鸭源鸡杆菌抗体消长规律研究及抗脂多糖单抗杂交瘤细胞株的建立
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摘要
鸭源鸡杆菌(Gallibacterium anatis)是巴氏杆菌科,鸡杆菌属的代表种。该菌可参与感染产蛋母鸡,引发一系列慢性、进行性生殖道疾病,如输卵管炎、输卵管囊肿,是引起产蛋量下降的一个重要病原体。近年来,国内外对于鸭源鸡杆菌的研究主要是针对其致病性和发病机理,对于鸭源鸡杆菌的血清学研究较少。
本研究利用实验室保存的鸭源鸡杆菌菌株YU-PDS-RZ-1-SLG制备超声波裂解抗原,建立了可以检测鸭源鸡杆菌所有血清型抗体的间接ELISA方法:包被抗原浓度为10μg·mL~(-1),包被条件为37℃2h,然后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,酶标二抗工作浓度为1:1000,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3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1:10稀释后的OD450,根据感染后不同阶段所测OD450绘制抗体消长曲线,大致了解了其抗体消长的规律:抗体水平在感染后2个月左右达到高峰,但维持时间较短,随后逐渐下降。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、免疫程序的制定及免疫效果的评价提供了手段。
利用实验室保存的鸭源鸡杆菌菌株YU-PDS-RZ-1-SLG提取鸭源鸡杆菌脂多糖(lipopolysaccharides,LPS)作为检测抗原,确定ELISA方法的最佳工作条件:包被抗原的浓度为2.77μg·mL~(-1),包被条件为37℃1.5h,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1:1600,酶标二抗工作浓度为1:3000,底物显色时间为15min。
将鸭源鸡杆菌裂解抗原及提取的脂多糖用PBS稀释到适当浓度,加入弗氏完全/不完全佐剂制成乳化抗原免疫Balb/c小鼠。按常规方法进行融合,用间接ELISA方法进行筛选,采用有限稀释法,经过4次亚克隆,最终获得3株抗鸭源鸡杆菌脂多糖的杂交瘤细胞株,分别命名为1H11、1B12和4D4。经过长期的体外培养和长期的冻存复苏后,3株杂交瘤细胞分泌抗体的能力略有降低,但仍能均一分泌抗体。间接ELISA方法检测3株杂交瘤细胞上清效价分别为:1:3200、1:6400、1:3200,小鼠腹水效价分别为1:80000、1:100000和1:80000。交叉试验结果显示,所制备的3株单抗与多杀性巴氏杆菌、羊布氏杆菌、鸡福氏志贺菌及大肠杆菌均无交叉反应。经鉴定,3株单抗的亚型分别为IgM、IgG2a、IgG2a。制备的3株能够分泌抗鸭源鸡杆菌脂多糖单克隆抗体的杂交瘤细胞株,为鸭源鸡杆菌病原的快速诊断方法的建立及其他相关科学研究提供了技术保障。
Gallibacterium anatis (G.anatis) is the type species of Gallibacterium which is grouped as a new genus within the family Pasteurellaceae. As a important pathogen to affect the egg production, G.anatis could infect layers to cause a series of chronic and progressive diseases of reproductive tracts, such as salpingitis and oviduct cysts. The researches mainly studied on the pathogenicity and etiopathogenesis of G.anatis in recent years, but there is a little study on serology of G. anatis.
A indirect enzyme-linked immunosorbent assay (ELISA) was developed in this study for detection of all serotypes antibodies against G.anatis in chickens. The coating antigen was made by ultrasonie method with the strain YU-PDS-RZ-1-SLG (1-S) of G.anatis. The optimum G.anatis antigen concentration was found to be 10μg·mL~(-1), coating time was 2 hours at 37℃and then overnight at 4℃. The blocking material was 1% gelatin and blocking time was 1 hour at 37℃. The dilution of the positive or negative sera was 1:100, and the optimum dilution of rabbit-anti-chicken IgG labelled with horseradish peroxidase was 1:1000. The optimum reaction time of the enzyme-substrate was determined as 15 min. The indirect ELISA was confirmed to be reproducible and specific by cross-assay, blocking-assay and reproductive assay. The coefficient of variation within plate was 2.01%~5.75%, and the coefficient of variation with between plates was 2.43%~6.20%. The sensitivity of indirect ELISA was 25 to 100 times than micro-agglutination test. Three groups of 20 three-day-old SPF chinkens were used and divided into 3 groups, one group was inoculated with 1.4×10~6 colony-forming units of G.anatis strain 1-S, the other two groups were used as cohabitating group and negative control group. The indirect ELISA technique was used for testing the antibodies against G.anatis from these chickens in different time after infection. The response curve was established when the OD450nm values were varies along with the time, the changes of the antibodies were observed that the antibody level reached the peak in two months post-infection,but the lasting duration of the antibodies was short,and then the antibody level dropped gradually. The results showed that the indirect ELISA could be used for detecting the sera of chickens from clinical cases and to predict the efficiency of vaccination.
Lipopolysaccharide(LPS) obtained from G.anatis strain 1-S was used as coating antigen for detection of antibody against G.anatis, and the optimum work conditions for indirect ELISA were developed as follows: the concentration of coating antigen was 2.77μg·mL~(-1) ;coating time at 37℃was 1.5 hours and then at 4℃overnight; the blocking material was 1% gelatin and blocking time at 37℃was 1 hour; the dilution of the positive or negative sera was 1:1600; the optimum dilution of goat-anti-mouse IgG labelled horseradish peroxidase was 1:3000, and the optimum reaction time of the enzyme-substrate was determined as 15 min.
Three hybridoma cell strains secreting monoclonal antibodies (McAbs) against G. anatis LPS were developed by cell fusion after immunizing the Balb/c mice with G. anatis LPS antigen and named as 1H11, 1B12 and 4D4. These hybridoma cell strains can stably secrete McAbs after twenty-five serial passages and freezing-thawing three times within a three-month storage at -196℃.The ELISA titers of antibodies against G. anatis LPS in culture supernatant were 1:3200, 1:6400 and 1:3200 ,and in ascites were 1:80000, 1:100000, 1:80000 for strains 1H11, 1B12 and 4D4, respectively. Pathogen detection of indirect ELISA showed the McAb had no cross-reaction with Pasteurella multocida, Brucella melitensis, Chicken Sh. flexneri and E.coli.The subtypes of three McAbs were IgM, IgG2a and IgG2a, respectively. The development of the three hybridoma cell strains secreting McAbs against G.anatis LPS laid the foundation for rapid and accurate monitoring of pathogen diagnosis and relevant scientific research of G.anatis in chickens.
本研究利用实验室保存的鸭源鸡杆菌菌株YU-PDS-RZ-1-SLG制备超声波裂解抗原,建立了可以检测鸭源鸡杆菌所有血清型抗体的间接ELISA方法:包被抗原浓度为10μg·mL~(-1),包被条件为37℃2h,然后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,酶标二抗工作浓度为1:1000,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3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1:10稀释后的OD450,根据感染后不同阶段所测OD450绘制抗体消长曲线,大致了解了其抗体消长的规律:抗体水平在感染后2个月左右达到高峰,但维持时间较短,随后逐渐下降。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、免疫程序的制定及免疫效果的评价提供了手段。
利用实验室保存的鸭源鸡杆菌菌株YU-PDS-RZ-1-SLG提取鸭源鸡杆菌脂多糖(lipopolysaccharides,LPS)作为检测抗原,确定ELISA方法的最佳工作条件:包被抗原的浓度为2.77μg·mL~(-1),包被条件为37℃1.5h,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1:1600,酶标二抗工作浓度为1:3000,底物显色时间为15min。
将鸭源鸡杆菌裂解抗原及提取的脂多糖用PBS稀释到适当浓度,加入弗氏完全/不完全佐剂制成乳化抗原免疫Balb/c小鼠。按常规方法进行融合,用间接ELISA方法进行筛选,采用有限稀释法,经过4次亚克隆,最终获得3株抗鸭源鸡杆菌脂多糖的杂交瘤细胞株,分别命名为1H11、1B12和4D4。经过长期的体外培养和长期的冻存复苏后,3株杂交瘤细胞分泌抗体的能力略有降低,但仍能均一分泌抗体。间接ELISA方法检测3株杂交瘤细胞上清效价分别为:1:3200、1:6400、1:3200,小鼠腹水效价分别为1:80000、1:100000和1:80000。交叉试验结果显示,所制备的3株单抗与多杀性巴氏杆菌、羊布氏杆菌、鸡福氏志贺菌及大肠杆菌均无交叉反应。经鉴定,3株单抗的亚型分别为IgM、IgG2a、IgG2a。制备的3株能够分泌抗鸭源鸡杆菌脂多糖单克隆抗体的杂交瘤细胞株,为鸭源鸡杆菌病原的快速诊断方法的建立及其他相关科学研究提供了技术保障。
Gallibacterium anatis (G.anatis) is the type species of Gallibacterium which is grouped as a new genus within the family Pasteurellaceae. As a important pathogen to affect the egg production, G.anatis could infect layers to cause a series of chronic and progressive diseases of reproductive tracts, such as salpingitis and oviduct cysts. The researches mainly studied on the pathogenicity and etiopathogenesis of G.anatis in recent years, but there is a little study on serology of G. anatis.
A indirect enzyme-linked immunosorbent assay (ELISA) was developed in this study for detection of all serotypes antibodies against G.anatis in chickens. The coating antigen was made by ultrasonie method with the strain YU-PDS-RZ-1-SLG (1-S) of G.anatis. The optimum G.anatis antigen concentration was found to be 10μg·mL~(-1), coating time was 2 hours at 37℃and then overnight at 4℃. The blocking material was 1% gelatin and blocking time was 1 hour at 37℃. The dilution of the positive or negative sera was 1:100, and the optimum dilution of rabbit-anti-chicken IgG labelled with horseradish peroxidase was 1:1000. The optimum reaction time of the enzyme-substrate was determined as 15 min. The indirect ELISA was confirmed to be reproducible and specific by cross-assay, blocking-assay and reproductive assay. The coefficient of variation within plate was 2.01%~5.75%, and the coefficient of variation with between plates was 2.43%~6.20%. The sensitivity of indirect ELISA was 25 to 100 times than micro-agglutination test. Three groups of 20 three-day-old SPF chinkens were used and divided into 3 groups, one group was inoculated with 1.4×10~6 colony-forming units of G.anatis strain 1-S, the other two groups were used as cohabitating group and negative control group. The indirect ELISA technique was used for testing the antibodies against G.anatis from these chickens in different time after infection. The response curve was established when the OD450nm values were varies along with the time, the changes of the antibodies were observed that the antibody level reached the peak in two months post-infection,but the lasting duration of the antibodies was short,and then the antibody level dropped gradually. The results showed that the indirect ELISA could be used for detecting the sera of chickens from clinical cases and to predict the efficiency of vaccination.
Lipopolysaccharide(LPS) obtained from G.anatis strain 1-S was used as coating antigen for detection of antibody against G.anatis, and the optimum work conditions for indirect ELISA were developed as follows: the concentration of coating antigen was 2.77μg·mL~(-1) ;coating time at 37℃was 1.5 hours and then at 4℃overnight; the blocking material was 1% gelatin and blocking time at 37℃was 1 hour; the dilution of the positive or negative sera was 1:1600; the optimum dilution of goat-anti-mouse IgG labelled horseradish peroxidase was 1:3000, and the optimum reaction time of the enzyme-substrate was determined as 15 min.
Three hybridoma cell strains secreting monoclonal antibodies (McAbs) against G. anatis LPS were developed by cell fusion after immunizing the Balb/c mice with G. anatis LPS antigen and named as 1H11, 1B12 and 4D4. These hybridoma cell strains can stably secrete McAbs after twenty-five serial passages and freezing-thawing three times within a three-month storage at -196℃.The ELISA titers of antibodies against G. anatis LPS in culture supernatant were 1:3200, 1:6400 and 1:3200 ,and in ascites were 1:80000, 1:100000, 1:80000 for strains 1H11, 1B12 and 4D4, respectively. Pathogen detection of indirect ELISA showed the McAb had no cross-reaction with Pasteurella multocida, Brucella melitensis, Chicken Sh. flexneri and E.coli.The subtypes of three McAbs were IgM, IgG2a and IgG2a, respectively. The development of the three hybridoma cell strains secreting McAbs against G.anatis LPS laid the foundation for rapid and accurate monitoring of pathogen diagnosis and relevant scientific research of G.anatis in chickens.
引文
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