CN103792373A - Colloidal gold immunochromatography test paper for antibodies of pseudorabies viruses of gE, gB and gD and preparation method - Google Patents
Colloidal gold immunochromatography test paper for antibodies of pseudorabies viruses of gE, gB and gD and preparation method Download PDFInfo
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Abstract
The invention discloses a colloidal gold immunochromatography test paper for antibodies of pseudorabies viruses of gE, gB and gD and a preparation method. The test paper comprises a sample absorption area, a gold mark probe area, an immobilization antigen and antibody area, a water absorption area and a supporting plate, and the sample absorption area, the gold mark probe area, the immobilization antigen and antibody area and the water absorption area are laid on the supporting plate and are partially overlapped in sequence. The gold mark probe area is covered with gold mark probes which are mouse anti-swine IgG antibodies marked by colloidal gold. The immobilization antigen and antibody area is provided with a detection line T1 covered with pseudorabies virus gE protein, a detection line T2 covered with pseudorabies virus gB protein, a detection line T3 covered with pseudorabies virus gD protein and a control line C covered with goat anti-mouse IgG antibodies. The test paper is fast in detection, high in accuracy, strong in specificity, easy and convenient to carry and operate and capable of being used for differential diagnosis on PRV wild poisonous infection and vaccine immunity and evaluation on the PRV vaccine immunity effect at the same time.
Description
Technical field
The present invention relates to a kind of Test paper and preparation method, be specifically related to colloidal gold immunochromatographydetection detection test paper and the preparation method of a kind of PRV gE, gB and gD antibody.
Background technology
Pseudoabies (Pseudorabies) is a kind of acute infectious disease that multiple domestic animal and wild animal suffer from altogether that comprises being caused by Pseudorabies virus (Pseudorabies virus, PRV).Pig is the topmost reservoir host of pseudoabies and the infection sources.Health pig with sick pig, be with malicious pig directly to contact can to infect this disease.Adult Pig is often subclinical infection; There is the symptom such as miscarriage, stillborn foetus, weak tire and mummy tire in infected farrowing sow; There is heating and nervous symptoms in infected newborn piglet, even exhaustion death, and mortality ratio can reach 100%.
Found in the U.S. that oneself was popular in the world for this disease since pseudoabies first from 1902.China is since nineteen forty-seven is found pseudoabies first, and to 2006, existing 31 provinces (district), municipalization were about the report of pseudoabies.Pseudoabies has caused huge economic loss to the sound development of China and even global pig industry, becomes one of serious infectious diseases of serious harm pig industry sound development.
In PRV and host's interaction, gB and the gD membrane glycoprotein of virus play an important role.Virus envelope glycoprotein gB and gD not only mediate the infection of virus to target cell, are also the major antigens of infected host immune system identification.In herpesviral, gB albumen belongs to one of the most conservative glycoprotein, and this glycoprotein can induce body to produce the neutralizing antibody of high titre, and is the essential component in virus infection.PRV gD is the essential structural proteins of virus infections, and this albumen participates in the process that penetrates of virus, a kind of important in and antigen, be also the main target of protection antibody.Research shows, gD gene is the important target sequence of prevention porcine pseudorabies recombinant adenovirus vaccine and nucleic acid vaccine, can be used as serodiagnosis antigen.GE glycoprotein is a kind of very important glycoprotein of PRV, determining the viral important role such as virulence and neural preferendum, breeds necessary albumen but gE is not PRV, the candidate gene of Chang Zuowei disappearance.
At present, the method for conventional detection PRV comprises latex agglutination (LAT), enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) etc.Colloidal gold immunity chromatography is as a kind of day by day ripe experiment detection method, low, easy and simple to handle with its high specificity, cost, reliable results, can single part or measure in batch, do not need the advantages such as any instrument to be widely accepted.Currently reported PRV Test paper is many can only detect the antibody for single proteantigen (gE), and tester often need to could further determine in the situation that there is no wild virus infection whether have PRV antibody by secondary detection.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming that overcomes prior art, with not enough, provides the colloidal gold immunochromatographydetection detection test paper of a kind of PRV gE, gB and gD antibody.This test paper adopts the anti-pig IgG monoclonal antibody of mouse of colloid gold label to make probe, and coated PRV gE, gB and gD proteantigen and goat anti-mouse igg antibody carry out the detection of resisting pstudorabies poison gE, gB and gD antibody.
Another object of the present invention is to the preparation method of the colloidal gold immunochromatographydetection detection test paper that above-mentioned PRV gE, gB and gD antibody are provided.
A further object of the present invention is the using method of the colloidal gold immunochromatographydetection detection test paper that above-mentioned PRV gE, gB and gD antibody are provided.
Object of the present invention is achieved through the following technical solutions:
The colloidal gold immunochromatographydetection detection test paper of a kind of PRV gE, gB and gD antibody, comprise absorption of sample district, gold mark probe region, immobilization antigen and antibody district, suction zones and back up pad, absorption of sample district, gold mark probe region, immobilization antigen-antibody district and suction zones are laid in back up pad and mutually partly overlap successively; Described gold mark probe region coated gold mark probe, is the anti-pig IgG monoclonal antibody of mouse of colloid gold label, and labelled amount is preferably the anti-pig IgG monoclonal antibody of every mL colloid gold label 5～20 μ g mouse, and gold mark probe package amount is preferably 5～10 μ L; Described immobilization antigen and antibody district (from gold mark probe region to suction zones direction) have detection line T1, T2, T3 and control line C successively; Described detection line T1 is coated with PRV gE albumen, and package amount is preferably 0.5～5 μ g; Detection line T2 is coated with PRV gB albumen, and package amount is preferably 0.5～5 μ g; Detection line T3 is coated with PRV gD albumen, and package amount is preferably 0.5～5 μ g; Control line C is coated with goat anti-mouse igg antibody, and it is 1～10 μ g that package amount is preferably.
The material of described back up pad is preferably the polyethylene board that is stained with one deck Polyvinylchloride lining form; The material in described absorption of sample district is preferably glass fibre membrane; The material of described gold mark probe region is preferably polyester film; Described immobilization antigen and the material in antibody district are preferably nitrocellulose filter; The material of described suction zones is preferably absorbent filter.
Described PRV gE, gB and gD proteantigen can be prepared or express according to conventional gene engineering method, and these are all that those skilled in the art can grasp or understand thoroughly.
The preparation method of the colloidal gold immunochromatographydetection detection test paper of above-mentioned PRV gE, gB and gD antibody, comprises the steps: anti-the mouse of colloid gold label pig IgG monoclonal antibody to be sprayed on polyester film; The coated PRV gE albumen of coated detection line T1(successively on nitrocellulose filter), the coated PRV gB albumen of T2(), the coated PRV gD albumen of T3() and control line C(be coated with goat anti-mouse igg antibody); Glass fibre membrane, polyester film, nitrocellulose filter and absorbent filter are assembled into the colloidal gold immunochromatographydetection detection test paper that obtains PRV gE, gB and gD antibody in back up pad.
Preferred, the preparation method of described PRV gE, gB and the colloidal gold immunochromatographydetection detection test paper of gD antibody comprises the steps:
(1) method of the anti-pig IgG monoclonal antibody of colloid gold label mouse: getting respectively radius is that 10～40nm concentration is 0.01% collaurum 20mL and anti-pig IgG monoclonal antibody 100～400 μ g of mouse, under the condition of pH value 8.5～9.2, vibrate and make its combination by magnetic agitation, add bovine serum albumin(BSA) (BSA) and PEG 20000 (PEG20000) as stabilizing agent, and making BSA final mass concentration is 0.1～5%, PEG20000 final mass concentration is 0.01～0.1%, adopt centrifuge method to remove the anti-pig IgG monoclonal antibody of unconjugated mouse and unstabilized colloid gold particle and agglutinator thereof, peony in centrifuge tube bottom is precipitated as the anti-pig IgG monoclonal antibody of collaurum-mouse compound.
(2) anti-the mouse of colloid gold label pig IgG monoclonal antibody is sprayed on polyester film: the PBS(0.01M with 20mL containing 1%BSA, pH value 7.4) the anti-pig IgG monoclonal antibody of difference washing colloids gold-mouse compound, the centrifugal supernatant of removing, obtain peony precipitation, precipitation after purifying is dissolved containing the PBS of 1%BSA with 2mL, be applied on polyester film freeze-drying with spraying equipment.
(3) nitrocellulose filter is coated: at nitrocellulose filter successively coated detection line T1, T2, T3 and control line C; The amount of the coated PRV gE albumen of detection line T1 is 0.5～5 μ g; The amount of the coated PRV gB albumen of detection line T2 is 0.5～5 μ g; The amount of the coated PRV gD albumen of detection line T3 is 0.5～5 μ g; The amount of the coated goat anti-mouse igg antibody of control line C is 1～10 μ g; Every live width 1～3mm.
(4) test paper assembling: as prop carrier, lay successively polyester film, nitrocellulose filter and the absorbent filter of glass fibre membrane, gold mark probe with the polyethylene board that is stained with one deck Polyvinylchloride lining form above, outside is sealed and made with adhesive tape.
Except as otherwise noted, while the present invention relates to the number percent between liquid and liquid, described number percent is volume/volume number percent; While the present invention relates to the number percent between liquid and solid, described number percent is volume/weight (mL/g) number percent; While the present invention relates to the number percent between solid and liquid, described number percent is weight/volume (g/mL) number percent; All the other are weight/percentage by weight.
The using method of the colloidal gold immunochromatographydetection detection test paper of above-mentioned PRV gE, gB and gD antibody, comprise the steps: one end, absorption of sample district of this test paper to immerse in blood serum sample to be checked, sentence read result after 5～10 minutes: control line C occurs red, represents that test paper is effective; Detection line T1, T2, T3 occur red, show respectively Pseudorabies virus gE, gB, gD antibody positive; Detection line T1, T2, T3 occur without color, and showing does not have corresponding Pseudorabies virus gE, gB, gD antibody or antibody amount very low in serum.
The present invention has the following advantages and effect with respect to prior art tool:
(1) detect fast: only need 5～10 minutes detection time, can meet the needs of Site Detection.
(2) high, the high specificity of Detection accuracy: this test paper does not have cross reaction with other pig susceptible pathogen antigens, and detection sensitivity and ELISA are basic identical.
(3) easy to carry, easy and simple to handle: the present invention need to be by Other Instruments equipment, be applicable to veterinary hospitals at different levels, pig farm and individual and use.
(4) Test paper can be preserved at normal temperatures, without special equipment and instrument.Storage life can reach 2 years, and detects reproducible.
(5) can a step detect PRV gE, gB and tri-kinds of antibody of gD simultaneously, in differentiating wild virus infection, can evaluate immune effect of vaccine, learn investigation, vaccine effect evaluation and pig farm for testing sieve pig, pestilence the new efficient instrument that provides is provided.
Accompanying drawing explanation
Fig. 1 is the planar structure areal map of the colloidal gold immunochromatographydetection detection test paper of PRV gE, gB and gD antibody; Wherein, 1-absorption of sample district, 2-gold mark probe region, 3-immobilization antigen and antibody district, 31-detection line T1,32-detection line T2,33-detection line T3,34-control line C, 4-suction zones.
Fig. 2 is the colloidal gold immunochromatographydetection detection test paper profile structural drawing (representing regional with concrete material) of PRV gE, gB and gD antibody, wherein, 5-is stained with the polyethylene board (back up pad) of one deck Polyvinylchloride lining form, 6-glass fibre membrane (absorption of sample district), 7-polyester film (gold mark probe region), 8-nitrocellulose filter (immobilization antigen and antibody district), 9-absorbent filter (suction zones).
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is specifically described, embodiments of the invention are only for technical scheme of the present invention is described, and non-limiting essence of the present invention.
Embodiment 1
The colloidal gold immunochromatographydetection detection test paper planar structure areal map of PRV gE, gB and gD antibody as shown in Figure 1, test paper surface level is followed successively by from bottom to top: absorption of sample district 1, gold mark probe region 2, immobilization antigen and antibody district 3 and suction zones 4, immobilization antigen and antibody district 3 are coated with detection line T1 31, detection line T2 32, detection line T3 33 and control line C 34.
The colloidal gold immunochromatographydetection detection test paper profile structural drawing of PRV gE, gB and gD antibody (representing regional with concrete material) as shown in Figure 2, the polyethylene board 5 that is stained with one deck Polyvinylchloride lining form is back up pad, glass fibre membrane 6 is absorption of sample district, polyester film 7 is gold mark probe region, nitrocellulose filter 8 is immobilization antigen and antibody district, absorbent filter 9 is suction zones, is coated with gold mark probe on polyester film 7; Glass fibre membrane 6, polyester film 7, nitrocellulose filter 8, absorbent filter 9 are laid on polyethylene board 5 and mutually partly overlap successively.
The anti-pig IgG monoclonal antibody of mouse that gold mark probe is colloid gold label, labelled amount is every mL colloid gold label 5 μ g monoclonal antibodies, gold mark probe package amount is 10 μ L.The amount of detection line T1 31 coated PRV gE albumen is 0.5 μ g; The amount of detection line T2 32 coated PRV gB albumen is 0.5 μ g; The amount of detection line T3 33 coated PRV gD albumen is 0.5 μ g; The amount of control line C 34 coated goat anti-mouse igg antibodies is 1 μ g.
The colloidal gold immunochromatographydetection detection test paper preparation method of above-mentioned PRV gE, gB and gD antibody is as follows:
(1) described Pseudorabies virus gE albumen, gB albumen and gD protein preparation method comprise the steps:
1) extract PRV (PRV) genome, the PRV strain in China gene order of delivering with reference to GenGank, respectively for gE, gB and gD gene design primer, wherein,
GE upstream primer: 5 '-ATGGATTCATGCTGAGGGAGGCACCCCCG-3 ',
GE downstream primer: 5 '-CGAAGCTTTAATAACGGCCGGTCGCCCAC-3 ';
GB upstream primer: 5 '-GGATCCGCGCACGTGAACGACATG-3 ',
GB downstream primer: 5 '-AAGCCTGAGCGCGTGCAGCTGGTT-3 ';
GD upstream primer: 5 '-GGATCCATGCTGCTCGCAGCGCTAT-3 ';
GD downstream primer: 5 '-AAGCTTGTCAGGAATCGCATCACGT-3 ';
2) the gene design primer described in step 1) is increased respectively gE genetic fragment, gB genetic fragment and gD genetic fragment, again the gE genetic fragment of amplification, gB genetic fragment and gD genetic fragment are cloned into respectively on pGEM-T-Vector, again the genetic fragment after clone is inserted into respectively in pET-32a prokaryotic expression carrier, builds the recombinant expression carrier of pET-32a-gE, pET-32a-gD and pET-32a-gB;
3) recombinant expression carrier of the pET-32a-gE of structure, pET-32a-gD and pET-32a-gB is converted into respectively in e. coli bl21 (DE3), carries out abduction delivering;
4) expression product step 3) being made, after HisBind affinity chromatography purifying, obtains recombinant protein gE, gB and gD.
(2) method of the anti-pig IgG monoclonal antibody of colloid gold label mouse: getting respectively radius is that 40nm concentration is 0.01% collaurum 20mL and the anti-pig IgG monoclonal antibody 100 μ g of mouse, under the condition of pH8.5, vibrate and make its combination by magnetic agitation, add bovine serum albumin(BSA) (BSA) and PEG 20000 (PEG20000) as stabilizing agent, and making BSA final mass concentration is 0.1%, PEG20000 final mass concentration is 0.01%, adopt centrifuge method to remove the anti-pig IgG monoclonal antibody of unconjugated mouse and unstabilized colloid gold particle and agglutinator thereof, peony in centrifuge tube bottom is precipitated as the anti-pig IgG monoclonal antibody of collaurum-mouse compound.
(3) anti-the mouse of colloid gold label pig IgG monoclonal antibody is sprayed on polyester film: the PBS(0.01M with 20mL containing 1%BSA, pH value 7.4) the anti-pig IgG monoclonal antibody of difference washing colloids gold-mouse compound, the centrifugal supernatant of removing, obtain peony precipitation, precipitation after purifying is dissolved containing the PBS of 1%BSA with 2mL, be applied on polyester film freeze-drying with spraying equipment.
(4) nitrocellulose filter is coated: at nitrocellulose filter successively coated detection line T1, T2, T3 and control line C; The amount of the coated PRV gE albumen of detection line T1 is 0.5 μ g; The amount of the coated PRV gB albumen of detection line T2 is 0.5 μ g; The amount of the coated PRV gD albumen of detection line T3 is 0.5 μ g; The amount of the coated goat anti-mouse igg antibody of control line C is 1 μ g.Every live width 1～3mm.
(5) test paper assembling: use the polyethylene board that is stained with one deck Polyvinylchloride lining form as prop carrier, lay successively polyester film, nitrocellulose filter and the absorbent filter of glass fibre membrane, gold mark probe above, outside is cut into the wide test paper of 2～4mm and is made after sealing with adhesive tape.
Embodiment 2:
Except detection line T1 package amount is 5 μ g, detection line T2 package amount is 5 μ g, and detection line T3 package amount is 5 μ g, and control line C package amount is 10 μ g, and the anti-pig IgG monoclonal antibody of gold mark probe colloid gold label mouse labelled amount is every mL colloid gold label 20 μ g monoclonal antibodies, and gold mark probe package amount is 5 μ L, the method of colloid gold label porcine circovirus 2 type monoclonal antibody: getting respectively radius is that 10nm concentration is 0.01% collaurum 20mL and the anti-pig IgG monoclonal antibody 400 μ g of mouse, under the condition of pH9.0, vibrate and make its combination by magnetic agitation, add bovine serum albumin(BSA) (BSA) and PEG 20000 (PEG20000) as stabilizing agent, and making BSA final mass concentration is 2%, PEG20000 final mass concentration is 0.05%, adopt centrifuge method to remove the anti-pig IgG monoclonal antibody of unconjugated mouse and unstabilized colloid gold particle and agglutinator thereof, peony in centrifuge tube bottom is precipitated as the anti-pig IgG monoclonal antibody of collaurum-mouse compound, all the other are identical with embodiment 1.
Except detection line T1 package amount is 2 μ g, detection line T2 package amount is 2 μ g, and detection line T3 package amount is 2 μ g, and control line C package amount is 5 μ g, and the anti-pig IgG monoclonal antibody of gold mark probe colloid gold label mouse labelled amount is every mL colloid gold label 10 μ g monoclonal antibodies, and gold mark probe package amount is 8 μ L, the method of colloid gold label porcine circovirus 2 type monoclonal antibody: getting respectively radius is that 20nm concentration is 0.01% collaurum 20mL and the anti-pig IgG monoclonal antibody 200 μ g of mouse, under the condition of pH9.0, vibrate and make its combination by magnetic agitation, add bovine serum albumin(BSA) (BSA) and PEG 20000 (PEG20000) as stabilizing agent, and making BSA final mass concentration is 2%, PEG20000 final mass concentration is 0.05%, adopt centrifuge method to remove the anti-pig IgG monoclonal antibody of unconjugated mouse and unstabilized colloid gold particle and agglutinator thereof, peony in centrifuge tube bottom is precipitated as the anti-pig IgG monoclonal antibody of collaurum-mouse compound, all the other are identical with embodiment 1.
Embodiment 4
The using method of the colloidal gold immunochromatographydetection detection test paper of PRV gE, gB and gD antibody:
When detection, extract a small amount of blood serum sample of measured, with PBS(0.01M, pH 7.4) dilute one times, one end, absorption of sample district of this test paper is immersed in the sample of dilution to sentence read result after 5～10 minutes: control line C occurs red, represent that test paper is effective; Detection line T1, T2, T3 occur red, show respectively PRV gE, gB, gD antibody positive.Detection line T1 or T2 or T3 occur without color, show PRV gE or gB or gD negative antibody in serum.
The susceptibility of test paper of the present invention and specific test.
Sensitivity tests: according to the detection method described in embodiment 4, the detection paper PRV totivirus positive serum (positive serum 1) and the PRVgE disappearance positive serum (positive serum 2) that utilize embodiment 1,2,3 to make, and compare with indirect elisa method.PRV positive serum 1 and 2 is carried out after 1:100 dilution, carry out 2 times of doubling dilutions, the test paper of Application Example 1,2,3 preparations detects again, and detection limit is 1:3200 and 1:6400 respectively, application indirect elisa method detects and (is specially: coated gE proteantigen, detection positive serum 1; Coated gB proteantigen, detects that positive serum 2, two is anti-is anti-pig IgG ELIAS secondary antibody) detection limit of PRV positive serum 1 and 2 is 1:3200 and 1:6400 respectively.
Specific detection: according to the detection method described in embodiment 4, utilize the detection paper porcine pseudorabies gE deletion of vaccine immune serum that embodiment 1,2,3 makes, the pig PRV-gE antibody that pig PRV attacks malicious serum, purifying, the pig PRV-gB antibody of purifying, pig PRV-gD antibody, pig PCV2 positive serum, pig blue-ear disease poison Positive Sera, antibody against swine fever virus positive serum, pig parvoviral positive serum, pig encephalitis B virus positive serum and the pig PRV negative serum of purifying.The results are shown in Table 1.
The specific detection result of table 1. test paper of the present invention
Note: "+" represents positive; "-" represents negative.
Result shows, test paper of the present invention and PRV positive antibody serum have positive reaction, reacts negative with other swine diseases poison positive serums, and three detection lines have positive reaction for each albumen respectively, there is no cross reaction with other albumen.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Chopper Biology Co., Ltd.
Colloidal gold immunochromatographydetection detection test paper and the preparation method of <120> PRV gE, gB and gD antibody
<130> 1
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> gE upstream primer
<400> 1
atggattcat gctgagggag gcacccccg 29
<210> 2
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> gE downstream primer
<400> 2
cgaagcttta ataacggccg gtcgcccac 29
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> gB upstream primer
<400> 3
ggatccgcgc acgtgaacga catg 24
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> gB downstream primer
<400> 4
aagcctgagc gcgtgcagct ggtt 24
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> gD upstream primer
<400> 5
ggatccatgc tgctcgcagc gctat 25
<210> 6
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> gD downstream primer
<400> 6
aagcttgtca ggaatcgcat cacgt 25
Claims (6)
1. the colloidal gold immunochromatographydetection detection test paper of a PRV gE, gB and gD antibody, it is characterized in that: comprise absorption of sample district, gold mark probe region, immobilization antigen and antibody district, suction zones and back up pad, absorption of sample district, gold mark probe region, immobilization antigen-antibody district and suction zones are laid in back up pad and mutually partly overlap successively; Described gold mark probe region coated gold mark probe is the anti-pig IgG monoclonal antibody of mouse of colloid gold label; There are the detection line T2 of the detection line T1 of coated PRV gE albumen, coated PRV gB albumen, the coated detection line T3 of PRV gD albumen and the control line C of coated goat anti-mouse igg antibody in described immobilization antigen and antibody district.
2. the colloidal gold immunochromatographydetection detection test paper of PRV gE according to claim 1, gB and gD antibody, is characterized in that: the material of described back up pad is the polyethylene board that is stained with one deck Polyvinylchloride lining form; The material in described absorption of sample district is glass fibre membrane; The material of described gold mark probe region is polyester film; Described immobilization antigen and the material in antibody district are nitrocellulose filter; The material of described suction zones is absorbent filter.
3. the colloidal gold immunochromatographydetection detection test paper of PRV gE according to claim 1, gB and gD antibody, it is characterized in that: the labelled amount of described gold mark probe is the anti-pig IgG monoclonal antibody of every mL colloid gold label 5～20 μ g mouse, the package amount of gold mark probe is 5～10 μ L; The amount of the described coated PRV gE albumen of detection line T1 is 0.5～5 μ g, the amount of the coated PRV gB albumen of detection line T2 is 0.5～5 μ g, the amount of the coated PRV gD albumen of detection line T3 is 0.5～5 μ g, and the amount of the coated goat anti-mouse igg antibody of control line C is 1～10 μ g.
4. the preparation method of the colloidal gold immunochromatographydetection detection test paper described in claim 1-3 any one, is characterized in that comprising the steps: anti-the mouse of colloid gold label pig IgG monoclonal antibody is sprayed on polyester film; Coated detection line T1, T2, T3 and control line C on nitrocellulose filter; Glass fibre membrane, polyester film, nitrocellulose filter and absorbent filter are assembled into the colloidal gold immunochromatographydetection detection test paper that obtains PRV gE, gB and gD antibody in back up pad.
5. the preparation method of colloidal gold immunochromatographydetection detection test paper according to claim 4, is characterized in that comprising the steps:
(1) the anti-pig IgG monoclonal antibody of colloid gold label mouse: getting respectively radius is that 10～40nm concentration is 0.01% collaurum 20mL and anti-pig IgG monoclonal antibody 100～400 μ g of mouse, under the condition of pH value 8.5～9.2, vibrate and make its combination by magnetic agitation, add bovine serum albumin(BSA) and PEG 20000 as stabilizing agent, and making BSA final mass concentration is 0.1～5%, PEG20000 final mass concentration is 0.01～0.1%, adopt centrifuge method to remove the anti-pig IgG monoclonal antibody of unconjugated mouse and unstabilized colloid gold particle and agglutinator thereof, peony in centrifuge tube bottom is precipitated as the anti-pig IgG monoclonal antibody of collaurum-mouse compound,
(2) anti-the mouse of colloid gold label pig IgG monoclonal antibody is sprayed on polyester film: the PBS anti-pig IgG monoclonal antibody of the washing colloids gold-mouse compound respectively with 20mL containing 1%BSA, the centrifugal supernatant of removing, obtain peony precipitation, precipitation after purifying is dissolved containing the PBS of 1%BSA with 2mL, be applied on polyester film freeze-drying with spraying equipment;
(3) nitrocellulose filter is coated: at nitrocellulose filter coated detection line T1, T2, T3 and control line C; The amount of the coated PRV gE albumen of detection line T1 is 0.5～5 μ g; The amount of the coated PRV gB albumen of detection line T2 is 0.5～5 μ g; The amount of the coated PRV gD albumen of detection line T3 is 0.5～5 μ g; The amount of the coated goat anti-mouse igg antibody of control line C is 1～10 μ g; Every live width 1～3mm;
(4) test paper assembling: as prop carrier, lay successively polyester film, nitrocellulose filter and the absorbent filter of glass fibre membrane, gold mark probe with the polyethylene board that is stained with one deck Polyvinylchloride lining form above, outside is sealed and made with adhesive tape.
6. the preparation method of the colloidal gold immunochromatographydetection detection test paper described in claim 1-3 any one, it is characterized in that comprising the steps: by described in claim 1-3 any one one end, absorption of sample district immerse in blood serum sample to be checked, sentence read result after 5～10 minutes: control line C occurs red, represents that test paper is effective; Detection line T1, T2, T3 occur red, show respectively Pseudorabies virus gE, gB, gD antibody positive; Detection line T1, T2, T3 occur without color, and showing does not have corresponding Pseudorabies virus gE, gB, gD antibody or antibody amount very low in serum.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN104597255A (en) * | 2015-02-04 | 2015-05-06 | 华中农业大学 | Test paper card for detecting pseudorabies virus gE protein antibody in porcine serum as well as preparation method and application thereof |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4562147A (en) * | 1983-06-13 | 1985-12-31 | Regents Of The University Of Minnesota | Method and kit for diagnosis of pseudorabies |
| US5122447A (en) * | 1989-01-23 | 1992-06-16 | Iowa State University Research Foundation, Inc. | Method of detecting pseudorabies virus specific serum antibody by use of a universal diagnostic antigen |
| CN101216492A (en) * | 2008-01-10 | 2008-07-09 | 中国农业科学院哈尔滨兽医研究所 | Test strip for rapid detection of porcine pseudorabies antibody and preparation method thereof |
| CN102109520A (en) * | 2009-12-29 | 2011-06-29 | 北京库尔科技有限公司 | Herpes simplex virus I and II IgM antibody joint inspection kit and preparation method thereof |
| CN103308684A (en) * | 2012-03-14 | 2013-09-18 | 武汉中博生物股份有限公司 | ELISA (enzyme-linked immuno sorbent assay) detection kit of porcine pseudorabies virus IgM antibody |
| CN103399154A (en) * | 2013-07-29 | 2013-11-20 | 武汉中博生物股份有限公司 | Test paper for colloidal gold immunochromatograohic assay of IgG (immunoglobulin G) antibody of dog anti-rabies virus and preparation method of test paper |
-
2014
- 2014-03-12 CN CN201410089066.2A patent/CN103792373A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4562147A (en) * | 1983-06-13 | 1985-12-31 | Regents Of The University Of Minnesota | Method and kit for diagnosis of pseudorabies |
| US5122447A (en) * | 1989-01-23 | 1992-06-16 | Iowa State University Research Foundation, Inc. | Method of detecting pseudorabies virus specific serum antibody by use of a universal diagnostic antigen |
| CN101216492A (en) * | 2008-01-10 | 2008-07-09 | 中国农业科学院哈尔滨兽医研究所 | Test strip for rapid detection of porcine pseudorabies antibody and preparation method thereof |
| CN102109520A (en) * | 2009-12-29 | 2011-06-29 | 北京库尔科技有限公司 | Herpes simplex virus I and II IgM antibody joint inspection kit and preparation method thereof |
| CN103308684A (en) * | 2012-03-14 | 2013-09-18 | 武汉中博生物股份有限公司 | ELISA (enzyme-linked immuno sorbent assay) detection kit of porcine pseudorabies virus IgM antibody |
| CN103399154A (en) * | 2013-07-29 | 2013-11-20 | 武汉中博生物股份有限公司 | Test paper for colloidal gold immunochromatograohic assay of IgG (immunoglobulin G) antibody of dog anti-rabies virus and preparation method of test paper |
Non-Patent Citations (1)
| Title |
|---|
| YEW JOON TAM 等: "Development of a Solid-Based Paper Strip Assay for Rapid Diagnosis of Pseudorabies", 《TROP BIOMED》 * |
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