CN102384975A - Indirect enzyme-linked immunosorbent assay (ELISA) method for detecting duck flavivirus serum antibody - Google Patents
Indirect enzyme-linked immunosorbent assay (ELISA) method for detecting duck flavivirus serum antibody Download PDFInfo
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- CN102384975A CN102384975A CN2011102328587A CN201110232858A CN102384975A CN 102384975 A CN102384975 A CN 102384975A CN 2011102328587 A CN2011102328587 A CN 2011102328587A CN 201110232858 A CN201110232858 A CN 201110232858A CN 102384975 A CN102384975 A CN 102384975A
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Abstract
The invention relates to an indirect enzyme-linked immunosorbent assay (ELISA) method for detecting a duck flavivirus serum antibody, which is characterized in that: duck flavivirus is used as an envelop antigen, and an indirect ELISA method for detecting duck flavivirus serum antibody in duck blood serum is established. The method can effectively diagnose the duck flavivirus which is prevalent at present in China and can carry out large-scale investigation of blood serum epidemiology, and a quick, simple and convenient detection method can be provided for controlling the prevention and the prevalent situation of the duck flavivirus.
Description
Technical field
The present invention relates to a kind of indirect ELISA method that is used to detect duckling virosis serum antibody, belong to the epizootiology field.
Background technology
Since in April, 2010; The lay eggs New Development eqpidemic disease of rapid drawdown of a kind of kind that is caused by flavivirus (egg) duck takes place on China Jiangsu, Zhejiang, Shandong, Henan, Fujian and other places in succession, can cause that the laying ducks feed intake descends rapidly, laying rate descends rapidly even stops production and dead in various degree.The ill back of the duck crowd that laying rate is high was dropped rapidly to 20% ~ 30% from 80% ~ 95% of the peak of laying eggs in about 4 ~ 5 days, and serious can occur stopping production in morbidity in back about 7 days; Just opened produce kind of (egg) duck laying rate rise slowly or subtract egg, low laying rate or the nothing peak of laying eggs occurs for a long time.Different regions, the different cultivars duck crowd incidence of disease just differ, and the incidence of disease almost 100% in the crowd, and case fatality rate is 0 ~ 12%.Its clinical symptoms is except the rapid drawdown of laying eggs, and a very important characteristic is the sick duck of duck ovarian hemorrhage to be taken place with shaking the head nervous symptoms such as turning round neck, and cuing open the inspection pathology, to be mainly membrana follicularis hemorrhage, congested, ovarian follicle distortion atrophy etc., and meningorrhagia appears in part.The researchist gathers its ovary, liver, brain etc. and carries out virus and separate from kind of the duck that dies of illness of rapid drawdown is laid eggs in performance, measure through the virus of being separated being carried out morphological observation, physicochemical property, RT-PCR detection and sequential analysis clearly this disease pathogen be flaviviridae (
Flaviviridae) a newcomer
[1-3]
The clear and definite cause of disease is in order to carry out preventing and controlling better.The task of top priority; The diagnosis of carrying out the duckling virus infections is the major issue of pendulum in face of researcher, the animal doctor of basic unit and egg duck raiser with the anti-system work of immunity; Set up duckling antiviral antibody detection method; Extremely meaningful to this sick active immunity effect assessment, seroepidemiological survey, flux is big, the result is definite, highly sensitive being favourably welcome and the ELISA method is because of it detects.At present, Su
[4]Deng tentatively setting up the ELISA method that detects duckling virosis antibody, but the method for being set up is comparatively coarse, and data are not clear, and various conditions are not optimized as yet, are inappropriate for clinical practice and popularization.
Summary of the invention
The purpose of this invention is to provide a kind of indirect ELISA method that can detect duckling virosis serum antibody fast, easily, this method also can be carried out quick diagnosis to the duckling virosis, to reduce the loss, to improve culture benefit.
The technical scheme that the present invention takes is following:
As envelope antigen, set up the indirect ELISA method that detects duckling antiviral antibody in the duck serum with duckling virus.
Said duckling virus is duckling virus
(Duck flavivirus)WR strain, preservation registration number are CGMCC NO.4906.
Said antigen is earlier through concentrating; Comprise the steps: to collect behind the embryo allantoic liquid that contains duckling virus in 4 ℃, centrifugal 30 min of 8000 r/min, supernatant is in centrifugal 4 ℃ of centrifugal 2 h of 45000 r/min, and it is resuspended with sterilization PBS to abandon the supernatant postprecipitation; Obtain viral concentrate, be stored in-70 ℃.
Said viral concentrate is further purified through sucrose gradient centrifugation, concentrates through ultracentrifugation to obtain.
The step that is further purified is: the sucrose balance liquid of 5 gradients such as preparation 10%, 20%, 30%, 40% and 50% is added in 10% sucrose liquid upper strata with viral concentrate, in 4 ℃, centrifugal 2 h of 40000 r/min levels; The result finds to have the virus band between 10%-20%, 30%-40%, 40%-50%; Collect and respectively be with centrifugal 2 h of 45000 r/min under back 4 ℃; It is resuspended with sterilization PBS to abandon the supernatant postprecipitation, and detects the viral level that each is with the RT-PCR method, maximum with 30%-40% band virus quantity; Obtain viral purification liquid, being stored in-70 ℃ is that antigen is subsequent use.
Said indirect ELISA method comprises: envelope antigen, sealing, application of sample, add ELIAS secondary antibody, colour developing, termination.
The best operating condition of said indirect ELISA method is: 3200 times of antigen diluents, and 160 times of diluted samples, the ELIAS secondary antibody dilute concentration is 1:3000, the negative and positive critical value is 0.43.
Remarkable advantage of the present invention: the indirect ELISA method of the detection duckling virosis antibody of foundation; This method can be carried out effective diagnosis to the popular duckling virosis of present China; And can carry out large-scale seroepidemiological survey, for the control of duckling virosis and the grasp of popularity provide a kind of quick, easy detection method.
Embodiment
Embodiment 1
Duckling viral antigen of the present invention obtains through following mode:
(1) antigen concentrates
One strain of cultivating this research department's isolation identification with half kind of embryo in a large number has good immunocompetent flavivirus WR strain (the preserving number CGMCC No.4906 of bacterial strain; Derive from a patent of invention of applicant's application: application number is 201110143407.6; A kind of duckling virus and inactivated vaccine thereof); Collection contains behind the embryo allantoic liquid of virus in 4 ℃, centrifugal 30 min of 8000 r/min, and supernatant is in centrifugal 4 ℃ of centrifugal 2 h of 45000 r/min, and it is resuspended with sterilization PBS to abandon the supernatant postprecipitation; Obtain viral concentrate, be stored in-70 ℃.
(2) purifying of antigen
The purifying of antigen is to concentrate acquisition through sucrose gradient centrifugation and ultracentrifugation.The sucrose balance liquid of preparation 5 gradients such as 10%, 20%, 30%, 40% and 50% adds viral concentrate on 10% sucrose liquid, behind 4 ℃, centrifugal 2 h of 40000 r/min levels.Between 10%-20%, 30%-40%, 40%-50%, finding has the virus band, collects and respectively is with centrifugal 2 h of 45000 r/min under back 4 ℃, and it is resuspended with sterilization PBS to abandon the supernatant postprecipitation.And with each viral level of being with of RT-PCR method detection, maximum with 30%-40% band virus quantity, obtain viral purification liquid, being stored in-70 ℃ is that antigen is subsequent use.
Embodiment 2
Duckling virosis positive serum of the present invention and negative serum obtain through following mode:
(1) positive serum
With WR strain virus refined solution (the viral purification liquid that embodiment 1 step (2) obtains) with carrying out emulsification with Freund's complete adjuvant in the 1:1 ratio behind the formalin-inactivated; Immunity is grown up and is opened laying ducks then; Immunity once more behind 15 d at interval; Three exempt from back heart blood sampling, centrifugal collection serum, after 56 ℃ of deactivations and through neutralization test, prove duckling virosis positive serum, subsequent use.
(2) negative serum
Collection do not have the infected duck flavivirus or immunity cross the whole blood of the healthy duck of duckling virosis vaccine, supernatant is collected in centrifugal back, in 56 ℃ of deactivations, and proves duckling virosis negative serum through neutralization test.
Embodiment 3
The righttest antigen-antibody concentration, ELIAS secondary antibody bulk concentration and the critical value of duckling virosis antibody indirect ELISA detection method of the present invention obtain through following mode:
(1) the righttest antigen-antibody concentration
Measure with matrix method.Antigen (viral purification liquid) with purifying dilutes by 1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800,1:25600 with coating buffer; Encapsulate in 96 hole Costar ELISA Plates, 4 ℃ spend the night after, seal 2 h with after the PBST washing 3 times for 37 ℃ with 5% skim milk; With PBST washing 3 times; Yin and yang attribute serum is with 20 times, 40 times, 80 times, 160 times, 320 times and 640 times of PBS dilutions, and each yin and yang attribute serum titer repeats 4 times, with envelope antigen effect 2 h; Reacting behind 1 h washing 3 times with 1000 times ELIAS secondary antibody after the PBST washing 3 times, TMB develops the color behind 15 min with 2 mol/L H
2The SO4 cessation reaction, and measure the OD450 value in every hole.Measure by the ratio (P/N value) of the mean value of each titre of yin and yang attribute serum and to obtain; 3200 times of antigen diluents, when serum diluted 160 times, the P/N value was the highest; Up to 6.5; And the OD450 value of positive serum is 1.092 (≈ 1), so the best dilute concentration of antigen-antibody is 3200 times of antigen diluents, and 160 times of serum dilutions.
(2) ELIAS secondary antibody bulk concentration
Antigen with purifying dilutes by 1:3200 with coating buffer; Encapsulate in 96 hole Costar ELISA Plates, 4 ℃ spend the night after, seal 2 h with after the PBST washing 3 times with 5% skim milk; With PBST washing 3 times; Yin and yang attribute serum is with 160 times of PBS dilutions, and each yin and yang attribute serum titer repeats 4 times, with envelope antigen effect 2 h; After reacting 1 h with ELIAS secondary antibody (1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800 and 1:25600 dilution) after the PBST washing 3 times, wash 3 times, TMB develops the color behind 15 min with 2 mol/L H
2The SO4 cessation reaction, and measure the OD450 value in every hole.Measured by the ratio (P/N value) of the mean value of each titre of yin and yang attribute serum and to obtain, when ELIAS secondary antibody dilution 1:1600 and 1:3200, the two P/N value is the highest, reaches 14 and 8.16 respectively.But the OD450 value of positive serum is respectively 2.661 and 0.922; Adjustment ELIAS secondary antibody concentration (1:1500,1:2000,1:2500,1:3000 and 1:3500) redeterminates the P/N value of each titre; Obtain the P/N value maximum (the P/N value is 9) of 1:3000 ELIAS secondary antibody concentration, and OD450 value 1.094.So the best dilute concentration of ELIAS secondary antibody is 1:3000.
(3) critical value
Obtain through measuring 30 parts of negative serums.Antigen with purifying dilutes by 1:3200 with coating buffer, encapsulate in 96 hole Costar ELISA Plates, 4 ℃ spend the night after; Seal 2 h with after the PBST washing 3 times for 37 ℃ with 5% skim milk; With PBST washing 3 times, negative serum is with 160 times of PBS dilutions, with envelope antigen effect 2 h; After reacting 1 h with ELIAS secondary antibody (1:3000 dilution) after the PBST washing 3 times, wash 3 times, TMB develops the color behind 15 min with 2 mol/L H
2The SO4 cessation reaction, and measure the OD450 value in every hole.Trying to achieve mean value
is 0.2096; Standard deviation SD=0.0719; So negative and positive critical value=
+3SD=0.2096+3 * 0.079=0.426 ≈ 0.43; If it is promptly positive therefore to judge that sample OD value is higher than critical value, the subcritical value is promptly negative.
(4) specificity check
Cross H9 subtype avian influenza vaccine, H5 subtype avian influenza vaccine, egg drop syndrome vaccine, duck plague vaccine, duck Escherichia coli disease vaccine duck crowd serum antibody with immunity and carry out the specificity check.Gather each 10 parts of the duck crowd whole bloods that immunity crosses H9 subtype avian influenza vaccine, H5 subtype avian influenza vaccine, egg drop syndrome vaccine, duck plague vaccine, duck Escherichia coli disease vaccine, it is subsequent use that serum is collected in centrifugal back; Antigen (refined solution that embodiment 1 step (2) obtains) with purifying dilutes by 1:3200 with coating buffer; Encapsulate in 96 hole Costar ELISA Plates; 4 ℃ spend the night after, seal 2 h with after the PBST washing 3 times with 5% skim milk, wash 3 times with PBST; H9 subtype avian influenza, H5 subtype avian influenza, egg drop syndrome, duck plague, duck colibacillosis serum, positive serum, negative serum are diluted 160 times with PBS; With envelope antigen effect 2 h, after reacting 1 h with ELIAS secondary antibody (1:3000 dilution) after the PBST washing 3 times, wash 3 times, TMB develops the color behind 15 min with 2 mol/L H
2The SO4 cessation reaction, and measure the OD450 value in every hole.The duck crowd's of the immune H9 subtype avian influenza excessively of result vaccine, H5 subtype avian influenza vaccine, egg drop syndrome vaccine, duck plague vaccine, duck Escherichia coli disease vaccine the serum and the OD450 value of negative serum all are lower than 0.43, and positive serum OD450 value is higher than 0.43.
(5) repeatability check
Repeat 3 detections to positive and negative serum.Antigen with purifying dilutes by 1:3200 with coating buffer, encapsulate in 96 hole Costar ELISA Plates, 4 ℃ spend the night after; Seal 2 h with after the PBST washing 3 times with 5% skim milk; With PBST washing 3 times, positive serum and negative serum are with 160 times of PBS dilutions, with envelope antigen effect 2 h; After reacting 1 h with ELIAS secondary antibody (1:3000 dilution) after the PBST washing 3 times, wash 3 times, TMB develops the color behind 15 min with 2 mol/LH
2The SO4 cessation reaction, and measure the OD450 value in every hole.The OD450 value of negative serum all is lower than 0.43 as a result, and positive serum OD450 value all is higher than 0.43.
(6) to the detection instance of clinical sample
Certain egg duck field is raised 1500, has inoculated H5 by the routine immunization program, the H5+H9 avian influenza vaccine.Answer the requirement of field side, we gather 20 parts of blood sample separation of serum before laying eggs, and H5 and H9 antibody are detected, and H5 and H9 serum antibody are all greater than 4log as a result
2Laying rate was 85% when the peak was laid eggs in this duck field; Laying rate dropped to 30% suddenly in 7 days afterwards; We gather 30 parts of blood sample separation of serum, add 20 parts of serum that detect H5 and H9 antibody simultaneously, detect by the duckling virosis serum antibody indirect ELISA detection method of having built up.Serum antibody OD450 all was lower than 0.43 before the result laid eggs, and the highest is 0.28; The descend OD450 value of back serum antibody of laying rate is uneven, only has 33% serum to be lower than 0.43, and other all is higher than critical value, is up to 1.582, diagnoses this duck crowd infected duck flavivirus with this.
List of references
(1) Cao Zhenzhen opens and deposits Huang Yu, Diao Youxiang, Ye Weicheng, Liu Yuehuan, Han Jingwen; Malaysian is bright, Zhang Dongdong, and Xu Feng, wangdan, Jiang Tiantian, Yuan Yuan thanks to light rain; Gao Xuhui, Tang Yi, Shi Shaohua, Wan Chun with, Zhang Chen, He Fen, Yang Mengjie; Lu Xinhao, Zhang Bing, Zhang Guozhong, Ma Xuejun magnifies third. the Primary Study [J] of duck hemorrhagic oaritis. Chinese animal doctor's magazine, 2010,46 (12): 3-6.
(2) Wan Chun and, Shi Shaohua, Cheng Longfei, Chen Hongmei, Fu Guanghua magnifies the third, Lin Fang, Lin Jiansheng, Huang Yu. a kind of kind of (egg) duck the lay eggs separation and Preliminary Identification [J] of rapid drawdown new virus of causing. Fujian agriculture journal, 2010,25 (6): 663-666.
(3) Wan Chun with, Shi Shaohua, Cheng Longfei, Chen Hongmei, Fu Guanghua, Peng Chunxiang, Lin Fang, Lin Jiansheng, Huang Yu. the foundation [J] of duck hemorrhagic oaritis virus RT-PCR detection method. Fujian agriculture journal, 2011,26 (1): 10-12.
（4）Jingliang?Su,?Shuang?Li，Xudong?Hu,?Xiuling?Yu,?Yongyue?Wang,?Peipei?Liu,?Xishan?Lu,?Guozhong?Zhang,?Xueying?Hu,?Di?Liu,?Xiaoxia?Li,?Wenliang?Su,?Hao?Lu,?Ngai?Shing?Mok,?Peiyi?Wang,?Ming?Wang,?Kegong?Tian,?George?F.?Gao.?Duck?Egg-Drop?Syndrome?Caused?by?BYD?Virus,?a?New?Tembusu-Related?Flavivirus?[J].PLoS?One.,2011?Mar?24;6(3):e18106
Claims (7)
1. an indirect ELISA method that detects duckling virosis serum antibody is characterized in that: as envelope antigen, set up the indirect ELISA method that detects duckling antiviral antibody in the duck serum with duckling virus.
2. the indirect ELISA method of detection duckling virosis serum antibody according to claim 1 is characterized in that: said duckling virus is duckling virus
(Duck flavivirus)WR strain, preservation registration number are CGMCC NO.4906.
3. the indirect ELISA method of detection duckling virosis serum antibody according to claim 1; It is characterized in that: said antigen is earlier through concentrating; Comprise the steps: to collect behind the embryo allantoic liquid that contains duckling virus in 4 ℃, centrifugal 30 min of 8000 r/min, supernatant is in centrifugal 4 ℃ of centrifugal 2 h of 45000 r/min, and it is resuspended with sterilization PBS to abandon the supernatant postprecipitation; Obtain viral concentrate, be stored in-70 ℃.
4. the indirect ELISA method of detection duckling virosis serum antibody according to claim 1 is characterized in that: said viral concentrate is further purified through sucrose gradient centrifugation, concentrates through ultracentrifugation to obtain again.
5. the indirect ELISA method of detection duckling virosis serum antibody according to claim 4; It is characterized in that: the step that is further purified is: the sucrose balance liquid of 5 gradients such as preparation 10%, 20%, 30%, 40% and 50%; Viral concentrate is added in 10% sucrose liquid upper strata, in 4 ℃, centrifugal 2 h of 40000 r/min levels; The result finds to have the virus band between 10%-20%, 30%-40%, 40%-50%; Collect and respectively be with centrifugal 2 h of 45000 r/min under back 4 ℃; It is resuspended with sterilization PBS to abandon the supernatant postprecipitation, and detects the viral level that each is with the RT-PCR method, maximum with 30%-40% band virus quantity; Obtain viral purification liquid, being stored in-70 ℃ is that antigen is subsequent use.
6. the indirect ELISA method of detection duckling virosis serum antibody according to claim 1, it is characterized in that: said indirect ELISA method comprises: envelope antigen, sealing, application of sample, add ELIAS secondary antibody, colour developing, termination.
7. the indirect ELISA method of detection duckling virosis serum antibody according to claim 6; It is characterized in that: the best operating condition of said indirect ELISA method is: 3200 times of antigen diluents; 160 times of diluted samples, ELIAS secondary antibody dilute concentration are 1:3000, and the negative and positive critical value is 0.43.
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| CN103320541A (en) * | 2013-07-11 | 2013-09-25 | 广西壮族自治区兽医研究所 | Duck flavivirus and duck plague virus duplex RT-PCR kit |
| CN103675275A (en) * | 2013-12-30 | 2014-03-26 | 山东滨州博莱威生物技术有限公司 | Duck flavivirus detection reagent kit |
| CN103777011A (en) * | 2014-02-07 | 2014-05-07 | 贵州大学 | Indirect ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting duck plague virus IgG antibody and preparation method |
| CN103837682A (en) * | 2014-02-28 | 2014-06-04 | 北京市兽医实验诊断所 | Duck novel barly yellow dwarf virus (BYDV) antibody enzyme linked immunosorbent assay (ELISA) detection kit |
| CN103901213A (en) * | 2014-03-31 | 2014-07-02 | 山东农业大学 | Detection method for distinguishing generation of antibody through activated vaccine and inactivated vaccine of tembusu virus |
| CN105543178A (en) * | 2015-12-24 | 2016-05-04 | 天津市中升挑战生物科技有限公司 | Agar gel precipitin antigen of duck hepatitis virus, preparation method for agar gel precipitin antigen and application of agar gel precipitin antigen |
| CN110208518A (en) * | 2019-05-28 | 2019-09-06 | 福建省农业科学院畜牧兽医研究所 | Indirect ELISA detection method and its detection kit based on 3 type adenovirus antibody of Fiber1 Protein Detection duck |
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| CN103320541A (en) * | 2013-07-11 | 2013-09-25 | 广西壮族自治区兽医研究所 | Duck flavivirus and duck plague virus duplex RT-PCR kit |
| CN103320541B (en) * | 2013-07-11 | 2016-06-08 | 广西壮族自治区兽医研究所 | Duck flavivirus and duck plague virus duplex RT-PCR detection kit |
| CN103675275A (en) * | 2013-12-30 | 2014-03-26 | 山东滨州博莱威生物技术有限公司 | Duck flavivirus detection reagent kit |
| CN103777011A (en) * | 2014-02-07 | 2014-05-07 | 贵州大学 | Indirect ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting duck plague virus IgG antibody and preparation method |
| CN103837682A (en) * | 2014-02-28 | 2014-06-04 | 北京市兽医实验诊断所 | Duck novel barly yellow dwarf virus (BYDV) antibody enzyme linked immunosorbent assay (ELISA) detection kit |
| CN103837682B (en) * | 2014-02-28 | 2016-08-10 | 北京市兽医实验诊断所 | A kind of duck novel banzi virus BYDV antibody ELISA detection kit |
| CN103901213A (en) * | 2014-03-31 | 2014-07-02 | 山东农业大学 | Detection method for distinguishing generation of antibody through activated vaccine and inactivated vaccine of tembusu virus |
| CN103901213B (en) * | 2014-03-31 | 2016-04-13 | 山东农业大学 | A kind of tembusu virus poison of living of distinguishing produces the detection method of antibody with inactivated vaccine |
| CN105543178A (en) * | 2015-12-24 | 2016-05-04 | 天津市中升挑战生物科技有限公司 | Agar gel precipitin antigen of duck hepatitis virus, preparation method for agar gel precipitin antigen and application of agar gel precipitin antigen |
| CN110208518A (en) * | 2019-05-28 | 2019-09-06 | 福建省农业科学院畜牧兽医研究所 | Indirect ELISA detection method and its detection kit based on 3 type adenovirus antibody of Fiber1 Protein Detection duck |
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