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

将SP2骨髓瘤细胞与小鼠脾融合，制备杂交瘤细胞，经酶联免疫吸附试验和间接免疫荧光试验(IFAT)证实抗体产生能力较强。采用IFAT和ELISA法对杂交瘤细胞进行筛选，检测抗体产生情况。用产生高效、特异抗体的杂交瘤细胞免疫SCID小鼠，以扩大腹水中抗体的产生。用IFAT、ELISA法和免疫印迹法进一步检测腹水中的单抗。从360个克隆中筛选出3个克隆，在检测微小弧菌卵囊、裂解物抗原和/或rCpGP15方面具有较高的特异性和精确度。然而，我们的主要目标将集中在使用免疫层析测试(ICT)来检测牛粪中的微小弧菌抗原来评估这些单抗。我们开发的信息通信技术的验证应用于以前用商业信息通信技术测试的牛的粪便样本。用活卵囊免疫小鼠制备的一种单抗，即D1-8，对检测自然感染的牛粪样本显示出中等效果，商业ICT和光学和荧光显微镜证实了这一点。ICT的条带强度与高浓度的旧卵囊裂解液呈正相关，对PBS、RIPA缓冲液无反应。由于经济和公共卫生的重要性，成功开发用于检测小牛粪便中微小隐孢子虫的ICT将是对隐孢子虫研究的重要补充，因为它提供了关于ICT技术方法和准备的新知识。

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.