Preparation Of Monoclonal Antibodies Against Capsid Protein Of Cryptosporidium Parvum Virus And Its Primary Application

To establishment of double-antibody sandwich ELISA, prokaryotic expression of S-dsRNA gene of CSpV should be put into effect first, then to develop monoclonal antibody against recombinant protein.Clone,prokaryotic expression and purification of S-dsRNA gene Total RNA was extracted from C.parvum and S-dsRNA gene was amplified by RT-PCR. The PCR product was cloned into pET-28a(+) expression vector. The recombinant plasmid pET-28a(+)-S was transformed into BL21(DE3) and the transformed bacteria were induced by IPTG. SDS-PAGE showed that the recombinant plasmid pET-28a(+)-S expressed a fusion protein(37 kDa). High level expression of recombinant protein was found at 1 mmol/L IPTG condition after incubation for 4h at 37℃and reached up to 72.61% of total E.coli proteins. It was recognized by the sera against C.parvum.The S-dsRNA gene has been successfully expressed with adequate reactionogenicity. Recombinant protein was purified by Ni-NTA affinity column and SDS-PAGE showed a single band is 37 kDa.Development and purification of monoclonal antibodies against recombinant protein Recombinant protein was purified as coating antigen so as to develop an indirect ELISA for capsid protein antibody of CSpV. The optimal antigen concentration for coating was 0.25μg/hole, and the optimal dilutions of the sera of C.parvum and enzyme-labeled the second antibody were 1:200 and 1:2000 respectively,blocking agent was the PBST solution of 5% nonfat dry milk by chessboard titration. The developed indirect ELISA had no cross reaction with positive serum of mouse against C.parvum, C.andersoni, E.tenella, G.lamblia and T.gondii. BALB/c mice were immunized with the purified recombinant protein. splenocytes were fused with SP2/0 until the specific antibody level of mice serum was reached 105 by indirect ELISA. The supernatant hybridoma clones were screened by indirect ELISA. Two hybridoma cell lines stably secreting mAbs against recombinant protein were obtained, named 1A9 and 23A7.The immunoglobulin subclass of 1A9 and 23A7 were IgG1 and IgG. respectively. The antibody titers of ascitic fluid both were 1:106. Western blotting and indirect immunofluorescence assay showed that 1A9 was specific mAbs against recombinant protein. The ascite of 1A9 was purified by HiTrap Protein G HP. SDS-PAGE showed that antibodies were purified well, the light chain and the heavy chain were seen clearly.Establishment of double-antibody sandwich ELISA A double-antibody sandwich ELISA was developed to detect capsid potein of Cyptosporidium parvum virus, which using purified monoclonal antibody as the capture antibody and purified monoclonal antibody labeled by HRP as the detection antibody. It was shown that the optimal dilution of monoclonal antibody for coating was 1:200. the optimal dilution of detection antibody was 1:800, the optimal blocking buffer was 1% BSA. There was no crossing reaction with the stool sample containing C.anderson. The detective sensitivity is 40-fold dilution of floatation supernatant, about 25 oocysts per gram stool. Eight positive stool samples were detected C.parvum by this way, the coincidence was 100%. The double-antibody sandwich ELISA was a rapid, simple method for detection of C.hominis and C. parvum, and suitable for monitoring human Cryptosporidiosis.