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鸭源鸡杆菌抗体间接ELISA检测方法的建立及应用 被引量:3

Development and primary application of indirect ELISA for detecting antibody against G. anatis
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摘要 利用鸭源鸡杆菌YU-PDS-RZ-1-SLG分离株制备超声波裂解抗原,建立了可以检测鸭源鸡杆菌多个血清型抗体的间接ELISA方法。包被抗原质量浓度为10mg/L,包被条件为37℃2h,再4℃过夜;封闭液为1%明胶,封闭条件为37℃1h;阴、阳性血清最佳稀释度为1∶100,酶标二抗工作滴度为1∶1 000;底物显色时间为15min。经交叉性试验、阻断试验和重复性试验证实建立的ELISA方法重复性好,特异性强。板内变异系数为2.01%~5.75%,板间变异系数为2.43%~6.20%。间接ELISA方法的灵敏度是微量凝集试验的25~100倍。利用所建立的ELISA方法检测了人工感染鸭源鸡杆菌的4日龄SPF鸡在感染后不同阶段感染组、同居组和空白对照组的血清抗体,并根据感染后不同阶段所测D450值绘制抗体消长曲线,其抗体水平在感染后32~47d开始上升,60d时达到高峰,但维持时间较短,2周后迅速下降。建立的间接ELISA方法可以用于临床病例的血清学快速检测,为进行鸭源鸡杆菌的血清流行病学调查提供了手段。 Indirect enzyme-linked immunosorbent assay(ELISA) was developed for detection of antibodies against all serotype Gallibacterium anatis (G. anatis) in chickens. The coating antigen was prepared by ultrasonic method with G. anatis strain YU-PDS-RZ-1-SLG. The optimum G. anatis antigen concentration was 10 mg/L, and coating time was 2 hours at 37℃ and then over night at 4℃. The blocking material was 1% gelatin and blocking time was 1 hour at 37℃. The dilution of the positive and negative sera was 1 : 100, and the optimum dilution of rabbit-anti-chicken IgG labeled with horseradish peroxidase was 1 : 1 000. The optimum reaction time of the enzyme-substrate was 15 min. The indirect ELISA was confirmed reproducible and specific by cross-assay,blocking-assay and reproductive as- say. The coefficient of variation within plate was 2.01 %-5.75 % ,and the coefficient of variation between plates was 2.43%-6.20%. The sensitivity of indirect ELISA was 25 to 100 times than that of micro-agglutination test. Twenty 4-day-old SPF chickens were used and divided into 3 groups,one group was inoculated with 1.4 × 106 colony-forming units of G. anatis strain, the other two groups were used as cohabitating group and negative control group, separate- ly. The indirect ELISA was used for testing the antibody against G. anatis from these chickens in different time after infection. The response curve was established when the D450 nm values varied along with the time. The antibody level reached the peak at the 60th day post-infection, and then dropped rapidly. The results showed that the indirect ELISA could be used for detecting the serum antibody of chickens in the epidemiology survey of G. anatis.
出处 《中国兽医学报》 CAS CSCD 北大核心 2012年第8期1130-1135,共6页 Chinese Journal of Veterinary Science
基金 中德(Boehringer Ingelheim Vetmedica)合作资助项目(43006167)
关键词 鸭源鸡杆菌 间接ELISA 人工感染 抗体测定 G. anatis indirect ELISA experimental infection antibody detection
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参考文献13

  • 1Christensen H, Bisgaard M,Bojesen A M, et al. Genetic relationships among avian isolates classified as Pasteu- rella haemolytica , "Actinobacillus salpingitidis " or Pasteurella anatis with proposal of Gallibacterium anatis gen. nov. , comb. nov. and description of addi- tional genomospecies within Gallibacterium gen. nov [J]. Int J Syst Evol Microbiol,2003,53:275-287.
  • 2Bisgaard M, Korczak B, Busse H, et al. Classification of the taxon 2 and taxon 3 complex of bisgaard within Gallibacterium and description of Gallibacterium melopsittaci sp. nov. ,Gallibacterium trehalosifermen- tans sp. nov. and Gallibacterium salpingitidis sp. nov [J]. Int J Syst Evol Microbiol, 2009,59 : 735-744.
  • 3Bojesen A M,Vazquez M E,Bager R J ,et al. Antimicro bial susceptibility and tetracycline resistance determi nant genotyping of Gallibacterium anatis [J]. Vet Mi crobiol, 2011,148(1) : 105-110.
  • 4Bojesen A M,Nielsen O L,Christensen J P,et al. In vi- vo studies of Gallibacterium anatis infection in chick- ens[J]. Avian Pathol, 2004,33 (2) : 145-152.
  • 5Bisgaard M. Ecology and significance of pasteurellaceae in animals[J]. Zentralblatt fur Bakteriologie, 1993,279 (1) :7-26.
  • 6Bojesen A M, Nielsen S S, Bisgaard M. Prevalence and transmission of haemolytic Gallibacterium species in chicken production systems with different biosecurity levels[J]. Avian Pathol,2003,32(5) :503-510.
  • 7杨立国 胡少昶 魏平华.酶免疫测定技术[M].南京:南京大学出版社,1998.189-284.
  • 8汪家政,范明,主编.蛋白质技术手册[M].北京:科学出版社,2001.42-47.
  • 9王川庆,陈陆,杨霞,皇甫和平,刘红英,彭志峰,郑鹿平,徐雪,刘惠敏,付仁一,郭轮涛.蛋鸡群卡氏杆菌感染情况的初步研究[J].河南农业科学,2008,37(3):97-100. 被引量:43
  • 10陈陆,付仁一,杨霞,王新卫,皇甫和平,刘慧敏,郑鹿平,郭伦涛,徐雪,王川庆.一种新禽病——禽卡氏杆菌病的研究进展[J].中国兽医科学,2010,40(2):215-219. 被引量:4

二级参考文献57

  • 1刘亚刚,刘内生,岳华,袁圣蓉,汤承,马蓉,廖品丽,刘斌,王文聪,陈自勤,蒋朝龙,余琼,李建,朱锦,毛依哈.安宁河流域鸭瘟鸭霍乱综合防制措施的研究[J].四川畜牧兽医,1996,23(1):10-12. 被引量:2
  • 2刘亚刚,刘内生,岳华,汤承,袁圣蓉,马蓉,李建,余琼,蒋朝龙,李志忠,杨新智,顾志堂.安宁河流域鸭瘟、鸭霍乱综合防制研究[J].中国兽医学报,1996,16(4):356-358. 被引量:4
  • 3刘亚刚,刘内生,岳华,袁圣蓉,汤承,马蓉,李建,余琼,蒋朝龙,马正忠,刘斌,廖品丽,撒建军.鸭多杀性巴氏杆菌母源抗体及其类型的研究[J].中国兽医科技,1996,26(6):24-25. 被引量:2
  • 4BISGAARD M.Ecology and significance of pasteurellaceae in animals[J].Zentralblal fur Bakteriologie,1993,279(1):7-26.
  • 5BOJESEN A M,NIELSEN S S,BISGAARD M.Prevalence and transmission of haemolytic Gallibacterium species in chicken production systems with different biosecurity levels[J].Avian Pathol,2003,32(5):503-510.
  • 6BOJESEN A M,NIELSEN O I,CHRISTENSEN J P,et al.In vivo studies of Gallibacterium anatis infection in chickens[J].Avian Pathol,2004,33(2):145-152.
  • 7MUTTERS R,IHM P,POHL S,et al.Reclassification of the genus Pasteurella trevisan 1887 on the basis of deoxyribonucleic acid homology,with proposals for the new species Pasteurella dag mat is,Pasteurella canis,Pasteurella stomatis,Pasteurella anatis,and Pasteurella langaa[J].Int J Syst Bacteriol,1985,35(3):309-322.
  • 8CHRISTENSEN H,BISGAARD M,BOJESEN A M,et al.Genetic relationships among avian isolates classified as Pasteurella haemolytica,Actinobacillus salpingitidis' or Pasteurella anatis with proposal of Gallibacterium anatis gen.nov,comb.nov.and description of additional genomospecies within Gallibacterium gen.nov.[J].Int J Syst Evol Microbiol,2003,53(1):275-287.
  • 9BISGAARD M,KORCZAK B,BUSSE H J,et al.Classification of the taxon 2 and taxon 3 complex of Bisgaard within Gallibacterium and description of Gallibacterium melopsittaci sp.nov,Gallibacterium trehalosifermentans sp.nov.and Gallibacterium salpingitidis sp.nov.[J].Int J Syst Evol Microbiol,2009,59(4):735-744.
  • 10BOJESEN A M,TORPDAHL M,CHRISTENSEN H,et al.Genetic diversity of Gallibacterium anatis isolates from different chicken flocks[J].J Clin Microbiol,2003,41(6):2737-2740.

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