Preparation Andapplication Of Monoclonal Antibodies Against Cryptosporidium Suis

The Cryptosporidium suis isolated from a pig farm of zhengzhou was identified by PCR-RFLP and zoogenetic infection. Oocysts were collected and then purified by sucrose gradient centrifugation. BALB/c mice were immunized with purified and sonicated oocysts, and hybridoma cell lines against C.suis were established by cell fusion. The methods of Monoclonal antibody Mediated IFA was established for Detecting Cryptosporidium Oocysts.The morphology and size of oocysts isolated were measured by saturated sucrose flotation method and modified acid-fast staining. Oocysts are spherical or overlap in size, oocysts wall are smooth and colorless. Oocysts are pink in saturated sucrose solution and rosy red by modified acid-fast staining using microscope. The average size of oocystis 4.74μm×4.25μm,oocysts shape index(length/width)is 1～1.10. The Small-Subunit rRNA(SSU rRNA) specific fragments of the isolate was amplified by Nested PCR. The PCR products was sequenced.Then the homology of sequence were comparatined and analysed by Clustal X1.81 software. The result showed that the homology was 100% compared with reference sequence of C.suis. The 7 day old piglets were inoculated showed mild diarrhea. The prepatent period was 3 to 4days after oocysts were ingested.The duration of oocysts excretion was more than 35 days and the peaks of occysts shedding appeared in the DAI 6～21days. Based on the results obtained above showed that the isolate was C.suis.Oocysts were collected and then purified by sucrose gradient centrifugation. BALB/c mice were immunized for five times with the purified, freeze-thawed and sonicated oocyst antigens of C.suis. Spleen cells of immunized mice were selected to fuse with SP2/0cells at the role of PEG1000. The positive screening were performed by indirect ELISA, and then three times generation of cloning were done by limiting dilution method, finally three hybridoma cell lines were obtained. The McAbs against C. suis were prepared and the characterization of McAbs was studied by means of indirect immunofluorescent assay (IFA) and indirect ELISA. Three clones( nominated 1E1C5, 1E11A5 and 2B7A1,respectively) of hybridoma cell lines secreting McAbs against oocyst antigens of C.suis were obtained, which were classified as IgM ,IgG3 and IgG2a subtype, respectively. Among the three McAbs, the titers of supernatant of cell cultures and ascites fluids ranged from 1:1 600 to 1:3 200, 1:80000 to 1∶160000, respectively. The ELISA superposition results showed that all McAbs could recognize two different antigenic sites and 1E1C5 demonstrated a higher affinity.The IFA results indicated that three McAbs could only conjugate the oocyst walls of C. suis, but had cross-reactions with Eimeria and C.hominis. The result indicates three McAbs that could react specifically against antigens of C. suis have been obtained, which lay a foundation for further establishing rapid diagnostic methods for C.suis.An indirect immunofluorescence assay for detection of C. suis oocysts was established using 1E1C5 monoclonal antibody which against C. suis oocyst wall as first antibody and fluorescein isothiocyanate conjugate of goat anti-mouse immunoglobulin as second antibody. The first antibody diluted with 1%BSA at 1:400 and the best reaction time was 60min. The best concentration of second antibody was 1:400 and the best incubated time was 45min. Specificity testing of the IFA revealed that there are no cross reaction with Cyclospora, C.andersoni, C.parvum, C.bailey oocyst and only weak reactivity with Eimeria and C.hominis oocysts. Sensitivity of the IFA indicate that the detection limit was 250 C.suis oocysts Per Milliter samples. The indirect fluorescence assay (IFA) was compared with the modified acid-fast stain (MAFS) for detection of Cryptosporidium oocysts in 50 pig fecal specimens. The sensitivity of the IFA was 100%; all 9 specimens positive by MAFS exhibited fluorescence. There were 41 specimens negative for Cryptosporidium sp. by MAFS; of these, 38 were negative by IFA. This discrepancy may reflect an increased sensitivity of the IFA to detect oocysts that were not visualized by MAFS because of faint staining or a paucity of organisms. The IFA offers a reasonable alternative because of its high sensitivity and specificity, the simplicity of performing it and its capability of providing a definitive diagnosis of Cryptosporidium oocysts.