摘要
利用猫瘟热病毒重组VP2蛋白作为包被抗原,以辣根过氧化物酶(HRP)标记的SPA作为广谱第二抗体,初步建立适用于2种猫科动物猫瘟热病毒抗体检测的SPA-ELISA方法。用建立的SPA-ELISA方法对临床30份猫血清样本、86份虎血清样本进行检测,同时与HI、间接ELISA方法进行比较。结果显示,确定纯化重组VP2蛋白以2.67 mg/L稀释质量浓度包被,脱脂乳以15%的浓度封闭90 min,待检血清以1∶50稀释度孵育60 min,HRP-SPA以1∶4 000稀释度作用45 min,可使SPA-ELISA获得最佳检测效果。SPA-ELISA批内及批间重复试验变异系数均小于10%,对临床30份猫血清样本、86份虎血清样本的对比检测表明,SPA-ELISA方法检出率较高,大于HI法的检出率,与间接ELISA法接近。3种检测方法检测猫血清的总体符合率为96.7%。在虎血清检测中,SPA-ELISA方法的阳性检出率亦高于HI方法,两者总体符合率为94.2%。
Based on the purified recombinant VP2 protein of tiger feline panleukopenia virus,an ELISA using SPA as conjugate（SPA-ELISA） was developed to detect FPV antibodies in clinical samples of cats and tigers.The assay was optimized that antigen coating concentration was 2.67 mg/L and a serum dilution was 1∶50,with a standard incubation time of 60 min.Blocking concentration of blocking reagent（skim milk） was 15%,with a standard incubation time of 90 min.HRP-SPA dilution was 1∶4 000,with a standard incubation time of 45 min.variation coefficient of intraassay and interassay about SPA-ELISA were all less than 10%.The contrast detection of the 30 serum samples of cats and 86 serum samples of tigers showed that the detection rate by SPA-ELISA was higher than HI,and close to indirect ELISA.The total coincidence rate of the three detecting methods was 96.7% by detecting 30 serum samples of cats.The coincidence rate of the SPA-ELISA and HI was 94.2% by detecting 86 serum samples of tigers.All these results indicated that SPA-ELISA could be a good method for serological detection of FPV antibodies in many species of felines.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2010年第11期1418-1421,共4页
Chinese Journal of Veterinary Science
基金
国家林业局野生动植物保护管理项目(2007)
