摘要
为建立以红色聚苯乙烯纳米微球为载体的犬细小病毒(CPV)抗体检测的间接凝集方法,本研究表达纯化了CPV-2VP2截短的重组蛋白rsVP2,并以rsVP2为致敏原与纳米微球偶联制备了免疫彩色纳米微球;通过对反应条件的优化,建立了一种快速检测CPV-2抗体的间接凝集方法。特异性试验结果显示,致敏微球仅与CPV-2阳性血清发生凝集反应,不与犬瘟热病毒、狂犬病病毒和犬腺病毒的阳性血清发生凝聚反应,特异性强;该凝集方法检测血清中和抗体的最低效价为1∶256;同一批次制备的3份致敏微球、3个不同批次制备的致敏微球以及4℃保存90d的致敏微球与CPV-2阳性血清的凝集反应特异性一致,凝集程度无明显差异,重复性稳定性均较好。该间接凝集检测方法对40份血清样品的检测结果与ImmunoComb■ Canine VacciCheck试剂盒(Dot-ELISA)检测结果的阳性符合率为97%。本研究建立的间接凝集方法为幼犬CPV-2母源抗体或免疫犬CPV-2抗体检测提供了一种快速、简便、准确、经济的方法。
To detect the maternal antibodies against canine parvovirus in puppies prior to vaccination or the seroconversion in vaccinated dogs, we tried to develop an indirect agglutination test based on immuno-colored nanospheres bound with recombinant antigen Truncated recombinant VP2 protein (rsVP2) of canine parvovirus type 2 (CPV-2) was expressed, purified, and bound with colored polyphenylethylene nanospheres. The indirect agglutination test was established by optimization of reaction conditions, the tests of specificity, sensitivity, stability, and detection of clinical samples. The agglutination reaction was only observed between sensitized nanospheres and positive serum against CPV-2, but was not with positive serum against CDV, CAV, and RBAV. The minimum neutralizing antibody titer to CPV-2 at 1∶256 was detected by this agglutination test, while the reaction displayed the agglutination degree of 50%(++). Three samples of sensitized nanospheres prepared in the same batch, or from three different batches, or stored at 4℃ for 90 days, had the same specificity, stability and reproducibility, and there was no significant difference in agglutination degree. 40 clinical samples displayed positive results with coincidence rate of 97% as compared to the commercial kit (ImmunoCombR Canine VacciCheck). These results indicated that the indirect agglutination test established in this study may be a rapid, simple, accurate and economical method, to determine the serum antibodies status against CPV-2 in puppies.
作者
唐毓
苏瑞红
侯美佳
孙思萌
冯启峥
董秀梅
张萍
师东方
TANG Yu;SU Rui-hong;HOU Mei-jia;SUN Si-meng;FENG Qi-zheng;DONG Xiu-mei;ZHANG Ping;SHI Dong-fang(College of Veterinary Medicine,Northeast Agriculture University,Harbin 150030,China;The State Northeastern Scientific Observation Station on Veterinary Etiological Biology,Harbin 150030,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2019年第6期605-610,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
农业部动物疫病病原生物学东北科学观测实验站观测项目
黑龙江省科技计划项目(GC12B303)
关键词
犬细小病毒
彩色聚苯乙烯纳米微球
间接凝集试验
抗体检测
canine parvovirus type 2
color polyphenylethylene nanospheres
indirect agglutination test
detection of antibodies
