Development of rapid immunochromatographic assay as a diagnostic tool for bovine cryptosporidiosis

快速免疫层析检测作为牛隐孢子虫病诊断工具的开发

基本信息

批准号：
19F19107
负责人：
西川義文
金额：
$ 1.47万
依托单位：
Obihiro University of Agriculture and Veterinary Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for JSPS Fellows
财政年份：
2019
资助国家：
日本
起止时间：
2019-04-25 至 2021-03-31
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-19F19107/
关键词：
クリプトスポリジムウシ下痢症診断

项目摘要

Hybridoma cells were prepared by fusion of SP2 myeloma cells with the spleen of mice showing high production of antibody as confirmed by ELISA and indirect immunofluorescence test (IFAT). Hybridoma cells were screened by IFAT and ELISA to detect antibody production. Hybridoma cells producing potent and specific antibody were used for immunization of SCID mice for expansion of antibody production in ascites. Further testing of monoclonal antibody (mAb) in ascites was applied using IFAT, ELISA and western blotting. 3 clones out from 360 showed high proficiency and specificity in detection of C. parvum oocysts, lysate antigen and/or rCpGP15. However, our main goal will be focused on evaluation of these mAbs using immunochromatographic test (ICT) to detect C. parvum antigens in cattle feces. Validation of our developed ICTs was applied against fecal samples of cattle previously tested with commercial ICT. Only 1 monoclonal antibody namely D1-8 that prepared from immunization of mice with viable oocysts showed a moderate efficacy for detection of fecal samples from naturally infected calves as confirmed by commercial ICT and light and fluorescent microscopy. The band intensity of ICT was correlated positively with high concentration of used oocyst lysate, with no reactivity to PBS, RIPA buffers. Due to economic and public health importance, the successful development of ICT for detection of C. parvum in calf feces will be an essential addition to Cryptosporidium research because of providing novel knowledge concerning the methodology and preparation of ICT technology.

通过ELISA和间接免疫荧光试验（IFAT）证实，通过与显示高产生抗体产生的小鼠脾脏的SP2骨髓瘤细胞融合制备杂交瘤细胞。通过IFAT和ELISA筛选杂交瘤细胞以检测抗体的产生。产生有效抗体和特异性抗体的杂交瘤细胞用于对SCID小鼠的免疫，以扩大腹水中抗体的产生。使用IFAT，ELISA和Western印迹对腹水中单克隆抗体（MAB）的进一步测试。 360的3个克隆在检测到卵囊卵囊，裂解物抗原和/或RCPGP15方面表现出很高的熟练程度和特异性。但是，我们的主要目标将使用免疫色谱测试（ICT）来评估这些mAB，以检测牛粪中的parvum抗原。对我们发达的ICT的验证是针对先前用商业ICT测试的牛的粪便样本。只有1种单克隆抗体，即通过活卵囊的小鼠免疫制备的D1-8显示出适度的疗效，用于检测来自自然感染犊牛的粪便样品，如商业ICT和光和荧光显微镜所证实。 ICT的带强度与高浓度的用过的卵囊裂解物成正相关，对PBS，RIPA缓冲液没有反应。由于经济和公共卫生的重要性，由于提供了有关ICT技术的方法和准备的新知识，因此成功地开发了C. parvum在小牛粪便中检测C. parvum的成功开发。