检测禽β-防御素6夹心ELISA法的建立及应用  

Establishment and Application of Sandwich ELISA Method for Detecting Avian Beta-defensin 6

作　　者：梁瑞[1] 梁盈[1] 祁克宗[1] 王爱荣[2] 彭开松[1] 

机构地区：[1]安徽农业大学动物科技学院,合肥230036 [2]河北省唐山市丰润区农业畜牧水产局,河北唐山064000

出　　处：《中国农业科技导报》2014年第4期176-180,共5页Journal of Agricultural Science and Technology

基　　金：国家自然科学基金青年基金项目(30800811);安徽农业大学引进与稳定人才基金项目(yj2006-13)资助

摘　　要：为定量检测禽β-防御素6（avian beta-defensin6,AvBD6）,建立了AvBD6的双抗体夹心ELISA法,并应用于鸡血清中AvBD6浓度的检测。研究结果显示,包被抗体（鼠抗AvBD6 B细胞线性表位的多克隆抗体）、检测抗体（兔抗AvBD6氮端的多克隆抗体）和辣根过氧化酶（HRP）标记羊抗兔IgG的最佳工作稀释倍数分别为2 500、2 000和10 000,AvBD6在20-320 ng/mL的线性范围内,标准曲线回归方程为：y=12 024x-2 583,（R^2=0.984 2）。对不同状态下鸡血清AvBD6浓度检测结果显示：ACTH（25 IU/kgBW）注射1 d后,对照组AvBD6浓度高于ACTH组;连续注射ACTH 5 d,对照组AvBD6浓度低于ACTH组,但二者间差异均不显著（P〉0.05）。大肠杆菌感染后24 h和沙门氏菌感染后3 h、24 h,感染组AvBD6浓度均显著地高于对照组（P〈0.05）,表明AvBD6参与对大肠杆菌和沙门氏菌系统感染的抵抗。To detect avian beta-defensin 6（AvBD6） quantitatively,a sandwich ELISA method was established and applied to detect the concentration of AvBD6 in serum. The results showed that the optimal dilution of capture antibody（ mouse anti-B cell linear epitopes of AvBD6 polyclonal antibody）,detecting antibody（ rabbit anti-Nterminal of AvBD6 polyclonal antibody） and horseradish peroxidase（ HRP）-conjugated goat anti-rabbit IgG was2 500,2 000 and 10 000,respectively. The standard curve regression equation was y = 12 024x- 2 583,（R^2= 0. 9842） in the linear detection range from 20 to 320 ng /mL. The serum AvBD6 concentration of control group was slightly higher than that of ACTH group（ P〉0. 05） after 1 day of ACTH（ 25 IU /kg BW） injection,but the AvBD6 concentration of the control group was slightly lower than that of ACTH group（P〉0. 05） after 5 d of ACTH injection;and the effect was not significant. The serum AvBD6 concentration of infected group after 24 h of Escherichia coli exposure or 3 h and 24 h of salmonella exposure were significantly higher than that of the control group（P〈0. 05）.Our results indicated that AvBD6 involved in the resistance to system infection of Escherichia coli and salmonella.

关 键 词：AvBD6 夹心ELISA ACTH 大肠杆菌 沙门氏菌 

分 类 号：Q511[生物学—生物化学] R392-33[医药卫生—免疫学]