CN104991057A - Kit for quickly detecting aleutian mink disease virus antibody through ELISA, and preparation method for kit - Google Patents
Kit for quickly detecting aleutian mink disease virus antibody through ELISA, and preparation method for kit Download PDFInfo
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- CN104991057A CN104991057A CN201510380894.6A CN201510380894A CN104991057A CN 104991057 A CN104991057 A CN 104991057A CN 201510380894 A CN201510380894 A CN 201510380894A CN 104991057 A CN104991057 A CN 104991057A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- 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 kit for quickly detecting an aleutian mink disease virus antibody through the ELISA, and a preparation method for the kit. The preparation method comprises the following steps: (1) aleutian virus culture; (2) aleutian virus purification; (3) preparation of a rabbit anti-mink secondary antibody and an enzyme-labeled secondary antibody; (4) preparation of the quick detection kit. Through the adoption of the kit, the aleutian mink disease virus antibody can be quickly, sensitively and accurately detected, and the problems in the double-flow electrophoresis method and the PCR method which are mainly used for detecting the aleutian mink disease virus antibody in China are solved.
Description
Technical field
The present invention relates to a kind of Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit and preparation method thereof, belong to technical field of immunoassay.
Background technology
Mink A Liushen is acute, the progressive infectious disease of the one caused by Aleutian Mink Disease Parvovirus.Along with the prolongation of time, recessiveness becomes dominant, and to ermine, group causes heavy losses, therefore sets up periodic purge system and is very important.
At present, the detection method of Aleutian Mink Disease Parvovirus antibody has double fluid to electrophoresis and PCR method, and double fluid needs double fluid to immunoelectrophoresis antigen to electrophoresis, antigen must be preserved below-20 DEG C, storage life was less than 1 year, and preservation condition is harsh, and the false positive of testing result is higher; PCR method needs expensive PCR detector, and detection time is long, 3-5 hour, and need technical professional to operate, with Aleutian disease double fluid to compared with electrophoresis, kit of the present invention can quick, sensitive, detect Aleutian Mink Disease Parvovirus antibody horizontal accurately, long shelf-life, sensitivity is higher; Compared with PCR detection kit, this kit does not need the PCR instrument of A Gui, and cost is low, and detection time is short, is easy to operation.
Summary of the invention
The invention provides a kind of Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit and preparation method thereof, for quick, sensitive, detect Aleutian Mink Disease Parvovirus antibody accurately and decrease time and cost.
The present invention is realized by following technical scheme:
A preparation method for Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit, comprises the following steps:
(1) aleutian disease virus is cultivated:
By young for 10 ages in days ermine, taking-up testis and kidney prepare cell suspension, and cell density is 1 ~ 5 × 10
4individual/ml, 37 DEG C of cultivations, nutrient solution adopts DMEM to cultivate base (Xi Bao bio tech ltd, Shanghai, model is R10-013-CV), the volume cultivating base by DMEM adds 5% NBCS (Tianjin Kang Yuan Bioisystech Co., Ltd), after cell grew up to individual layer through 4 days, discard nutrient solution, add the degerming third generation reaction poison continuing through ermine body, connecing poison amount is 1%, and add maintenance medium, maintenance medium adopts the DMEM nutrient solution of not increase serum, within one week, occur pathology and collect virus after connecing poison, freezing freeze thawing three times is stand-by;
(2) aleutian disease virus purifying:
(Shanghai is great along bio tech ltd to take gel dry powder G-200, model is GR-F1031) 1 0-12 gram, pour in the distilled water of 20 times of gel volumes lentamente, soak three hours at ambient temperature or soak 4-5 hour in boiling water, stir gently, in room temperature static 30 minutes, supernatant liquid is inclined to, wash 4 times with distilled water is floating again, then fill post application of sample; Every pipe eluent be arranged in order during collection, make specific assay by part by S P A thalline (+) serum reagent, collect according to specificity and peak value, first peak is the AD toxalbumin of purifying, and the second peak is non-specific protein; After s e p h a d e x-G 200 is separated, sample is loaded in bag filter, be condensed into the protein content of requirement, dialysis, balance, packing, 4 DEG C of preservations with PEG 20000 (Yongtai, Jinan Chemical Co., Ltd.), obtain the viral antigen of purifying;
(3) preparation that rabbit water resistant ermine two is anti-and the preparation of ELIAS secondary antibody
Mink serum caprylic acid-ammonium is purified, the ratio of the serum of purifying and 501 adjuvants 1:0.5-1.5 is by volume mixed, preferred volume ratio is 1:1, immunity is carried out to 2-3 monthly age New Zealand white rabbit, leg muscle is injected, only, once every immunity in two weeks, immunizing dose, position are the same later for 1mg/; 5 exempt from rear Culling heart blood, namely obtain rabbit water resistant ermine two and resist, then use protein A column purification rabbit anteserum, calculate protein concentration, prepare ELIAS secondary antibody by sodium periodate oxidizing process, concrete steps are as follows: take 10mg horseradish peroxidase (Sigma company, HRP), be dissolved in 1mL pure water; Add the freshly prepared 0.06mol/L sodium periodate solution of 1mL (Beijing chemical reagents corporation), room temperature lucifuge stirs 30min; Add 1mL and dissolved the anti-0.1mol/L carbonate buffer solution of the rabbit water resistant ermine after 5mg purifying two, room temperature lucifuge gently stirs 2-3h; Add freshly prepared 0.2mL 5mg/mL sodium borohydride (Beijing chemical reagents corporation), place 2h, with stable bond thing for 4 DEG C; Move into bag filter again, be the PBS dialysed overnight of 7.2 in pH ,-20 DEG C save backup;
(4) preparation of quick detection kit
1. the preparation of ELISA Plate:
At the viral antigen of the upper pre-coated purifying of polyethylene micropore plate (Shanghai raw work), this bag by use pH be 9.6 10mM carbonate buffer solution (Beijing chemical reagents corporation), wrap at 4 DEG C and spent the night; Then use PBST to wash twice, PBST is 10mMPBS+0.05%Tween20, then adds confining liquid, and confining liquid is 10mM PBS+2% bovine serum albumin(BSA), BSA, and close 2 hours for 37 DEG C, dry final vacuum is drained and is packaged into finished product kit ELISA Plate;
2. the configuration of other compositions of kit:
A) ELIAS secondary antibody: be mixed with 1:500(V with enzyme dilution (10mM PBS+1% bovine serum albumin(BSA)+0.05%Tween20)
eLIAS secondary antibody: V
enzyme dilution) working concentration;
B) sample diluting liquid: 10mM PBS;
C) positive control: 10mM PBS+ positive serum;
D) negative control: 10mM PBS+ negative serum;
E) developer A liquid: 0.5% superoxide urea+5mM PBS;
F) developer B liquid: 1% 3,3', 5,5'-tetramethyl benzidine+5mM PBS;
G) stop buffer: 5mM sulfuric acid;
H) 20 × concentrated cleaning solution: 1M PBS+0.2%Tween20.
Protection scope of the present invention also comprises Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit and the application thereof of preparation.
The present invention compared with prior art has the following advantages:
The present invention can quick, sensitive, detect Aleutian Mink Disease Parvovirus antibody accurately, overcome domestic detection mink A Liushen antibody mainly with the problem of double fluid to electrophoresis and PCR method.
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.
The preparation method of embodiment 1 one kinds of Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit, comprises the following steps:
(1) aleutian disease virus is cultivated:
By young for 10 ages in days ermine, taking-up testis and kidney prepare cell suspension, and cell density is 1 ~ 5 × 10
4individual/ml, 37 DEG C of cultivations, nutrient solution adopts DMEM to cultivate base (Xi Bao bio tech ltd, Shanghai, model is R10-013-CV), the volume cultivating base by DMEM adds 5% NBCS (Tianjin Kang Yuan Bioisystech Co., Ltd), after cell grew up to individual layer through 4 days, discard nutrient solution, add the degerming third generation reaction poison continuing through ermine body, connecing poison amount is 1 %, and add maintenance medium, maintenance medium adopts the DMEM nutrient solution of not increase serum, within one week, occur pathology and collect virus after connecing poison, freezing freeze thawing three times is stand-by;
(2) aleutian disease virus purifying:
(Shanghai is great along bio tech ltd to take gel dry powder G-200, model is GR-F1031) 1 0-12 gram, pour in the distilled water of 20 times of gel volumes lentamente, soak three hours at ambient temperature or soak 4-5 hour in boiling water, stir gently, in room temperature static 30 minutes, supernatant liquid is inclined to, wash 4 times with distilled water is floating again, then fill post application of sample; Every pipe eluent be arranged in order during collection, make specific assay by part by S P A thalline (+) serum reagent, collect according to specificity and peak value, first peak is the AD toxalbumin of purifying, and the second peak is non-specific protein; After s e p h a d e x-G 200 is separated, sample is loaded in bag filter, be condensed into the protein content of requirement, dialysis, balance, packing, 4 DEG C of preservations with PEG 20000 (Yongtai, Jinan Chemical Co., Ltd.), obtain the viral antigen of purifying;
(3) preparation that rabbit water resistant ermine two is anti-and the preparation of ELIAS secondary antibody
Mink serum caprylic acid-ammonium is purified, the ratio of purified blood serum and 501 adjuvants 1:0.5-1.5 is by volume mixed, immunity is carried out to 2-3 monthly age New Zealand white rabbit, leg muscle is injected, only, once every immunity in two weeks, immunizing dose, position are the same later for 1mg/; 5 exempt from rear Culling heart blood, namely obtain rabbit water resistant ermine two and resist, then use protein A column purification rabbit anteserum, calculate protein concentration, prepare ELIAS secondary antibody by sodium periodate oxidizing process;
(4) preparation of quick detection kit
1. the preparation of ELISA Plate:
At the viral antigen of the upper pre-coated purifying of polyethylene micropore plate (Shanghai raw work), this bag by use pH be 9.6 10mM carbonate buffer solution (Beijing chemical reagents corporation), wrap at 4 DEG C and spent the night; Then use PBST to wash twice, PBST is 10mMPBS+0.05%Tween20, then adds confining liquid, and confining liquid is 10mM PBS+2% bovine serum albumin(BSA), BSA, and close 2 hours for 37 DEG C, dry final vacuum is drained and is packaged into finished product kit ELISA Plate;
2. the configuration of other compositions of kit:
A) ELIAS secondary antibody: be mixed with 1:500(V with enzyme dilution (10mM PBS+1% bovine serum albumin(BSA)+0.05%Tween20)
eLIAS secondary antibody: V
enzyme dilution) working concentration;
B) sample diluting liquid: 10mM PBS;
C) positive control: 10mM PBS+ positive serum;
D) negative control: 10mM PBS+ negative serum;
E) developer A liquid: 0.5% superoxide urea+5mM PBS;
F) developer B liquid: 1% 3,3', 5,5'-tetramethyl benzidine+5mM PBS;
G) stop buffer: 5mM sulfuric acid;
H) 20 × concentrated cleaning solution: 1M PBS+0.2%Tween20.
Embodiment 2 is on the basis of embodiment 1, and prepare ELIAS secondary antibody by sodium periodate oxidizing process, concrete steps are as follows:
Take 10mg horseradish peroxidase (Sigma company, HRP), be dissolved in 1mL pure water; Add the freshly prepared 0.06mol/L sodium periodate solution of 1mL (Beijing chemical reagents corporation), room temperature lucifuge stirs 30min; Add 1mL and dissolved the anti-0.1mol/L carbonate buffer solution of the rabbit water resistant ermine after 5mg purifying two, room temperature lucifuge gently stirs 2-3h; Add freshly prepared 0.2mL 5mg/mL sodium borohydride (Beijing chemical reagents corporation), place 2h, with stable bond thing for 4 DEG C; Move into bag filter again, be the PBS dialysed overnight of 7.2 in pH ,-20 DEG C save backup.
The detection method of test example 1 quick detection kit of the present invention and result judge
(1) two holes are set and add 100 μ L positive controls respectively;
(2) two holes are separately set and add 100 μ L negative controls respectively;
(3) get 100 μ L to add in other holes through the measuring samples that 1:100 has diluted, and make a record;
Hatch 20min for (4) 37 DEG C;
(5) abandon into waste liquid barrel by the liquid in each hole, the cleansing solution that every hole adds 250 μ L carries out washing plate, and repeat 3 times, each 30s, pats dry residual liquid in hole with towel or paper handkerchief;
(6) every hole adds 100 μ L enzyme mark bonds;
Hatch 20min for (7) 37 DEG C; Repeat step (5);
(8) every hole first adds 50 μ L substrate solution A, then adds 50 μ L substrate solution B;
Hatch the colour developing of 10min(lucifuge for (9) 37 DEG C); Every hole adds 50 μ L stop buffer cessation reactions again;
(10) absorbance in each hole is measured at once in 450nm wavelength place, i.e. OD
450value.
Test validity judges:
(1) negative control: under normal circumstances, negative control hole OD450 value≤0.3;
(2) positive control: under normal circumstances, Positive control wells OD450 value > 0.3;
(3) P/N=sample OD/ N value, if negative control average is less than or equal to 0.1, then N value=0.2+ negative control average; If negative control average is greater than 0.1, then N value=negative control average.
(4) result judges: P/N >=2, judge that this detection sample is as the positive; 2≤P/N < 3, judges that this detection sample is as the weak positive, P/N >=3, judges that this detection sample is as strong positive.
Test example 2 quick detection kit of the present invention carries out Sensitivity and Specificity contrast to immunoelectrophoresis for Aleutian Mink Disease Parvovirus antibody with double fluid, and comparing result is in table 1 and table 2
Table 1 carries out susceptibility contrast with mink A Liushen double fluid to immunoelectrophoresis (CIE)
Table 2 carries out specificity contrast with mink A Liushen double fluid to immunoelectrophoresis (CIE)
Note: above+to represent the positive;-represent feminine gender.
Claims (8)
1. a preparation method for Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit, is characterized in that, described preparation method comprises the following steps:
(1) aleutian disease virus is cultivated:
By young for 10 ages in days ermine, taking-up testis and kidney prepare cell suspension, and cell density is 1 ~ 5 × 10
4individual/ml, 37 DEG C of cultivations, nutrient solution adopts DMEM to cultivate base, and the volume cultivating base by DMEM adds 5% NBCS, after cell grew up to individual layer through 4 days, discard nutrient solution, add the degerming third generation reaction poison continuing through ermine body, connecing poison amount is 1 %, add maintenance medium, maintenance medium adopts the DMEM nutrient solution of not increase serum, within one week, occurs pathology and collects virus after connecing poison, and freezing freeze thawing three times is stand-by;
(2) aleutian disease virus purifying:
Take gel dry powder G-200 10-12 gram, pour in the distilled water of 20 times of gel volumes lentamente, soak three hours at ambient temperature or soak 4-5 hour in boiling water, stir gently, in room temperature static 30 minutes, supernatant liquid is inclined to, wash 4 times with distilled water is floating again, then fill post application of sample; Every pipe eluent be arranged in order during collection, make specific assay by part by S P A thalline (+) serum reagent, collect according to specificity and peak value, first peak is the AD toxalbumin of purifying, and the second peak is non-specific protein; After s e p h a d e x-G 200 is separated, sample is loaded in bag filter, be condensed into the protein content of requirement, dialysis, balance, packing, 4 DEG C of preservations with PEG 20000, obtain the viral antigen of purifying.
2. the preparation method of Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit according to claim 1, is characterized in that, the preparation that in described quick detection kit, rabbit water resistant ermine two is anti-and the preparation of ELIAS secondary antibody:
Mink serum caprylic acid-ammonium is purified, the ratio of purified blood serum and 501 adjuvants 1:0.5-1.5 is by volume mixed, immunity is carried out to 2-3 monthly age New Zealand white rabbit, leg muscle is injected, only, once every immunity in two weeks, immunizing dose, position are the same later for 1mg/; 5 exempt from rear Culling heart blood, namely obtain rabbit water resistant ermine two and resist, then use protein A column purification rabbit anteserum, calculate protein concentration, prepare ELIAS secondary antibody by sodium periodate oxidizing process.
3. the preparation method of Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit according to claim 2, is characterized in that, prepares the concrete steps of ELIAS secondary antibody as follows in described quick detection kit by sodium periodate oxidizing process:
Take 10mg horseradish peroxidase, be dissolved in 1mL pure water; Add the freshly prepared 0.06mol/L sodium periodate solution of 1mL, room temperature lucifuge stirs 30min; Add 1mL and dissolved the anti-0.1mol/L carbonate buffer solution of the rabbit water resistant ermine after 5mg purifying two, room temperature lucifuge gently stirs 2-3h; Add freshly prepared 0.2mL 5mg/mL sodium borohydride, place 2h, with stable bond thing for 4 DEG C; Move into bag filter again, be the PBS dialysed overnight of 7.2 in pH ,-20 DEG C save backup.
4. the preparation method of Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit according to claim 2, is characterized in that, purified by mink serum caprylic acid-ammonium, the ratio of the serum of purifying and 501 adjuvants 1:1 is by volume mixed.
5. the preparation method of Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit according to claim 1, is characterized in that, the preparation of ELISA Plate in described quick detection kit:
The viral antigen of pre-coated purifying on polyethylene micropore plate, this bag by use pH be 9.6 10mM carbonate buffer solution, wrap at 4 DEG C and spent the night; Then use PBST to wash twice, PBST is 10mMPBS+0.05%Tween20, then adds confining liquid, and confining liquid is 10mM PBS+2% bovine serum albumin(BSA), BSA, and close 2 hours for 37 DEG C, dry final vacuum is drained and is packaged into finished product kit ELISA Plate.
6. the preparation method of Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit according to claim 1, is characterized in that, the configuration of other compositions in described quick detection kit:
A) ELIAS secondary antibody: be mixed with 1:500(V with enzyme dilution (10mM PBS+1% bovine serum albumin(BSA)+0.05%Tween20)
eLIAS secondary antibody: V
enzyme dilution) working concentration;
B) sample diluting liquid: 10mM PBS;
C) positive control: 10mM PBS+ positive serum;
D) negative control: 10mM PBS+ negative serum;
E) developer A liquid: 0.5% superoxide urea+5mM PBS;
F) developer B liquid: 1% 3,3', 5,5'-tetramethyl benzidine+5mM PBS;
G) stop buffer: 5mM sulfuric acid;
H) 20 × concentrated cleaning solution: 1M PBS+0.2%Tween20.
7. the Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit that the preparation method as described in any one of claim 1-6 obtains.
8. Aleutian Mink Disease Parvovirus antibody ELISA quick detection kit as claimed in claim 7 is in the application of the context of detection of Aleutian Mink Disease Parvovirus antibody.
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| CN201510380894.6A CN104991057A (en) | 2015-07-02 | 2015-07-02 | Kit for quickly detecting aleutian mink disease virus antibody through ELISA, and preparation method for kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105242042A (en) * | 2015-09-22 | 2016-01-13 | 山东省滨州畜牧兽医研究院 | ELISA rapid detection kit for Aleutian mink disease virus antibody and preparation method thereof |
| CN105801673A (en) * | 2016-04-12 | 2016-07-27 | 北京瑞鹰生物技术有限公司 | Mink ADV (aleutian disease virus) antigen as well as preparation method and detection kit thereof |
| CN107365360A (en) * | 2017-08-10 | 2017-11-21 | 吉林大学 | Antigenic Peptide microballoon and preparation method and the application in Aleutian disease detection |
-
2015
- 2015-07-02 CN CN201510380894.6A patent/CN104991057A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105242042A (en) * | 2015-09-22 | 2016-01-13 | 山东省滨州畜牧兽医研究院 | ELISA rapid detection kit for Aleutian mink disease virus antibody and preparation method thereof |
| CN105801673A (en) * | 2016-04-12 | 2016-07-27 | 北京瑞鹰生物技术有限公司 | Mink ADV (aleutian disease virus) antigen as well as preparation method and detection kit thereof |
| CN105801673B (en) * | 2016-04-12 | 2019-04-30 | 北京纳百生物科技有限公司 | Aleutian Mink Disease Parvovirus antigen and preparation method thereof and detection kit |
| CN107365360A (en) * | 2017-08-10 | 2017-11-21 | 吉林大学 | Antigenic Peptide microballoon and preparation method and the application in Aleutian disease detection |
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Application publication date: 20151021 |