摘要
将待检家畜血纸浸出液点在硝酸纤维素膜上，以金标记的血吸虫虫卵可溶性抗原为探针（以下简称血吸虫抗原胶体金），建立了检测家畜血吸虫抗体的二步金标免疫渗滤法（Two-step Dot Immunogold Filtration Assay，T—DIGFA）。家畜血吸虫病阳性血样在50-90S形成肉眼可观察的红色斑点，可进行快速诊断。该法可以检测出人工感染血吸虫尾蚴7d和7d以上的阳性牛和兔血纸抗体，与肝片吸虫、锥虫、蛔虫病之间无交叉反应。T—DIGFA检测粪孵血吸虫毛蚴阳性牛血纸139份、阴性牛血纸130份，与粪孵法阳性符合率为100％，阴性符合率为99．2％；T—DIGFA与斑点酶联SPA法阳性符合率和阴性符合率完全相同。血吸虫抗原胶体金在4～8℃保存期可达6个月、室温保存期为3个月。试验证实，T-DIGFA有很高的敏感性、特异性、重复性和稳定性，适合于基层单位和现场进行家畜血吸虫病抗体的快速诊断、普查和检疫。
A two-step dot immunogold filtration assay（T-DIGFA） was established to detect antibodies against schistosome in livestock. In this method, 1 μL lixivium from blood paper for examination was dotted on a nitrocellulose membrane（1.3 cm× 1.3 cm） in a plastic box with a hole （φ0. 6 cm） on it＇s face center, 50s-90s later, 100 μL extract of schistosome egg labelled with colloidal gold were added on nitrocellulose membrane in the hole. If there were anti-schistosome antibodies in blood paper lixivium, a red mark would appear. If there were no anti-schistosome antibodies in the lixivium, no red mark could be seen. In this study 139 blood paper positive samples from cattle with schistosomiasis diagnosed by feces hatching miracidium method and 130 blood paper negative samples from cattle without schistosomiasis were detected with T-DIGFA, the results showed that schistosomiasis positive coincidence rate was 100% and negative coincidence rate was 99.2 % compared with feces hatching miracidium method. The detected result was according to that using HRP-SPA-Dot-ELISA method completely. T-DIGFA could also detect the antibodies against schistosome in lixivium of blood paper from cattle and rabbits infected with schistosome cercaria artificially after 7 days （including 7 days） and from the cattle infected by one twain of schistosome naturally. There was no cross reaction with the antibodies of the Faciola hepatica, Trypanosoma evansi and ascariasis. The same 125 samples of blood paper were detected by five operators with five colloidal gold prepared on different time, respectively, no different results were found between operators or between various batches of colloidal gold. T-DIGFA reagent kit that were stored in 4--8 ℃ for 6 months or kept in room temperature for 3 months still kept valid. The tests verified that T-DIGFA method have high sensitivity, specificity and stability. It is suitable for fast diagnosis and general survey for schistosmiasis of animals.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2006年第7期687-692,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
浙江省重大攻关项目(2004C12027)
关键词
二步金标免疫渗滤法
血吸虫病
抗体
家畜
two-step dot immunogold filtration assay
schistosomiasis
antibody
livestock
