CN105486861A - Rabbit pestivirus antibody rapid detection card and preparation method thereof - Google Patents

Rabbit pestivirus antibody rapid detection card and preparation method thereof Download PDF

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CN105486861A
CN105486861A CN201510380893.1A CN201510380893A CN105486861A CN 105486861 A CN105486861 A CN 105486861A CN 201510380893 A CN201510380893 A CN 201510380893A CN 105486861 A CN105486861 A CN 105486861A
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rabbit
pestivirus
cell
antibody
serum
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CN105486861B (en
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武玉香
沈志强
曲光刚
唐世云
孟卫芹
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Binzhou, Shandong Province animal and veterinary research institute
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Shandong Lvdu Bio Sicience & Technology Co Ltd
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses

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Abstract

The invention relates to a rabbit pestivirus antibody rapid detection card and a preparation method thereof. The preparation method comprises the following concrete steps: (1) preparing an anti-rabbit-pestivirus VP60 monoclonal antibody, concretely performing immunization program, cell fusion, hybridomas screening and cloning, and antibody preparation and purification; and (2) preparing the rabbit pestivirus antibody rapid detection card, concretely decocting a colloidal gold solution, marking the anti-rabbit-pestivirus VP60 monoclonal antibody, processing a colloidal-gold pad, processing a sample pad, and determining C and T lines. The rabbit pestivirus antibody rapid detection card is capable of rapidly, sensitively and accurately detecting rabbit pestivirus antibody, and overcomes the problems that domestic rabbit pestivirus antibody detection mainly employs erythrocyte hemagglutination and ELISA 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 technical field of immunoassay.
Background technology
Rabbit pest are that the one caused by virus is acute, hot, septic and destructive infectious disease.All can occur throughout the year, the equal susceptible of various rabbit.Young rabbits more than 3 monthly ages and adult rabbits M & M the highest (can up to more than 95%).
At present, the detection method of Rabbit pest virus antibody has Hemagglutination Method, RT-PCR method, euzymelinked immunosorbent assay (ELISA), wherein, RT-PCR method, euzymelinked immunosorbent assay (ELISA) need large-scale instrument and professional, and Hemagglutination Method is simple to operate, but need human red blood cell, red blood cell price is 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, for quick, sensitive, detect Rabbit pest virus antibody accurately and decrease time and cost.
Summary of the invention
The invention provides a kind of Rabbit pest virus antibody rapid detection card and preparation method thereof, the present invention be quick, sensitive, detect Rabbit pest virus antibody accurately and decrease time and cost.Technical scheme of the present invention is as follows:
A preparation method for Rabbit pest virus antibody rapid detection card, concrete steps are:
(1) preparation of anti-rabbit pestivirus VP60 monoclonal antibody
1. immune programme for children
The ratio of insect cell expression albumen VP60 immunogene and 501 adjuvants 1:0.5-1.5 is by volume mixed, be preferably the ratio mixing of 1:0.8-1.3 by volume, immunity is carried out to 8-10 Balb/c mouse in age in week, leg muscle is injected, 50 μ g/ only, later every immunity in two weeks once, immunizing dose, position are the same; 5 exempt from rear booster immunization, lumbar injection, and do not add adjuvant, dosage doubles.
2. Fusion of Cells
Intraabdominal to splenocyte in Balb/c Mice Body and its myeloma cell is mixed according to the ratio of cell quantity 20:0.5-1.5, the centrifugal 5-8min of 800rpm, preferably Intraabdominal to the splenocyte in Balb/c Mice Body and its myeloma cell is mixed according to the ratio of cell quantity 20:0.8-1.3, the centrifugal 6min of 800rpm, supernatant discarded, 1mL50%PEG(W/W is added) in 30S, leave standstill 1min, the DMEM nutrient culture media of 1mL serum-free is slowly added in first 1min, the DMEM nutrient culture media of 2mL serum-free is slowly added in second 1min, the DMEM nutrient culture media of 2mL serum-free is slowly added in the 3rd 1min, the DMEM nutrient culture media of 5mL serum-free is slowly added in the 4th 1min, the DMEM nutrient culture media of 10mL serum-free is slowly added in the 5th 1min, the centrifugal 10min of 800rpm, abandon supernatant, with containing 20% calf serum (purchased from Gibico company, article No. H1565) complete medium re-suspended cell, cell count is 3-6 × 10 5individual/mL is laid on containing feeder cells (2-6 × 10 4individual/mL) Tissue Culture Plate, be placed in CO 2in incubator.
3. hybridoma screening and cloning
When cell grows at the bottom of hole 1/3, indirect competitive ELISA is adopted to measure cell conditioned medium liquid, screen positive hole, limiting dilution assay is adopted to carry out subclone in positive hole, 6th day, get cell conditioned medium to detect, finally choose anti-rabbit pestivirus VP60 hybridoma cell strain 2F4, by Cryopreservation of Hybridoma Cells in liquid nitrogen container, described hybridoma cell strain 2F4 is preserved in China typical culture collection center on June 11st, 2015, deposit number is CCTCCC201582, and preservation address is Wuhan, China Wuhan University.
4. antibody preparation and purifying
The production of monoclonal antibody: adopt ascites in animal body to induce method;
The purifying of monoclonal antibody: adopt SPA post method to carry out purifying to ascites, obtain anti-rabbit pest VP60 monoclonal antibody of the present invention.
(2) preparation of Rabbit pest virus antibody rapid detection card
1. the boiling of colloidal gold solution
Test card of the present invention adopts trisodium citrate reduction method to prepare collaurum, method is the round-bottomed flask of the ultrapure water loading 250mL measuring 99mL, put into stirrer, be placed on magnetic stirring apparatus, add 1% chlorauric acid solution 1mL, suitable speed stirs, open heater switch, disposablely rapidly 1.6mL1% citric acid three sodium solution is added after solution boiling, chlorauric acid solution gradually becomes claret by grey, heating 10min is continued after colour stable, 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 antibody
With the K of 0.lmol/L 2cO 3collaurum pH value is regulated to be 9.0.Every 1mL colloidal gold solution adds 6 μ g anti-rabbit pestivirus VP60 monoclonal antibodies, under room temperature, 120rpm shakes 30min, add 10% bovine serum albumin(BSA) 20 μ L, under room temperature, 120rpm shakes 30min, the centrifugal 20min of 12000rpm, abandon supernatant, use 0.01MPBS dissolution precipitation, namely obtain the anti-rabbit pestivirus VP60 monoclonal antibody marked.
3. gold mark pad process
Select polyester fiber 6613 as gold-marking binding pad.Gold is marked pad and be immersed in 5min in treating fluid A, treating fluid A is 0.01Mpbs, pH7.4,0.2%TritonX-100, takes out 37 DEG C of oven dry, for subsequent use.
4. sample pad process
Selection DL42 is sample pad, sample pad is immersed in 5min in treating fluid B, and treating fluid B is 0.01Mpbs, pH7.4,1%TritonX-100,1%BSA+0.05%NaN 3), 5min, takes out 37 DEG C of oven dry, for subsequent use.
5. C, T line is determined
Select SartoriusCn140 film, respectively using Rabbit pest virus VP60 albumen with sheep anti mouse two is anti-draws on NC film as T line and C line, concentration is respectively 1.3mg/mL and 0.6mg/mL.
The present invention also comprises the Rabbit pest virus antibody rapid detection card prepared by said method.
The present invention compared with prior art has the following advantages:
The present invention can quick, sensitive, detect Rabbit pest virus antibody accurately, overcome domestic detection rabbit pest antibody mainly with red cell hemagglutination, the problem of enzyme linked immunosorbent detection.
Accompanying drawing explanation
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 process decision chart of the present invention.
Symbol description:
1. sample pad, 2.PVC plate, 3. gold pad, 4. detection line, 5. nature controlling line, 6.NC film, 7. adsorptive pads.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But embodiment is only exemplary, does not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1 one kinds of Rabbit pest virus antibody rapid detection card preparation methods
(1) preparation of anti-rabbit pestivirus VP60 monoclonal antibody
1. immune programme for children
By insect cell expression albumen VP60 immunogene (Binzhou, Shandong Province animal and veterinary research institute) and 501 adjuvant (Shandong Lvdu Bio Sicience & Technology Co., Ltd., article No. LD003) by volume 1:1 ratio mixing, immunity is carried out to 8-10 Balb/c mouse in age in week (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.), leg muscle is injected, 50 μ g/ only, later every immunity in two weeks once, immunizing dose, position are the same; 5 exempt from rear booster immunization, lumbar injection, and do not add adjuvant, dosage doubles;
2. Fusion of Cells
Intraabdominal to splenocyte in Balb/c Mice Body and its myeloma cell is mixed according to the ratio of cell quantity 20:1, the centrifugal 7min of 800rpm, supernatant discarded, 1mL50%PEG(W/W is added) in 30S, leave standstill 1min, the DMEM nutrient culture media of 1mL serum-free is slowly added (purchased from Gibico company in first 1min, article No. H390), the DMEM nutrient culture media of 2mL serum-free is slowly added in second 1min, the DMEM nutrient culture media of 2mL serum-free is slowly added in the 3rd 1min, the DMEM nutrient culture media of 5mL serum-free is slowly added in the 4th 1min, the DMEM nutrient culture media of 10mL serum-free is slowly added in the 5th 1min, the centrifugal 10min of 800rpm, abandon supernatant, with containing 20% calf serum (purchased from Gibico company, article No. H1565) complete medium re-suspended cell, cell count is 3-6 × 10 5individual/mL is laid on containing feeder cells (2-6 × 10 4individual/mL) Tissue Culture Plate, be placed in CO 2in incubator.
3. hybridoma screening and cloning
When cell grows at the bottom of hole 1/3, indirect competitive ELISA is adopted to measure cell conditioned medium liquid, screen positive hole, limiting dilution assay is adopted to carry out subclone in positive hole, 6th day, get cell conditioned medium to detect, finally choose anti-rabbit pestivirus VP60 hybridoma cell strain 2F4, by Cryopreservation of Hybridoma Cells in liquid nitrogen container, described hybridoma cell strain 2F4 is preserved in China typical culture collection center on June 11st, 2015, deposit number is CCTCCC201582, and preservation address is Wuhan, China Wuhan University.
4. antibody preparation and purifying
The production of monoclonal antibody: adopt ascites in animal body to induce method;
The purifying of monoclonal antibody: adopt SPA post (purchased from Niu Long bio tech ltd, Hangzhou) method to carry out purifying to ascites, obtain anti-rabbit pest VP60 monoclonal antibody of the present invention.
(2) preparation of Rabbit pest virus antibody rapid detection card
1. the boiling of colloidal gold solution
The ultrapure water measuring 99mL loads the round-bottomed flask of 250mL, put into stirrer, be placed on magnetic stirring apparatus, add 1% gold chloride (the outstanding Bioisystech Co., Ltd in Shanghai, article No. JY-SJ111) solution 1mL, suitable speed stirs, open heater switch, disposablely rapidly 1.6mL1% citric acid three sodium solution is added after solution boiling, chlorauric acid solution gradually becomes claret by grey, heating 10min is continued after colour stable, 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 antibody
With the K of 0.lmol/L 2cO 3collaurum pH value is regulated to be 9.0.Every 1mL colloidal gold solution adds 6 μ g anti-rabbit pestivirus VP60 monoclonal antibody (Shandong Lvdu Bio Sicience & Technology Co., Ltd., article No. LDANTI-VP60), under room temperature, 120rpm shakes 30min, add 10% bovine serum albumin(BSA) 20 μ L, under room temperature, 120rpm shakes 30min, and the centrifugal 20min of 12000rpm, abandons supernatant, use 0.01MPBS dissolution precipitation, namely obtain the anti-rabbit pestivirus VP60 monoclonal antibody marked.
3. gold mark pad process
Select polyester fiber 6613(Shanghai Jie Ning biotinylated biomolecule Science and Technology Ltd., article No. polyester fiber 6613) as gold-marking binding pad.Gold is marked pad and is immersed in treating fluid A(0.01Mpbs, pH7.4,0.2%TritonX-100) 5min, take out 37 DEG C of oven dry, for subsequent use.
4. sample pad process
Select DL42(Shanghai Jinbiao Bio-Tech Co., Ltd., article No. DL42) be sample pad, sample pad is immersed in treating fluid B(0.01MpbspH7.4,1%TritonX-100,1%BSA, 0.05%NaN 3), 5min, takes out 37 DEG C of oven dry, for subsequent use.
5. C, T line is determined
Select SartoriusCn140 film (Sartorius AG, article No. Cn140), respectively by Rabbit pest virus VP60 albumen (Shandong Lvdu Bio Sicience & Technology Co., Ltd., article No. LDVP60) and the anti-(Shandong Lvdu Bio Sicience & Technology Co., Ltd. of sheep anti mouse two, article No. LDks001) to draw on NC film as T line and C line, concentration is respectively 1.3mg/mL and 0.6mg/mL.
Embodiment 2 one kinds of Rabbit pest virus antibody rapid detection card preparation methods
(1) preparation of anti-rabbit pestivirus VP60 monoclonal antibody
1. immune programme for children
By insect cell expression albumen VP60 immunogene (animal and veterinary research institute gives by Binzhou, Shandong Province) and 501 adjuvant (Shandong Lvdu Bio Sicience & Technology Co., Ltd., article No. LD003) by volume 1:0.6 ratio mixing, immunity is carried out to 8-10 Balb/c mouse in age in week (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.), leg muscle is injected, 50 μ g/ only, later every immunity in two weeks once, immunizing dose, position are the same; 5 exempt from rear booster immunization, lumbar injection, and do not add adjuvant, dosage doubles.
2. Fusion of Cells
Intraabdominal to splenocyte in Balb/c Mice Body and its myeloma cell is mixed according to the ratio of cell quantity 20:0.7, the centrifugal 8min of 800rpm, supernatant discarded, 1mL50%PEG(W/W is added) in 30S, leave standstill 1min, the DMEM nutrient culture media of 1mL serum-free is slowly added (purchased from Gibico company in first 1min, article No. H390), the DMEM nutrient culture media of 2mL serum-free is slowly added in second 1min, the DMEM nutrient culture media of 2mL serum-free is slowly added in the 3rd 1min, the DMEM nutrient culture media of 5mL serum-free is slowly added in the 4th 1min, the DMEM nutrient culture media of 10mL serum-free is slowly added in the 5th 1min, the centrifugal 10min of 800rpm, abandon supernatant, with containing 20% calf serum (purchased from Gibico company, article No. H1565) complete medium re-suspended cell, cell count is 3-6 × 10 5individual/mL is laid on containing feeder cells (2-6 × 10 4individual/mL) Tissue Culture Plate, be placed in CO 2in incubator.
3. hybridoma screening and cloning
When cell grows at the bottom of hole 1/3, indirect competitive ELISA is adopted to measure cell conditioned medium liquid, screen positive hole, limiting dilution assay is adopted to carry out subclone in positive hole, 6th day, get cell conditioned medium to detect, finally choose anti-rabbit pestivirus VP60 hybridoma cell strain 2F4, by Cryopreservation of Hybridoma Cells in liquid nitrogen container, described hybridoma cell strain 2F4 is preserved in China typical culture collection center on June 11st, 2015, deposit number is CCTCCC201582, and preservation address is Wuhan, China Wuhan University.
4. antibody preparation and purifying
The production of monoclonal antibody: adopt ascites in animal body to induce method;
The purifying of monoclonal antibody: adopt SPA post (purchased from Niu Long bio tech ltd, Hangzhou) method to carry out purifying to ascites, obtain anti-rabbit pest VP60 monoclonal antibody of the present invention.
(2) preparation of Rabbit pest virus antibody rapid detection card
1. the boiling of colloidal gold solution
Method is the round-bottomed flask of the ultrapure water loading 250mL measuring 99mL, put into stirrer, be placed on magnetic stirring apparatus, add 1% gold chloride (the outstanding Bioisystech Co., Ltd in Shanghai, article No. JY-SJ111) solution 1mL, suitable speed stirs, open heater switch, disposablely rapidly 1.6mL1% citric acid three sodium solution is added after solution boiling, chlorauric acid solution gradually becomes claret by grey, heating 10min is continued after colour stable, 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 antibody
With the K of 0.lmol/L 2cO 3collaurum pH value is regulated to be 9.0.Every 1mL colloidal gold solution adds 6 μ g anti-rabbit pestivirus VP60 monoclonal antibody (Shandong Lvdu Bio Sicience & Technology Co., Ltd., article No. LDANTI-VP60), under room temperature, 120rpm shakes 30min, add 10% bovine serum albumin(BSA) 20 μ L, under room temperature, 120rpm shakes 30min, and the centrifugal 20min of 12000rpm, abandons supernatant, use 0.01MPBS dissolution precipitation, namely obtain the anti-rabbit pestivirus VP60 monoclonal antibody marked.
3. gold mark pad process
Select polyester fiber 6613(Shanghai Jie Ning biotinylated biomolecule Science and Technology Ltd., article No. polyester fiber 6613) as gold-marking binding pad.Gold is marked pad and is immersed in treating fluid A(0.01Mpbs, pH7.4,0.2%TritonX-100) 5min, take out 37 DEG C of oven dry, for subsequent use.
4. sample pad process
Select DL42(Shanghai Jinbiao Bio-Tech Co., Ltd., article No. DL42) be sample pad, sample pad is immersed in treating fluid B(0.01MpbspH7.4,1%TritonX-100,1%BSA, 0.05%NaN3), 5min, takes out 37 DEG C of oven dry, for subsequent use.
5. C, T line is determined
Select SartoriusCn140 film (Sartorius AG, article No. Cn140), respectively by Rabbit pest virus VP60 albumen (Shandong Lvdu Bio Sicience & Technology Co., Ltd., article No. LDVP60) and the anti-(Shandong Lvdu Bio Sicience & Technology Co., Ltd. of sheep anti mouse two, article No. LDks001) to draw on NC film as T line and C line, concentration is respectively 1.3mg/mL and 0.6mg/mL.
Embodiment 3 one kinds of Rabbit pest virus antibody rapid detection card preparation methods
(1) preparation of anti-rabbit pestivirus VP60 monoclonal antibody
1. immune programme for children
By insect cell expression albumen VP60 immunogene (animal and veterinary research institute gives by Binzhou, Shandong Province) and 501 adjuvant (Shandong Lvdu Bio Sicience & Technology Co., Ltd., article No. LD003) by volume 1:1.3 ratio mixing, immunity is carried out to 8-10 Balb/c mouse in age in week (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.), leg muscle is injected, 50 μ g/ only, later every immunity in two weeks once, immunizing dose, position are the same; 5 exempt from rear booster immunization, lumbar injection, and do not add adjuvant, dosage doubles;
2. Fusion of Cells
Intraabdominal to splenocyte in Balb/c Mice Body and its myeloma cell is mixed according to the ratio of cell quantity 20:1.3, the centrifugal 8min of 800rpm, supernatant discarded, 1mL50%PEG(W/W is added) in 30S, leave standstill 1min, the DMEM nutrient culture media of 1mL serum-free is slowly added (purchased from Gibico company in first 1min, article No. H390), the DMEM nutrient culture media of 2mL serum-free is slowly added in second 1min, the DMEM nutrient culture media of 2mL serum-free is slowly added in the 3rd 1min, the DMEM nutrient culture media of 5mL serum-free is slowly added in the 4th 1min, the DMEM nutrient culture media of 10mL serum-free is slowly added in the 5th 1min, the centrifugal 10min of 800rpm, abandon supernatant, with containing 20% calf serum (purchased from Gibico company, article No. H1565) complete medium re-suspended cell, cell count is 3-6 × 10 5individual/mL is laid on containing feeder cells (2-6 × 10 4individual/mL) Tissue Culture Plate, be placed in CO 2in incubator.
3. hybridoma screening and cloning
When cell grows at the bottom of hole 1/3, indirect competitive ELISA is adopted to measure cell conditioned medium liquid, screen positive hole, limiting dilution assay is adopted to carry out subclone in positive hole, 6th day, get cell conditioned medium to detect, finally choose anti-rabbit pestivirus VP60 hybridoma cell strain 2F4, by Cryopreservation of Hybridoma Cells in liquid nitrogen container, described hybridoma cell strain 2F4 is preserved in China typical culture collection center on June 11st, 2015, deposit number is CCTCCC201582, and preservation address is Wuhan, China Wuhan University.
4. antibody preparation and purifying
The production of monoclonal antibody: adopt ascites in animal body to induce method;
The purifying of monoclonal antibody: adopt SPA post (purchased from Niu Long bio tech ltd, Hangzhou) method to carry out purifying to ascites, obtain anti-rabbit pest VP60 monoclonal antibody of the present invention.
(2) preparation of Rabbit pest virus antibody rapid detection card
1. the boiling of colloidal gold solution
The ultrapure water measuring 99mL loads the round-bottomed flask of 250mL, put into stirrer, be placed on magnetic stirring apparatus, add 1% gold chloride (the outstanding Bioisystech Co., Ltd in Shanghai, article No. JY-SJ111) solution 1mL, suitable speed stirs, open heater switch, disposablely rapidly 1.6mL1% citric acid three sodium solution is added after solution boiling, chlorauric acid solution gradually becomes claret by grey, heating 10min is continued after colour stable, 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 antibody
With the K of 0.lmol/L 2cO3 regulates collaurum pH value to be 9.0.Every 1mL colloidal gold solution adds 6 μ g anti-rabbit pestivirus VP60 monoclonal antibody (Shandong Lvdu Bio Sicience & Technology Co., Ltd., article No. LDANTI-VP60), under room temperature, 120rpm shakes 30min, add 10% bovine serum albumin(BSA) 2 μ L, under room temperature, 120rpm shakes 30min, and the centrifugal 20min of 12000rpm, abandons supernatant, use 0.01MPBS dissolution precipitation, namely obtain the anti-rabbit pestivirus VP60 monoclonal antibody marked.
3. gold mark pad process
Select polyester fiber 6613(Shanghai Jie Ning biotinylated biomolecule Science and Technology Ltd., article No. polyester fiber 6613) as gold-marking binding pad.Gold is marked pad and is immersed in treating fluid A(0.01Mpbs, pH7.4,0.2%TritonX-100) 5min, take out 37 DEG C of oven dry, for subsequent use.
4. sample pad process
Select DL42(Shanghai Jinbiao Bio-Tech Co., Ltd., article No. DL42) be sample pad, sample pad is immersed in treating fluid B(0.01MpbspH7.4,1%TritonX-100,1%BSA, 0.05%NaN3), 5min, takes out 37 DEG C of oven dry, for subsequent use.
5. C, T line is determined
Select SartoriusCn140 film (Sartorius AG, article No. Cn140), respectively by Rabbit pest virus VP60 albumen (Shandong Lvdu Bio Sicience & Technology Co., Ltd., article No. LDVP60) and the anti-(Shandong Lvdu Bio Sicience & Technology Co., Ltd. of sheep anti mouse two, article No. LDks001) to draw on NC film as T line and C line, concentration is respectively 1.3mg/mL and 0.6mg/mL.
The use of test example 1 Rabbit pest virus antibody of the present invention rapid detection card
The structure of test card of the present invention as shown in Figure 1, when using of the present invention, gathers whole blood 0.1ml, drips in the sample well in test card, judged result during 5-10 minute with suction pipe.
Result judges as Fig. 2,3 and 4.
(1) positive (Fig. 3): if the colour developing of C line, and T line does not develop the color, and is judged to the positive, reaches and can prevent strong virus attack antibody horizontal.
(2) negative (Fig. 2): if the colour developing of C line, T line develops the color simultaneously, is judged to feminine gender or antibody horizontal is low.
(3) invalid (Fig. 4): in viewport, if C line does not develop the color, then result is invalid, and suggestion retests.
Test example 2 test card susceptibility of the present invention and accuracy test
Extract the rabbit anteserum that 100 parts of rabbit anteserums injecting rabbit pestilence seedling and 100 parts did not inject rabbit pestilence seedling, carry out detection by blood coagulation tests and this test card respectively and check.Result shows, and both susceptibility coincidence rates are 98%, and accuracy is 99%.

Claims (8)

1. a preparation method for Rabbit pest virus antibody rapid detection card, concrete steps are:
(1) preparation of anti-rabbit pestivirus VP60 monoclonal antibody
1. immune programme for children
The ratio of insect cell expression albumen VP60 immunogene and 501 adjuvants 1:0.5-1.5 by volume mixed, carry out immunity to 8-10 Balb/c mouse in age in week, leg muscle is injected, and only, once every immunity in two weeks, immunizing dose, position are the same later for 50 μ g/; 5 exempt from rear booster immunization, lumbar injection, and do not add adjuvant, dosage doubles;
2. Fusion of Cells
Intraabdominal to splenocyte in Balb/c Mice Body and its myeloma cell is mixed according to the ratio of cell quantity 20:0.5-1.5, the centrifugal 5-8min of 800rpm, supernatant discarded, 1mL50%PEG(W/W is added) in 30S, leave standstill 1min, the DMEM nutrient culture media of 1mL serum-free is slowly added in first 1min, the DMEM nutrient culture media of 2mL serum-free is slowly added in second 1min, the DMEM nutrient culture media of 2mL serum-free is slowly added in the 3rd 1min, the DMEM nutrient culture media of 5mL serum-free is slowly added in the 4th 1min, the DMEM nutrient culture media of 10mL serum-free is slowly added in the 5th 1min, the centrifugal 10min of 800rpm, abandon supernatant, with the complete medium re-suspended cell containing 20% calf serum, cell count is 3-6 × 10 5individual/mL is laid on containing 2-6 × 10 4the Tissue Culture Plate of individual/mL feeder cells, is placed in CO 2in incubator,
3. hybridoma screening and cloning
When cell grows at the bottom of hole 1/3, indirect competitive ELISA is adopted to measure cell conditioned medium liquid, screen positive hole, limiting dilution assay is adopted to carry out subclone in positive hole, 6th day, get cell conditioned medium to detect, finally choose anti-rabbit pestivirus VP60 hybridoma cell strain 2F4, by Cryopreservation of Hybridoma Cells in liquid nitrogen container, described hybridoma cell strain 2F4 is preserved in China typical culture collection center on June 11st, 2015, deposit number is CCTCCC201582, and preservation address is Wuhan, China-Wuhan University;
4. antibody preparation and purifying
The production of monoclonal antibody: adopt ascites in animal body to induce method;
The purifying of monoclonal antibody: adopt SPA post method to carry out purifying to ascites, obtain anti-rabbit pest VP60 monoclonal antibody of the present invention;
(2) preparation of Rabbit pest virus antibody rapid detection card
1. the boiling of colloidal gold solution
Trisodium citrate reduction method is adopted to prepare collaurum;
2. the mark of anti-rabbit pestivirus VP60 monoclonal antibody
With the K of 0.lmol/L 2cO 3collaurum pH value is regulated to be 9.0, every 1mL colloidal gold solution adds 6 μ g anti-rabbit pestivirus VP60 monoclonal antibodies, under room temperature, 120rpm shakes 30min, add 10% bovine serum albumin(BSA) 20 μ L, under room temperature, 120rpm shakes 30min, and the centrifugal 20min of 12000rpm, abandons supernatant, use 0.01MPBS dissolution precipitation, namely obtain the anti-rabbit pestivirus VP60 monoclonal antibody marked;
3. gold mark pad process
Select polyester fiber 6613 as gold-marking binding pad, gold is marked pad and be immersed in 5min in treating fluid A, treating fluid A is 0.01Mpbs, pH7.4,0.2%TritonX-100, takes out 37 DEG C of oven dry, for subsequent use;
4. sample pad process
Selection DL42 is sample pad, sample pad is immersed in 5min in treating fluid B, and treating fluid B is 0.01Mpbs, pH7.4,1%TritonX-100,1%BSA, 0.05%NaN 3), 5min, takes out 37 DEG C of oven dry, for subsequent use;
5. C, T line is determined
Select SartoriusCn140 film, respectively using Rabbit pest virus VP60 albumen with sheep anti mouse two is anti-draws on NC film as T line and C line, concentration is respectively 1.3mg/mL and 0.6mg/mL.
2. method according to claim 1, is characterized in that, the ratio of insect cell expression albumen VP60 immunogene and 501 adjuvants 1:0.8-1.3 is by volume mixed in described step (1).
3. method according to claim 2, is characterized in that, the ratio of insect cell expression albumen VP60 immunogene and 501 adjuvants 1:1 is by volume mixed in described step (1).
4. method according to claim 1, is characterized in that, Intraabdominal to the splenocyte in Balb/c Mice Body and its myeloma cell is mixed according to the ratio of cell quantity 20:1 in described step (1), the centrifugal 7min of 800rpm.
5. method according to claim 4, is characterized in that, Intraabdominal to the splenocyte in Balb/c Mice Body and its myeloma cell is mixed according to the ratio of cell quantity 20:0.8-1.3 in described step (1), the centrifugal 6min of 800rpm.
6. method according to claim 1, it is characterized in that, in described step (1), hybridoma cell strain 2F4 is preserved in China typical culture collection center on June 11st, 2015, and deposit number is CCTCCC201582, and preservation address is Wuhan, China-Wuhan University.
7. method according to claim 1, is characterized in that, the concrete steps adopting trisodium citrate reduction method to prepare collaurum in described step (2) are:
Method is the round-bottomed flask of the ultrapure water loading 250mL measuring 99mL, put into stirrer, be placed on magnetic stirring apparatus, add 1% chlorauric acid solution 1mL, suitable speed stirs, open heater switch, disposablely rapidly 1.6mL1% citric acid three sodium solution is added after solution boiling, chlorauric acid solution gradually becomes claret by grey, heating 10min is continued, after solution cooling, with filtering with microporous membrane after colour stable, 4 DEG C save backup, and UV scanning obtains the colloid gold particle that maximum absorption band is 523nm.
8. the Rabbit pest virus antibody rapid detection card that as described in any one of claim 1-7 prepared by method.
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