摘要
用适宜摩尔浓度的异型双功能交联剂SPDP将R-藻红蛋白与纯化的禽流感病毒H9亚型多克隆抗体交联,并经高效液相色谱纯化制备R-藻红蛋白标记抗体荧光探针。以溴化氰活化的琼脂糖微球为固相载体,采用双抗体夹心法建立固相免疫荧光检测法,以制备的R-藻红蛋白标记抗体荧光探针检测禽流感病毒H9尿囊毒。结果表明,阳性微球在蓝、绿光激发下发射桔黄色荧光,荧光明亮,无背景光干扰,特异性好,灵敏度高,可检测蛋白浓度为1.85×10-5mg.mL-1的H9亚型尿囊毒。R-藻红蛋白标记探针的检测灵敏度高于FITC标记探针,R-藻红蛋白可替代FITC或与FITC等荧光染料一起用于多色荧光免疫检测。
R-phycoerythrin was an excellent fluorescent dye because of a high fluorescence quantum yield and large Stokes shift. R-phycoerythrin was conjugated with purified multiple-clone antibody of avian influenza virus （AIV） of H9 subtype by means of optimum concentration of SPDP, and subsequently purified with HPLC. Then the R-phyeoerythrin-labeled antibody probe was applied to detect H9-subtype AIV, by use of solid-phase immunofluorescenee detection method on a CNBr-activated sepharose spheres. The system showed orange-yellowed emission fluorescence with high sensitivity and high specificity, and the detection sensitivity attained 1.85 × 10^-5 mg · mL^-1, which was more sensitive than that of FITC-labeled antibody probe. R-phycoerythrin can be used in multiple immunofluorescence detection combined with other dyes. The solid-phase immunofluorescence detection method is hoped to be applied for rapid detection of AIV or other viruses.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2009年第2期92-96,共5页
Journal of Nanjing Agricultural University
基金
国家高技术研究发展计划资助项目(2002AA302213)
兽医生物技术国家重点实验室开放基金项目(SKLVBF200909)
关键词
R-藻红蛋白
荧光标记
固相免疫荧光检测
禽流感病毒
R-phycoerythrin
fluorescence labeling
solid-phase immunofluorescence detection
avian influenza virus
