Preparation Of Monoclonal Antibodies Against Cryptosporidum Andersoni And Development Of Sandwich ELISA

An epidemiology of cryptosporidia infection in cows was surveyed with both saturated sucrose solution floatation and modified Ziehl-Neelsen staining (acid-fast stain) technique on five dairy farms in Zhengzhou, Shangqiu, and Luoyang,Henan Province. 582 fecal samples were individually collected from these farms and 64 samples were positive, the total infectious rate of Cryptosporidium was 11%. Two types of oocyst were found, one is of 7.3μm×6.3μm with shape index of 1.22, and another is of 3.32μm×3.16μm with shape index of 1.05. They were identified as Cryptosporidium parvum with infection rate of 3.1 % ( 20/582 ) and C. andersoni (7.9%, 44/582) respectively according to their morphological characteristics. The infectious rate of calves is much higher than that of both beefs and cows. There were no symptoms in infected cattle. Neonatal calves were challenged with oocysts C. parvum. Results showed that the patent period was 7 days, and peak numbers of oocysts were observed at the 16 days post infection, which persisted for 5 days. Evident diarrhea appeared in calves after challenged. The whole digestive tract mucosa were stained with H.E after dissection , oocysts were detected only in middle ileum under light microscope and electron microscope.The oocyts of C.andersoni strain were purified by discontinuous sucrose gradients, then treated by filtration and centrifugation, freeze-thawed and sonicated. Balb/c mice were immunized for four times with purified oocyst antigens of C. andersoni. Spleen cells of the mice were selected to fuse with SP2/0 cells. The positive wells were selected by indirect ELISA, and after 3 to 4 generations of clone by limited dilution, 5 hybridized cell clones ( 1A3, 3B10, 3F4, 4B3, 4Fll)against C andersoni were obtained that chromosome numbers ranged between 96 and 98 . The solution titers ranged from 1: 100 to 1: 12,800, and ascites titers between 1: 12,800 and 1:204,800. The monoclonal ascites were examined with indirect immunofluorescence assay, and 4 of 5 were anti-oocyst wall , the other one was anti-sporozoite. Three ascites clones were purified and passed through SDS- PAGE...