鸡鲍氏志贺菌疫苗免疫抗体间接ELISA定量检测方法的建立及应用
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摘要
鸡鲍氏志贺氏菌病是近年所发现的鸡的一种新发传染病,该病是由鸡鲍氏志贺氏菌引起的一种急性传染病,可使不同品种、不同日龄的鸡发生腹泻,并可引起幼雏大批发病和死亡。且有关研究结果已表明：志贺氏菌可在人禽间造成交互感染。因此,对该病展开研究不但具有重要的兽医卫生学意义,而且具有重大的医学公共卫生学意义。本研究通过建立间接ELISA方法,并应用于鸡鲍氏志贺氏菌灭活疫苗免疫鸡血清抗体的检测,为正确评价疫苗免疫效果、制定合理免疫程序、有效预防和控制本病提供了准确可靠的血清学检测手段和方法。研究结果如下：1.间接ELISA方法的建立
本研究以甲醛灭活的鸡鲍氏志贺氏菌全菌体为包被抗原,以辣根过氧化物酶(HRP)标记的兔抗鸡IgG为二抗,成功建立了检测鸡鲍氏志贺氏菌灭活疫苗免疫抗体的间接ELISA方法,并确定了ELISA的最佳工作条件：抗原包被浓度为108cfu/ml,最佳包被温度为4℃12h；最佳封闭条件为1%BSA37℃封闭2h；血清最佳稀释度为1：160；二抗最佳稀释度为1：4000,最佳工作条件为37℃1h；底物最佳工作条件为37℃20min。通过交叉试验、特异性试验、敏感性试验、重复性试验等结果表明该方法具有特异性强、灵敏度高、重复性好等优点。其中板内变异系数0.36%～0.64%,板间变异系数为1.05%～1.85%；包被的全菌体抗原与其他腹泻病原的抗体无交叉反应,阻断结果为阳性；间接ELISA方法灵敏度是微量凝集试验的80-160倍。
分别检测20份阳性鸡血清不同稀释度的OD值,发现阳性血清OD值与血清中ELISA效价倒数的对数之间均存在线性相关性。根据拟和的标准曲线,确定回归方程为：log[ET倒数]=1.776+1.921x。回归方程的建立为临床免疫后根据血清OD值来预测和分析抗体水平提供了数学模型。
2.间接ELISA方法应用于鸡鲍氏志贺氏菌灭活疫苗免疫鸡血清抗体检测
为了评价鸡鲍氏志贺氏菌灭活疫苗的体液免疫效果,应用建立的间接ELISA方法对疫苗免疫后的鸡血清抗体进行检测,结果表明免疫组鸡群的免疫抗体效价明显高于同期的未免疫对照组(p<0.05),二免后的抗体水平明显高于一免(p<0.05)。鸡群在一免后抗体水平迅速升高,第2周即可达到最高峰,在二免后第9周仍高于一免(p<0.05)。一免后第4周二免组和一免后第6周二免组二者同期抗体水平差异不明显(p>0.05)。结果表明,鸡鲍氏志贺氏菌灭活疫苗能够显著提高机体的体液免疫功能；所建立的间接ELISA方法能够准确的评价鸡鲍氏志贺氏菌疫苗的免疫效果；该疫苗评价方法为鸡鲍氏志贺氏菌疫苗免疫抗体水平的测定、免疫程序的合理制定、有效预防和控制本病提供了一个良好的技术手段,便宜于在生产中推广应用。
The S.boydii disease is a new infectious disease Against chickens, which found in recent years.The disease is an acute disease caused by Shigella.It can lead to lots of different breed and day-old chicken occurre diarrhea and died. The disease has possibility of cross-infection between human and birds,which has strong public health significance.In this study, an indirect ELISA method was established and applied to detecting serum antibody angainst inactivated chicken S.boydii vaccine.It provides an serological detection method for evaluating the immune effect of chicken S.boydii vaccine to develop the reasonable immunization procedure for preventing or controlling disease effectively. The results are as follows:
1.The establishment of indirect ELISA method.
In this study, Chicken S.boydii whole thallus was used as a coated antigen inactivated by formaldehyde.Rabbit anti-chicken IgG labeled by horseradish peroxidase (HRP) was used as the second antibody. The indirect ELISA method was established for the detection of inactivated vaccine immune antibody of chicken S.boydii, and the best work of the ELISA conditions were determined:the concentration of coated antigen was 108 cfu/ml,the best temperature and time for coating was at 4℃in 12h; the best conditions for closing was used 1% BSA at 37℃in 2h; the best dilution of serum was 1:160;the best dilution of second antibody was 1:4000, and the best working conditions was at 37℃in lh; the best working conditions of substrate was at 37℃in 20min. The cross-testing, specificity, sensitivity tests, and repeatability tests show that the method has good reproducibility, great specificity, high sensitivity. The coefficient of variation of one-plate was 0.36%～0.64%, and 1.05%～1.85% of inter-plate; the coated whole cell antigen and other diarrheal diseases had no cross-reactivity, the results of blocking were positive. The sensitivity of indirect ELISA method is 80-160 times as much as micro-agglutination test.
The OD values of 20 different positive chicken serum in different dilution were detected and found that linear correlation existed between the OD value of positive serum and the logarithm of ELISA titer reciprocal of serum.The regression equation of log [ET countdown]= .776+1.921 x was determined under the proposed standard curve.The establishment of the regression equation provided a mathematical model for forecasting and analyzing antibody levels by the serum OD values after clinical immunity.
2.Indirect ELISA used in chicken S.boydii inactivated vaccine serum antibody detection.
In order to evaluate the humoral immune effects of chicken S.boydii inactivated vaccine, the indirect ELISA method were applied to testing the post-vaccination antibody. The results showed that the immune antibody titer of immunity class was significantly higher than non immunity class (p<0.05)in the same period, the antibody levels of the second immunization were significantly higher than the first immunization (p<0.05).The antibody levels of immunity class increased rapidly after immunization and reached a peak in the two weeks.Nine weeks after the second immunization was still higher than the first immunization (p<0.05).The antibody levels of the second immunization in the forth week or the sixth weeks after the first immunization didn't have obvious difference in the same period (p> 0.05).The results showed that chicken S.boydii inactivated vaccine can improve the body's humoral immune function significantly;The established indirect ELISA method can evaluate the effects of chicken S.boydii vaccine truly; the vaccine and evaluation methods provide a good technical for testing S.boydii vaccine antibody and establishing the rational immune program.It will provide a good technical for preventing or controlling the disease.It is suit for applying and popularizing in production cheaply.
本研究以甲醛灭活的鸡鲍氏志贺氏菌全菌体为包被抗原,以辣根过氧化物酶(HRP)标记的兔抗鸡IgG为二抗,成功建立了检测鸡鲍氏志贺氏菌灭活疫苗免疫抗体的间接ELISA方法,并确定了ELISA的最佳工作条件：抗原包被浓度为108cfu/ml,最佳包被温度为4℃12h；最佳封闭条件为1%BSA37℃封闭2h；血清最佳稀释度为1：160；二抗最佳稀释度为1：4000,最佳工作条件为37℃1h；底物最佳工作条件为37℃20min。通过交叉试验、特异性试验、敏感性试验、重复性试验等结果表明该方法具有特异性强、灵敏度高、重复性好等优点。其中板内变异系数0.36%～0.64%,板间变异系数为1.05%～1.85%；包被的全菌体抗原与其他腹泻病原的抗体无交叉反应,阻断结果为阳性；间接ELISA方法灵敏度是微量凝集试验的80-160倍。
分别检测20份阳性鸡血清不同稀释度的OD值,发现阳性血清OD值与血清中ELISA效价倒数的对数之间均存在线性相关性。根据拟和的标准曲线,确定回归方程为：log[ET倒数]=1.776+1.921x。回归方程的建立为临床免疫后根据血清OD值来预测和分析抗体水平提供了数学模型。
2.间接ELISA方法应用于鸡鲍氏志贺氏菌灭活疫苗免疫鸡血清抗体检测
为了评价鸡鲍氏志贺氏菌灭活疫苗的体液免疫效果,应用建立的间接ELISA方法对疫苗免疫后的鸡血清抗体进行检测,结果表明免疫组鸡群的免疫抗体效价明显高于同期的未免疫对照组(p<0.05),二免后的抗体水平明显高于一免(p<0.05)。鸡群在一免后抗体水平迅速升高,第2周即可达到最高峰,在二免后第9周仍高于一免(p<0.05)。一免后第4周二免组和一免后第6周二免组二者同期抗体水平差异不明显(p>0.05)。结果表明,鸡鲍氏志贺氏菌灭活疫苗能够显著提高机体的体液免疫功能；所建立的间接ELISA方法能够准确的评价鸡鲍氏志贺氏菌疫苗的免疫效果；该疫苗评价方法为鸡鲍氏志贺氏菌疫苗免疫抗体水平的测定、免疫程序的合理制定、有效预防和控制本病提供了一个良好的技术手段,便宜于在生产中推广应用。
The S.boydii disease is a new infectious disease Against chickens, which found in recent years.The disease is an acute disease caused by Shigella.It can lead to lots of different breed and day-old chicken occurre diarrhea and died. The disease has possibility of cross-infection between human and birds,which has strong public health significance.In this study, an indirect ELISA method was established and applied to detecting serum antibody angainst inactivated chicken S.boydii vaccine.It provides an serological detection method for evaluating the immune effect of chicken S.boydii vaccine to develop the reasonable immunization procedure for preventing or controlling disease effectively. The results are as follows:
1.The establishment of indirect ELISA method.
In this study, Chicken S.boydii whole thallus was used as a coated antigen inactivated by formaldehyde.Rabbit anti-chicken IgG labeled by horseradish peroxidase (HRP) was used as the second antibody. The indirect ELISA method was established for the detection of inactivated vaccine immune antibody of chicken S.boydii, and the best work of the ELISA conditions were determined:the concentration of coated antigen was 108 cfu/ml,the best temperature and time for coating was at 4℃in 12h; the best conditions for closing was used 1% BSA at 37℃in 2h; the best dilution of serum was 1:160;the best dilution of second antibody was 1:4000, and the best working conditions was at 37℃in lh; the best working conditions of substrate was at 37℃in 20min. The cross-testing, specificity, sensitivity tests, and repeatability tests show that the method has good reproducibility, great specificity, high sensitivity. The coefficient of variation of one-plate was 0.36%～0.64%, and 1.05%～1.85% of inter-plate; the coated whole cell antigen and other diarrheal diseases had no cross-reactivity, the results of blocking were positive. The sensitivity of indirect ELISA method is 80-160 times as much as micro-agglutination test.
The OD values of 20 different positive chicken serum in different dilution were detected and found that linear correlation existed between the OD value of positive serum and the logarithm of ELISA titer reciprocal of serum.The regression equation of log [ET countdown]= .776+1.921 x was determined under the proposed standard curve.The establishment of the regression equation provided a mathematical model for forecasting and analyzing antibody levels by the serum OD values after clinical immunity.
2.Indirect ELISA used in chicken S.boydii inactivated vaccine serum antibody detection.
In order to evaluate the humoral immune effects of chicken S.boydii inactivated vaccine, the indirect ELISA method were applied to testing the post-vaccination antibody. The results showed that the immune antibody titer of immunity class was significantly higher than non immunity class (p<0.05)in the same period, the antibody levels of the second immunization were significantly higher than the first immunization (p<0.05).The antibody levels of immunity class increased rapidly after immunization and reached a peak in the two weeks.Nine weeks after the second immunization was still higher than the first immunization (p<0.05).The antibody levels of the second immunization in the forth week or the sixth weeks after the first immunization didn't have obvious difference in the same period (p> 0.05).The results showed that chicken S.boydii inactivated vaccine can improve the body's humoral immune function significantly;The established indirect ELISA method can evaluate the effects of chicken S.boydii vaccine truly; the vaccine and evaluation methods provide a good technical for testing S.boydii vaccine antibody and establishing the rational immune program.It will provide a good technical for preventing or controlling the disease.It is suit for applying and popularizing in production cheaply.
引文
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