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%。