Monoclonal antibody technology applied to the detection of iCryptosporidium parvum/ioocysts in human and cattle fecal samples

摘要

With the discovery that the coccidian parasite Cryptosporidium sp. can cause severe symptoms in humans, many diagnostic techniques were quickly implemented such as microscopic visualization, immunofluorescence and PCR. Currently, there is no effective drug treatment and none of the current diagnostic methods is 100% accurate. In this study, a BALB/C mouse was subcutaneously immunized with Cryptosporidium parvum oocysts extract. The spleen was removed and the splenocytes were fused with SP2/0 myeloma cells in order to obtain hybridoma cells secreting antibodies specific to C. parvum antigens. Human and cattle fecal samples previously characterized by microscopy [Ziehl-Neelsen staining (ZN) and Lugol] and PCR for the presence of C. parvum and Giardia duodenalis, were analyzed by indirect immunofluorescence, using the developed hybridomas supernatants. The study shows that the selected hybridomas supernatants identify C. parvum oocysts in fecal samples in correlation with C. parvum oocysts identified using ZN/PCR. No false positive results were obtained and the two best supernatants gave 20% -30% of false negative results. No cross reaction with G. duodenalis was observed. By comparing our results with those obtained with an immunofluorescence commercial kit, it suggests the potential use of the monoclonal antibodies present in two of the hybridomas supernatants as a detection tool of C. parvum. With a reliability of 80.8% and 73.1% versus ZN and PCR methods for IFI, compared with a reliability of 76.9% and 92.3% versus ZN and PCR for commercial DIF kit, the supernatant 4.1D5 seems to be the most promising subject to further study its usefulness for C. parvum detection.