloidal gold solutions, and 4 DEG C are kept in dark place,
Step 4: gold labeling antibody solution is obtained using 20nm colloidal gold solutions label Mink Parvovirus monoclonal antibody, specifically
Method is：
(1) the colloidal gold solution 1mL of 20nm is taken, with the K of 0.1mol/L2CO3Solution is adjusted to best pH value 8.0, stirs and evenly mixs；
(2) 12 μ g of Mink Parvovirus (MEV) monoclonal antibody after purification are added in, mixing is stored at room temperature 30min；
(3) it adds in PEG-20000 and BSA solution to stir and evenly mix, stands 30min；
(4) labeled good colloidal gold probe 1500r/min is centrifuged into 30min, removal precipitation；Supernatant 12000r/min is centrifuged
30min abandons supernatant；The colloidal gold probe of precipitation is suspended and centrifuged with BSA and sodium azide solution, will finally be precipitated with 1mL's
The mixture of BSA and sodium azide solution is dissolved, and obtains gold labeling antibody solution, 4 DEG C are kept in dark place；
Step 5: glass film is after pretreatment, gold labeling antibody solution is sprayed on glass film according to the point sample amount of 6 μ l/cm, is obtained
Gold-labelled pad；
The preprocessing process of glass film is：Glass fibre element film is cut into the strip of 5mm × 30cm, is with 0.01mol/L, PH
8.2 TB solution treatments are placed in 37 DEG C of drying；
Step 6: the Mink Parvovirus VP2 albumen of a concentration of 0.85mg/ml after purification is sprayed on the detection line of NC films
On, the sheep anti mouse secondary antibody of a concentration of 1mg/ml is sprayed on the nature controlling line of NC films；
In step 6, VP2 albumen after purification and sheep anti mouse secondary antibody are lined on NC films using BiodotXYZ3060 instruments；
Stroked parameters are：2 μ l/cm, C line of T lines, 1 μ l/cm, T lines and C lines interval 5mm；Biodot XYZ3060 instrument parameters are：T linear speeds
Spend 50mm/s, acceleration 1000mm/s2, Z axis decline 55mm, drop 40nl, pitch 0.4, on time 0.4；C linear velocities
50mm/s, acceleration 1000mm/s2, Z axis decline 55mm, drop 40nl, pitch 0.4, on time 0.4；
Step 7: NC films, gold-labelled pad, sample pad and absorption pad are assembled into colloidal gold colloidal gold detection test paper strip.
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