R-phycoerythrin标记抗猪瘟病毒荧光抗体的制备及其与FITC标记荧光抗体检测的比较研究  

Preparation of R-phycoerythrin-labeled Anti-CSFV Fluorescence Antibody and Comparison with FITC-labeled Anti-CSFV Fluorescence Antibody

作　　者：赵守山 颜世敢 朱丽萍[3] 崔道实 

机构地区：[1]山东省滨州市邹平县畜牧兽医局,山东滨州256207 [2]山东省农科院畜牧兽医研究所山东省动物疫病防控与繁育重点实验室,山东济南250100 [3]山东轻工业学院食品与生物工程学院,山东济南250353 [4]山东齐鲁动物保健品有限公司,山东济南250100

出　　处：《西南农业学报》2011年第5期1962-1966,共5页Southwest China Journal of Agricultural Sciences

基　　金：国家高技术研究发展计划(2002AA302213);兽医生物技术国家重点实验室开放基金(SKLVBF200909)

摘　　要：用摩尔比100∶1的化学交联剂SPDP分别将RPE、抗猪瘟病毒抗体衍生,两种衍生物再以摩尔比1∶1液相交联,HPLC纯化制备出RPE标记的抗猪瘟病毒荧光抗体。对制备的荧光抗体从荧光特性、抗体活性、稳定性及纯度等方面进行鉴定。以溴化氰活化的琼脂糖微球为固相载体,采用固相免疫荧光检测法,以制备的RPE、FITC分别标记的荧光抗体检测猪瘟病毒细胞毒。结果表明,RPE标记的荧光抗体检测阳性微球在蓝绿光激发下发射桔黄色荧光,荧光明亮,无本底光干扰,检测灵敏度高达2.67×10-6mg.mL-1,是FITC标记的荧光抗体检测灵敏度的10倍。RPE可替代FITC或与FITC等荧光染料一起用于多色荧光免疫检测。R-phycoerythrin from Polysiphonia urceolata was an excellent fluorescent dye with orange-yellowed bright fluorescence.Both of them have the same absorption peak,but the maximum fluorescence emission of R-phycoerythrin was 50 nm larger than that of FITC.Purified R-phycoerythrin and multiple-clone antibody of Classical Swine Fever virus was modified by SPDP at the molar ratio of 100 to 1,and then conjugated with each other at the molar ratio of 1 to 1,subsequently purified with HPLC.R-phycoerythrin-labeled antibody was applied to detect CSFV by use of solid-phase immunofluorescence detection method on a CNBr-activated sepharose spheres.The system showed orange-yellowed emission fluorescence with high sensitivity and high specificity,the detection sensitivity attained 2.67×10-6 mg·mL-1,which was ten times more sensitive than that of FITC-labeled antibody.R-phycoerythrin can substitute for FITC or be used in multiple immunofluorescence detection combined with other dyes.

关 键 词：R-藻红蛋白 荧光标记 固相免疫荧光检测 猪瘟病毒 

分 类 号：S852.659[农业科学—基础兽医学]