CN105486861B - A kind of Rabbit pest virus antibody rapid detection card and preparation method thereof - Google Patents

A kind of Rabbit pest virus antibody rapid detection card and preparation method thereof Download PDF

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CN105486861B
CN105486861B CN201510380893.1A CN201510380893A CN105486861B CN 105486861 B CN105486861 B CN 105486861B CN 201510380893 A CN201510380893 A CN 201510380893A CN 105486861 B CN105486861 B CN 105486861B
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rabbit
pestivirus
monoclonal antibodies
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antibody
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CN105486861A (en
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曲光刚
武玉香
沈志强
王玉茂
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Binzhou, Shandong Province animal and veterinary research institute
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Shandong Binzhou Animal Science & Veterinary Medicine Academy
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Abstract

The present invention relates to a kind of Rabbit pest virus antibody rapid detection card and preparation method thereof, concretely comprise the following steps：（1）The preparation of anti-rabbit pestivirus VP60 monoclonal antibodies, including the screening of immune programme for children, cell fusion, hybridoma and cloning, Antibody preparation and purifying；（2）The preparation of Rabbit pest virus antibody rapid detection card, including the boiling of colloidal gold solution, the processing of the mark of anti-rabbit pestivirus VP60 monoclonal antibodies, gold standard pad, sample pad processing, C, T lines are determined.The present invention can quick, sensitive, accurate detection Rabbit pest virus antibody, it is main with red cell hemagglutination to overcome domestic detection rabbit pest antibody, the problem of enzyme linked immunosorbent detection.

Description

A kind of Rabbit pest virus antibody rapid detection card and preparation method thereof
Technical field
The present invention relates to a kind of Rabbit pest virus antibody rapid detection card and preparation method thereof, belong to immunoassay technology neck Domain.
Background technology
Rabbit pest are a kind of acute, hot, septic and destructive infectious disease as caused by virus.It can send out throughout the year Raw, various rabbit are susceptible.3 young rabbit and adult rabbits morbidity and mortality the highest more than monthly age（Up to more than 95%）.
At present, the detection method of Rabbit pest virus antibody has Hemagglutination Method, RT-PCR methods, ELISA, wherein, RT-PCR Method, ELISA need large-scale instrument and professional, and Hemagglutination Method is simple to operate, but need human red blood cells, red blood cell valency Lattice are more expensive, should not preserve for a long time, this research and utilization immune colloid gold principle, develop a kind of Rabbit pest virus antibody rapid detection card Preparation method, is that quick, sensitive, accurate detection Rabbit pest virus antibody reduces time and cost.
The content of the invention
The invention provides a kind of Rabbit pest virus antibody rapid detection card and preparation method thereof, the present invention for it is quick, sensitive, Accurately detection Rabbit pest virus antibody reduces time and cost.Technical scheme is as follows：
A kind of preparation method of Rabbit pest virus antibody rapid detection card, is concretely comprised the following steps：
（1）The preparation of anti-rabbit pestivirus VP60 monoclonal antibodies
1. immune programme for children
By insect cell expression albumen VP60 immunogenes and 501 adjuvants by volume 1:0.5-1.5 ratio mixing, preferably For by volume 1:0.8-1.3 ratio mixing, 8-10 week old Balb/c mouse are immunized, leg muscle injection, 50 μ g/ Only, it was immunized once every two weeks later, immunizing dose, position are ibid；5 exempt from rear booster immunization, and abdominal cavity injection is not added with adjuvant, agent Amount is doubled.
2. cell fusion
By the myeloma cell intraperitoneal with it of the splenocyte in Balb/c Mice Bodies according to cell quantity 20：0.5-1.5 Ratio mixing, 800rpm centrifugation 5-8min, are preferably by the myeloma intraperitoneal with it of the splenocyte in Balb/c Mice Bodies Cell is according to cell quantity 20：0.8-1.3 ratio mixing, 800rpm centrifugation 6min, supernatant discarded, in addition in 30S 1mL50%PEG（W/W）, 1min is stood, the DMEM culture mediums of 1mL serum-frees are slowly added in first 1min, at second The DMEM culture mediums of 2mL serum-frees are slowly added in 1min, the DMEM cultures of 2mL serum-frees are slowly added in the 3rd 1min Base, is slowly added to the DMEM culture mediums of 5mL serum-frees in the 4th 1min, and 10mL is slowly added to without blood in the 5th 1min Clear DMEM culture mediums, 800rpm centrifugation 10min, abandon supernatant, with containing 20% calf serum（Purchased from Gibico companies, article No. H1565）Complete medium re-suspended cell, cell count be 3-6 × 105Individual/mL is laid on containing feeder cells（2-6×104Individual/ mL）Tissue Culture Plate, be placed in CO2In incubator.
3. hybridoma screening and cloning
When the 1/3 of cell length to bottom hole, cell supernatant, the positive hole of screening, by sun are determined using indirect competitive ELISA Property hole be subcloned using limiting dilution assay, the 6th day, take cell conditioned medium to detect, it is final to choose anti-rabbit pestivirus VP60 hybridization Tumor cell strain 2F4, by Cryopreservation of Hybridoma Cells in liquid nitrogen container, described hybridoma cell strain 2F4 was protected on June 11st, 2015 China typical culture collection center is hidden in, deposit number is CCTCC C201582, and preservation address is that Wuhan, China Wuhan is big Learn.
4. Antibody preparation and purifying
The production of monoclonal antibody：Method is induced using ascites in animal body；
The purifying of monoclonal antibody：Ascites is purified using SPA post methods, anti-rabbit pest VP60 monoclonals of the present invention are produced Antibody.
（2）The preparation of Rabbit pest virus antibody rapid detection card
1. colloidal gold solution is boiled
Present invention detection card is to prepare collaurum using trisodium citrate reduction method, and method is the ultra-pure water dress for measuring 99mL Enter 250 mL round-bottomed flask, be put into stirrer, be placed on magnetic stirring apparatus, add the mL of 1% chlorauric acid solution 1, appropriate speed Stirring, opens heater switch, disposably adds the citric acid three sodium solutions of 1.6 mL 1% after rapid after solution boiling, gold chloride is molten Liquid to be gradually become continue after claret, colour stable by grey heats 10 min, after after solution cooling, with filtering with microporous membrane, 4 DEG C save backup, UV scanning obtains the colloid gold particle that maximum absorption band is 523nm.
2. the mark of anti-rabbit pestivirus VP60 monoclonal antibodies
With 0.l mol/L K2CO3It is 9.0 to adjust collaurum pH value.Every 1 mL colloidal gold solutions add 6 μ g anti-rabbit seasonal febrile diseases Malicious VP60 monoclonal antibodies, at room temperature 120rpm shake 30 min, add the μ L of 10% bovine serum albumin(BSA) 20,120rpm shakes at room temperature 30 min, 12000rpm centrifuge 20 min, abandon supernatant, are dissolved and precipitated with 0.01MPBS, that is, obtain the anti-rabbit pestivirus marked VP60 monoclonal antibodies.
3. gold standard pad is handled
Selection polyester fiber 6613 is used as gold-marking binding pad.Gold standard pad is immersed in 5min in treatment fluid A, treatment fluid A is 0.01Mpbs, pH7.4,0.2%Triton X-100, take out 37 DEG C of drying, standby.
4. sample pad is handled
Selection DL42 is sample pad, and sample pad is immersed in into 5min in treatment fluid B, and treatment fluid B is 0.01 Mpbs, PH7.4,1%Triton X-100,1%BSA+0.05% NaN3）, 5min takes out 37 DEG C of drying, standby.
5. C, T lines are determined
The films of Sartorius Cn 140 are selected, Rabbit pest virus VP60 albumen and sheep anti mouse secondary antibody are regard as T lines and C lines respectively Draw on NC films, concentration is respectively 1.3mg/mL and 0.6mg/mL.
Present invention additionally comprises the Rabbit pest virus antibody rapid detection card prepared by the above method.
The present invention has advantages below compared with prior art：
The present invention quick, sensitive, accurate can detect Rabbit pest virus antibody, overcome domestic detection rabbit pest antibody main With red cell hemagglutination, the problem of enzyme linked immunosorbent detection.
Brief description of the drawings
Fig. 1 is Rabbit pest virus antibody rapid detection card structural representation of the present invention；
Fig. 2-Fig. 4 is Rabbit pest virus antibody rapid detection card result judgement figure of the present invention.
Symbol description：
1. sample pad, 2.PVC plates, 3. gold pads, 4. detection lines, 5. nature controlling lines, 6.NC films, 7. adsorptive pads.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and It is apparent.But embodiment is only exemplary, does not constitute any limitation to the scope of the present invention.Those skilled in the art should It should be appreciated that, the details and form of technical solution of the present invention can be repaiied without departing from the spirit and scope of the invention Change or replace, but these modifications and replacement are each fallen within protection scope of the present invention.
A kind of Rabbit pest virus antibody rapid detection card preparation method of embodiment 1
（1）The preparation of anti-rabbit pestivirus VP60 monoclonal antibodies
1. immune programme for children
By insect cell expression albumen VP60 immunogenes（Shandong Province Binzhou animal and veterinary research institute）With 501 adjuvants（Shandong Green City bio tech ltd, article No. LD003）By volume 1：1 ratio mixing, to 8-10 week old Balb/c mouse（Purchase From Beijing Vital River Experimental Animals Technology Co., Ltd.）It is immunized, leg muscle injection, 50 μ g/, later every two weeks It is immunized once, immunizing dose, position are ibid；5 exempt from rear booster immunization, and abdominal cavity injection is not added with adjuvant, dosage is doubled；
2. cell fusion
By the myeloma cell intraperitoneal with it of the splenocyte in Balb/c Mice Bodies according to cell quantity 20：1 ratio Mixing, 800rpm centrifugation 7min, supernatant discarded, in adding 1mL50%PEG in 30S（W/W）, 1min is stood, in first 1min Inside it is slowly added to the DMEM culture mediums of 1mL serum-frees（Purchased from Gibico companies, article No. H390）, slowly add in second 1min Enter the DMEM culture mediums of 2mL serum-frees, the DMEM culture mediums of 2mL serum-frees are slowly added in the 3rd 1min, at the 4th The DMEM culture mediums of 5mL serum-frees are slowly added in 1min, the DMEM trainings of 10mL serum-frees are slowly added in the 5th 1min Base is supported, 800rpm centrifugation 10min abandon supernatant, with containing 20% calf serum（Purchased from Gibico companies, article No. H1565）Complete training Base re-suspended cell is supported, cell count is 3-6 × 105Individual/mL is laid on containing feeder cells（2-6×104Individual/mL）Tissue Culture Plate, It is placed in CO2In incubator.
3. hybridoma screening and cloning
When the 1/3 of cell length to bottom hole, cell supernatant, the positive hole of screening, by sun are determined using indirect competitive ELISA Property hole be subcloned using limiting dilution assay, the 6th day, take cell conditioned medium to detect, it is final to choose anti-rabbit pestivirus VP60 hybridization Tumor cell strain 2F4, by Cryopreservation of Hybridoma Cells in liquid nitrogen container, described hybridoma cell strain 2F4 was protected on June 11st, 2015 China typical culture collection center is hidden in, deposit number is CCTCC C201582, and preservation address is that Wuhan, China Wuhan is big Learn.
4. Antibody preparation and purifying
The production of monoclonal antibody：Method is induced using ascites in animal body；
The purifying of monoclonal antibody：Using SPA posts（Purchased from Hangzhou Niu Long bio tech ltd）Method is carried out to ascites Purifying, produces anti-rabbit pest VP60 monoclonal antibodies of the present invention.
（2）The preparation of Rabbit pest virus antibody rapid detection card
1. colloidal gold solution is boiled
The ultra-pure water for measuring 99mL loads 250mL round-bottomed flask, is put into stirrer, is placed on magnetic stirring apparatus, adds 1% gold chloride（Shanghai one Bioisystech Co., Ltd of outstanding person, article No. JY-SJ111）Heating is opened in the mL of solution 1, appropriate speed stirring Switch, after the rapid disposable addition citric acid three sodium solutions of 1.6 mL 1% after solution boiling, chlorauric acid solution is gradually become by grey Continue to heat 10 min for claret, after colour stable, after after solution cooling, with filtering with microporous membrane, 4 DEG C save backup, purple Outer scanning obtains the colloid gold particle that maximum absorption band is 523nm.
2. the mark of anti-rabbit pestivirus VP60 monoclonal antibodies
With 0.l mol/L K2CO3It is 9.0 to adjust collaurum pH value.Every 1 mL colloidal gold solutions add 6 μ g anti-rabbit seasonal febrile diseases Malicious VP60 monoclonal antibodies (Shandong Lvdu Bio Sicience ＆ Technology Co., Ltd., article No. LDANTI-VP60), 120rpm shakes 30 at room temperature Min, adds the μ L of 10% bovine serum albumin(BSA) 20, and 120rpm shakes 30 min at room temperature, and 12000rpm centrifuges 20 min, abandons supernatant, Dissolved and precipitated with 0.01MPBS, that is, obtain the anti-rabbit pestivirus VP60 monoclonal antibodies marked.
3. gold standard pad is handled
Select polyester fiber 6613（Shanghai Jie Ning biotinylated biomolecules Science and Technology Ltd., article No. polyester fiber 6613）As Gold-marking binding pad.Gold standard pad is immersed in treatment fluid A（0.01Mpbs, pH7.4,0.2%Triton X-100）5min, takes out 37 DEG C of drying, it is standby.
4. sample pad is handled
Select DL42（Shanghai Jinbiao Bio-Tech Co., Ltd., article No. DL42）For sample pad, sample pad is immersed in Treatment fluid B（0.01 Mpbs pH7.4,1%Triton X-100,1%BSA, 0.05% NaN3）, 5min, 37 DEG C of drying of taking-up, It is standby.
5. C, T lines are determined
Select the films of Sartorius Cn 140（Sartorius AG, article No. Cn140）, respectively by Rabbit pest virus VP60 albumen (Shandong Lvdu Bio Sicience ＆ Technology Co., Ltd., article No. LDVP60) and sheep anti mouse secondary antibody（Shandong Green City biotechnology has Limit company, article No. LDks001）Drawn as T lines and C lines on NC films, concentration is respectively 1.3mg/mL and 0.6mg/mL.
A kind of Rabbit pest virus antibody rapid detection card preparation method of embodiment 2
（1）The preparation of anti-rabbit pestivirus VP60 monoclonal antibodies
1. immune programme for children
By insect cell expression albumen VP60 immunogenes（By Shandong Province Binzhou, animal and veterinary research institute gives）With 501 adjuvants （Shandong Lvdu Bio Sicience ＆ Technology Co., Ltd., article No. LD003）By volume 1:0.6 ratio mixing, to 8-10 week old Balb/c Mouse（Purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.）It is immunized, leg muscle injection, 50 μ g/, after It was immunized once every two weeks, immunizing dose, position are ibid；5 exempt from rear booster immunization, and abdominal cavity injection is not added with adjuvant, dosage is doubled.
2. cell fusion
By the myeloma cell intraperitoneal with it of the splenocyte in Balb/c Mice Bodies according to cell quantity 20:0.7 ratio Example mixing, 800rpm centrifugation 8min, supernatant discarded, in adding 1mL50%PEG in 30S（W/W）, 1min is stood, at first The DMEM culture mediums of 1mL serum-frees are slowly added in 1min（Purchased from Gibico companies, article No. H390）, delay in second 1min The slow DMEM culture mediums for adding 2mL serum-frees, are slowly added to the DMEM culture mediums of 2mL serum-frees in the 3rd 1min, the The DMEM culture mediums of 5mL serum-frees are slowly added in four 1min, 10mL serum-frees are slowly added in the 5th 1min DMEM culture mediums, 800rpm centrifugation 10min, abandon supernatant, with containing 20% calf serum（Purchased from Gibico companies, article No. H1565）'s Complete medium re-suspended cell, cell count is 3-6 × 105Individual/mL is laid on containing feeder cells（2-6×104Individual/mL）Cell Culture plate, is placed in CO2In incubator.
3. hybridoma screening and cloning
When the 1/3 of cell length to bottom hole, cell supernatant, the positive hole of screening, by sun are determined using indirect competitive ELISA Property hole be subcloned using limiting dilution assay, the 6th day, take cell conditioned medium to detect, it is final to choose anti-rabbit pestivirus VP60 hybridization Tumor cell strain 2F4, by Cryopreservation of Hybridoma Cells in liquid nitrogen container, described hybridoma cell strain 2F4 was protected on June 11st, 2015 China typical culture collection center is hidden in, deposit number is CCTCC C201582, and preservation address is that Wuhan, China Wuhan is big Learn.
4. Antibody preparation and purifying
The production of monoclonal antibody：Method is induced using ascites in animal body；
The purifying of monoclonal antibody：Using SPA posts（Purchased from Hangzhou Niu Long bio tech ltd）Method is carried out to ascites Purifying, produces anti-rabbit pest VP60 monoclonal antibodies of the present invention.
（2）The preparation of Rabbit pest virus antibody rapid detection card
1. colloidal gold solution is boiled
Method is the round-bottomed flask for the ultra-pure water loading 250mL for measuring 99mL, is put into stirrer, is placed in magnetic stirring apparatus On, add 1% gold chloride（Shanghai one Bioisystech Co., Ltd of outstanding person, article No. JY-SJ111）The mL of solution 1, appropriate speed stirring, Heater switch is opened, after the rapid disposable addition citric acid three sodium solutions of 1.6mL 1% after solution boiling, chlorauric acid solution is by ash Color, which to gradually become continue after claret, colour stable, heats 10 min, after after solution cooling, with filtering with microporous membrane, 4 DEG C of preservations Standby, UV scanning obtains the colloid gold particle that maximum absorption band is 523nm.
2. the mark of anti-rabbit pestivirus VP60 monoclonal antibodies
With 0.l mol/L K2CO3It is 9.0 to adjust collaurum pH value.6 μ g anti-rabbit seasonal febrile diseases are added per 1mL colloidal gold solutions Malicious VP60 monoclonal antibodies (Shandong Lvdu Bio Sicience ＆ Technology Co., Ltd., article No. LDANTI-VP60), 120rpm shakes 30 at room temperature Min, adds the μ L of 10% bovine serum albumin(BSA) 20, and 120rpm shakes 30 min at room temperature, and 12000rpm centrifuges 20 min, abandons supernatant, Dissolved and precipitated with 0.01MPBS, that is, obtain the anti-rabbit pestivirus VP60 monoclonal antibodies marked.
3. gold standard pad is handled
Select polyester fiber 6613（Shanghai Jie Ning biotinylated biomolecules Science and Technology Ltd., article No. polyester fiber 6613）As Gold-marking binding pad.Gold standard pad is immersed in treatment fluid A（0.01Mpbs, pH7.4,0.2%Triton X-100）5min, takes out 37 DEG C of drying, it is standby.
4. sample pad is handled
Select DL42（Shanghai Jinbiao Bio-Tech Co., Ltd., article No. DL42）For sample pad, sample pad is immersed in Treatment fluid B（0.01 Mpbs pH7.4,1%Triton X-100,1%BSA, 0.05% NaN3）, 5min, 37 DEG C of bakings of taking-up It is dry, it is standby.
5. C, T lines are determined
Select the films of Sartorius Cn 140（Sartorius AG, article No. Cn140）, respectively by Rabbit pest virus VP60 Albumen (Shandong Lvdu Bio Sicience ＆ Technology Co., Ltd., article No. LDVP60) and sheep anti mouse secondary antibody（The limited public affairs of Shandong Green City biotechnology Department, article No. LDks001）Drawn as T lines and C lines on NC films, concentration is respectively 1.3mg/mL and 0.6mg/mL.
A kind of Rabbit pest virus antibody rapid detection card preparation method of embodiment 3
（1）The preparation of anti-rabbit pestivirus VP60 monoclonal antibodies
1. immune programme for children
By insect cell expression albumen VP60 immunogenes（By Shandong Province Binzhou, animal and veterinary research institute gives）With 501 adjuvants （Shandong Lvdu Bio Sicience ＆ Technology Co., Ltd., article No. LD003）By volume 1：1.3 ratio mixing, to 8-10 week old Balb/c Mouse（Purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.）It is immunized, leg muscle injection, 50 μ g/, after It was immunized once every two weeks, immunizing dose, position are ibid；5 exempt from rear booster immunization, and abdominal cavity injection is not added with adjuvant, dosage is doubled；
2. cell fusion
By the myeloma cell intraperitoneal with it of the splenocyte in Balb/c Mice Bodies according to cell quantity 20：1.3 ratio Example mixing, 800rpm centrifugation 8min, supernatant discarded, in adding 1mL50%PEG in 30S（W/W）, 1min is stood, at first The DMEM culture mediums of 1mL serum-frees are slowly added in 1min（Purchased from Gibico companies, article No. H390）, delay in second 1min The slow DMEM culture mediums for adding 2mL serum-frees, are slowly added to the DMEM culture mediums of 2mL serum-frees in the 3rd 1min, the The DMEM culture mediums of 5mL serum-frees are slowly added in four 1min, 10mL serum-frees are slowly added in the 5th 1min DMEM culture mediums, 800rpm centrifugation 10min, abandon supernatant, with containing 20% calf serum（Purchased from Gibico companies, article No. H1565）'s Complete medium re-suspended cell, cell count is 3-6 × 105Individual/mL is laid on containing feeder cells（2-6×104Individual/mL）Cell Culture plate, is placed in CO2In incubator.
3. hybridoma screening and cloning
When the 1/3 of cell length to bottom hole, cell supernatant, the positive hole of screening, by sun are determined using indirect competitive ELISA Property hole be subcloned using limiting dilution assay, the 6th day, take cell conditioned medium to detect, it is final to choose anti-rabbit pestivirus VP60 hybridization Tumor cell strain 2F4, by Cryopreservation of Hybridoma Cells in liquid nitrogen container, described hybridoma cell strain 2F4 was protected on June 11st, 2015 China typical culture collection center is hidden in, deposit number is CCTCC C201582, and preservation address is that Wuhan, China Wuhan is big Learn.
4. Antibody preparation and purifying
The production of monoclonal antibody：Method is induced using ascites in animal body；
The purifying of monoclonal antibody：Using SPA posts（Purchased from Hangzhou Niu Long bio tech ltd）Method is carried out to ascites Purifying, produces anti-rabbit pest VP60 monoclonal antibodies of the present invention.
（2）The preparation of Rabbit pest virus antibody rapid detection card
1. colloidal gold solution is boiled
The ultra-pure water for measuring 99mL loads 250mL round-bottomed flask, is put into stirrer, is placed on magnetic stirring apparatus, adds 1% gold chloride（Shanghai one Bioisystech Co., Ltd of outstanding person, article No. JY-SJ111）Heating is opened in the mL of solution 1, appropriate speed stirring Switch, after the rapid disposable addition citric acid three sodium solutions of 1.6 mL 1% after solution boiling, chlorauric acid solution is gradually become by grey Continue to heat 10 min for claret, after colour stable, after after solution cooling, with filtering with microporous membrane, 4 DEG C save backup, purple Outer scanning obtains the colloid gold particle that maximum absorption band is 523nm.
2. the mark of anti-rabbit pestivirus VP60 monoclonal antibodies
With 0.l mol/L K2CO3 regulation collaurum pH value is 9.0.Every 1 mL colloidal gold solutions add 6 μ g anti-rabbit pest Viral VP60 monoclonal antibodies (Shandong Lvdu Bio Sicience ＆ Technology Co., Ltd., article No. LDANTI-VP60), 120rpm shakes 30 at room temperature Min, adds the μ L of 10% bovine serum albumin(BSA) 2, and 120rpm shakes 30 min at room temperature, and 12000rpm centrifuges 20 min, abandons supernatant, uses 0.01MPBS dissolving precipitations, that is, obtain the anti-rabbit pestivirus VP60 monoclonal antibodies marked.
3. gold standard pad is handled
Select polyester fiber 6613（Shanghai Jie Ning biotinylated biomolecules Science and Technology Ltd., article No. polyester fiber 6613）As Gold-marking binding pad.Gold standard pad is immersed in treatment fluid A（0.01Mpbs, pH7.4,0.2%Triton X-100）5min, takes out 37 DEG C of drying, it is standby.
4. sample pad is handled
Select DL42（Shanghai Jinbiao Bio-Tech Co., Ltd., article No. DL42）For sample pad, sample pad is immersed in Treatment fluid B（0.01 Mpbs pH7.4,1%Triton X-100,1%BSA, 0.05% NaN3）, 5min, 37 DEG C of bakings of taking-up It is dry, it is standby.
5. C, T lines are determined
Select the films of Sartorius Cn 140（Sartorius AG, article No. Cn140）, respectively by Rabbit pest virus VP60 albumen (Shandong Lvdu Bio Sicience ＆ Technology Co., Ltd., article No. LDVP60) and sheep anti mouse secondary antibody（Shandong Green City biotechnology has Limit company, article No. LDks001）Drawn as T lines and C lines on NC films, concentration is respectively 1.3mg/mL and 0.6mg/mL.
The use of the Rabbit pest virus antibody rapid detection card of the present invention of test example 1
The structure of present invention detection card is as shown in figure 1, when using the present invention, gathering whole blood 0.1ml, being dripped to suction pipe In sample well in detection card, judged result at 5-10 minutes.
Result judgement such as Fig. 2,3 and 4.
（1）It is positive（Fig. 3）：If C lines develop the color, and T lines do not develop the color, and are judged to the positive, and having reached can prevent strong virus attack to resist Body level.
（2）It is negative（Fig. 2）：If C lines develop the color, T lines develop the color simultaneously, are judged to negative or antibody level low.
（3）It is invalid（Fig. 4）：In peep hole, if C lines do not develop the color, result is invalid, it is proposed that retest.
The present invention of test example 2 detection card sensitiveness and accuracy test
100 parts of rabbit anteserums for injecting rabbit pestilence seedling and 100 parts of rabbit anteserums for not injecting rabbit pestilence seedling are extracted, point Do not carried out detection with blood coagulation tests and this detection card and checked.As a result show, both sensitiveness coincidence rates are 98%, accuracy is 99%。

Claims (4)

1. a kind of preparation method of Rabbit pest virus antibody rapid detection card, is concretely comprised the following steps：
（1）The preparation of anti-rabbit pestivirus VP60 monoclonal antibodies
Method production monoclonal antibody is induced using ascites in animal body, ascites is purified using SPA post methods, institute is prepared Anti-rabbit pestivirus VP60 monoclonal antibodies are stated, the hybridoma cell strain for producing the anti-rabbit pestivirus VP60 monoclonal antibodies 2F4 is preserved in China typical culture collection center on June 11st, 2015, and deposit number is CCTCC C201582, preservation Location is Wuhan, China-Wuhan University；
（2）The preparation of Rabbit pest virus antibody rapid detection card
1. colloidal gold solution is boiled：Collaurum is prepared using trisodium citrate reduction method；
2. the mark of anti-rabbit pestivirus VP60 monoclonal antibodies：With 0.l mol/L K2CO3It is 9.0 to adjust collaurum pH value, often 1 mL colloidal gold solutions add anti-rabbit pestivirus VP60 monoclonal antibodies described in 6 μ g, and 120rpm shakes 30 min at room temperature, add The μ L of 10% bovine serum albumin(BSA) 20, at room temperature 120rpm shake 30 min, 12000rpm centrifuges 20 min, abandons supernatant, uses 0.01M PBS dissolving precipitations, that is, obtain the anti-rabbit pestivirus VP60 monoclonal antibodies marked；
3. gold-marking binding pad is handled：Select polyester fiber 6613 as gold-marking binding pad, gold-marking binding pad is immersed in treatment fluid A Middle 5min, treatment fluid A are 0.01M PBS, pH7.4,0.2%Triton X-100, take out 37 DEG C of drying, standby；
4. sample pad is handled：Selection DL42 is sample pad, and sample pad is immersed in into 5min in treatment fluid B, and treatment fluid B is 0.01 M PBS, pH7.4,1%Triton X-100,1%BSA, 0.05% NaN3, 5min takes out 37 DEG C of drying, standby；
5. C, T lines are determined：Select Sartorius Cn140 films, respectively using Rabbit pest virus VP60 albumen and sheep anti mouse secondary antibody as T lines and C lines are drawn on NC films, and concentration is respectively 1.3mg/mL and 0.6mg/mL.
2. according to the method described in claim 1, it is characterised in that the step（2）Middle use trisodium citrate reduction method system For concretely comprising the following steps for collaurum：The ultra-pure water for measuring 99 mL loads 250 mL round-bottomed flask, is put into stirrer, is placed in magnetic On power agitator, the mL of 1% chlorauric acid solution 1 is added, appropriate speed stirring opens heater switch, after rapid one after solution boiling Secondary property adds the citric acid three sodium solutions of 1.6 mL 1%, and chlorauric acid solution gradually become by grey and continue after claret, colour stable 10 min are heated, after after solution cooling, with filtering with microporous membrane, 4 DEG C save backup, and UV scanning obtains maximum absorption band and is 523nm colloid gold particle.
3. a kind of Rabbit pest virus antibody rapid detection card, it is characterised in that it is the method as any one of claim 1-2 Prepare.
4. the hybridoma cell strain 2F4 for producing anti-rabbit pestivirus VP60 monoclonal antibodies, it is characterised in that the hybridoma Cell line 2F4 is preserved in China typical culture collection center on June 11st, 2015, and deposit number is CCTCC C201582, Preservation address is Wuhan, China-Wuhan University.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0704528A2 (en) * 1994-07-08 1996-04-03 Cyanamid Iberica, Sa Rabbit hemorrhagic disease virus (RHDV) recombinant capsids and proteins, diagnostic kits and vaccines containing them
WO2007008092A2 (en) * 2005-07-08 2007-01-18 Panstwowy Instytut Weterynaryjny - Panstwowy Instytut Badawczy W Pulawach Diagnostic kit for the examination of animal sera for the presence of anti-rhd antibodies and a diagnostic kit for detecting the rhd virus
CN101520459A (en) * 2009-04-08 2009-09-02 江苏省农业科学院 Rabbit hemorrhagic disease virus colloidal gold test strip
CN101533016A (en) * 2009-04-15 2009-09-16 江苏省农业科学院 Indirect ELISA reagent kit for detecting antibodies against rabbit hemorrhagic disease virus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0704528A2 (en) * 1994-07-08 1996-04-03 Cyanamid Iberica, Sa Rabbit hemorrhagic disease virus (RHDV) recombinant capsids and proteins, diagnostic kits and vaccines containing them
WO2007008092A2 (en) * 2005-07-08 2007-01-18 Panstwowy Instytut Weterynaryjny - Panstwowy Instytut Badawczy W Pulawach Diagnostic kit for the examination of animal sera for the presence of anti-rhd antibodies and a diagnostic kit for detecting the rhd virus
CN101520459A (en) * 2009-04-08 2009-09-02 江苏省农业科学院 Rabbit hemorrhagic disease virus colloidal gold test strip
CN101533016A (en) * 2009-04-15 2009-09-16 江苏省农业科学院 Indirect ELISA reagent kit for detecting antibodies against rabbit hemorrhagic disease virus

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
兔出血症病毒胶体金免疫层析试纸条诊断方法的建立及初步应用;蔡少平 等;《畜牧兽医学报》;20121231;第43卷(第11期);1795-1801 *
兔瘟病毒VP60蛋白原核表达及其应用;马力 等;《家畜生态学报》;20130531;第35卷(第5期);67-70 *

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