Production Of Monoclonal Antibodies Against Em10 Of Echinococcus And Their Pathological Application For Echinococcosis

Objective:(1)To develop and produce monoclonal antibodies(mAbs)againstEm10,asurfaceidenticalproteinofbothEchinococcus multilocularisandEchinococcusgranulosus;(2)Todeterminethe expression of Em10 in different developmental stages of the worms,especially the bidirectional development from protoscoleces of the worms by using mAbs which may indicate the functional role of Em10 in worm development;(3)To probe liver tissues from patients with alveolar echinococcosis and cystic echinococcosis patients in different types of echinococcosis for determining the role of mAbs against Em10 in histological identification of echinococcosis,to explore the tracer of the immunolabeling of mAbs to echinococcal cysts in vivo echinococcosis model and to determine the metabolism of the lableled mAbs in vivo,which is important for clinical molecular diagnosis of echinococcosis.Methods:(1)Immunization:BALB/cmicewereimmunizedwith recombinant Em10 antigen emulsified with Freunds adjuvants.Blood samples were collected from the tip of mouse tail,and the anti-Em10antibody titers were detected by ELISA.(2)Cell fusion:When serum titer of a mouse was more than 1:200,000,the spleen was removed to prepare suspension cells,which were fused with SP2/0 cells.The hybridoma cells were grown in the medium containing HAT for selection.Positive hybridoma cell lines secrecting antibodies against Em10 were further processed for cloning.The supernatant was collected and tested byELISAin96well-platescoatedwithrecombinantprotein Em10.(3)Cloning hybridoma cells secreting antibody against Em10:Cells from positive wells were diluted to one cell per three wells with each containing 10~4 support cells from mouse peritoneal cavity(peritoneal macrophages).After the cloned cell grew to 1/10 of well bottom,supernatant was collected and antibody against Em10 was detected by ELISA.The cells were expanded by culture and stored in liquid nitrogen.(4)Production of ascites:Female BALB/c mice were injected intraperitoneally with paraffin oil 0.5 mL/mouse.After 7 days,each mouse was peritoneally injected with 0.5mL 2×10~6/mL hybridoma cells.Ascites was colleted after 10-13 days of injection;(5)Identification of monoclonal antibodies:Monoclonal antibodies were identified by ELISA,Western Blotting and immunofluorescence;(6)Detection of Echinococcus from patients:Liver tissues from patients infected with AE(n=30)with different stages(P1-P4),CE(n=30)with different cyst types(CE1-CE5),30 cases with different liver diseases including liver cancer,hepatic cyst,cirrhosis patients)were probed with the monoclonal antibodies.All echinococcosis specimens were identified by PCR and DNA sequencing for confirming the infection with E.granulosus or E.multilocularis.Immunohistochemistry was used to determine the positive and negative identificationofechinococcosisbythemonoclonalantibodies.(7)Expression of Em10 in the bidirectional development of protoscoleces by Mab-Em10:Sheep livers were collected from sheep natureally infected with cystic echinococcosis and protoscoleces were collected aspirally from the cysts.After digested by pepsin,the protoscoleces were cultured in medium containing dog bile to adult development and without bile to cyst development.Samples were collected at different time points.The protein of the worm was extracted and the expression of Em10 was determined by Western Blotting.(8)Probing echinococcal cysts by Em10monoclonal antibodies:Mice were infected with microcysts cultured from protoscoleces for two months.After 12 months,0.05 mg of Cy3fluorescein-labeledmonoclonalantibodywasivinjected.The fluorecscein was detected by an in vivo imager.Results:(1)Recombinant Em10 showed good antigenicity and induced mice a serum antibody titer more than 200,000.Spleen cells were fused with mouse SP2/0 cells.After cloining processing,two cell lines were positive against Em10,which were named as Mab-Em10-8 and Mab-Em10-16.The antibody subtypes were IgG2a and IgG1,respectively.The cell supernatant titer was detected by ELISA showing more than 10,000.Hybridom ascites was prepared by using cloned monoclonal cells,and the antibody titer was more than 1 million.The primary antibody was identified by Western blotting,whichshowedthetwomonoclonalantibodiesstrongly recognized recombinant Em10 antigen and the natural proteins from different stages of the worm.(2)Mab-Em10-8 monoclonal antibody stronglyreactedwithproteinsfromadultworms(AW)and protoscolecesp(PSC)of E.granulosus;(3)Mab-Em10-16 monoclonal antibody strongly recognised proteins of AW,PSC,and cyst germinal cells(GL),but did not react with proteins in hydatid cy fluid(HCF).(4)These samples were detected by the two monoclonal antibodies in immunohistochemistry.The results showed that the monoclonal antibodies recognized and sequence-determined echinococcosis patients,and the sensitivity of the detection were 56.67%.The specificity was97%.Mab-Em10-8 was strongly reactive with AE lesions of P3 type,while Mab-Em10-8 recognized CE1-CE3 lesions and did not recognize CE4 and CE5 lesions.Mab-Em10-16 recognized P2-P4 type of AE lesion tissue,and P3 type showed the highest sensitivity.Mab-Em10-16recognized that CE lesions were similar to these samples by Mab-Em10-8;(5)Protoscoleces of Eg and Em were cultured in vitro in medium containing bile or not to induce protoscoleced developing adult worms or cysts.For cyst formation,the two parasites were different in term of formation of cyst wall.The wall of Em was thicker than Eg.(6)We observed that the fluorescein-labeled monoclonal antibody was not obvious in mice by the small animal imaging.Conclusion:Two mAbs against Em10 were successfully produced.Mab-Em10-16 can recognize late P3 and P4 vesicular echinococcosis lesions,and Mab-Em10-8recognized P2,P3,P4 of AE,and CE1-CE3 of cystic echinococcosis lesions.The study provides fundmental reagents for the molecular diagnosis of echinococcosis.