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

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.