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

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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.