00000ctm a22000004a 4500 UP-1685523046125428658 Buklod 20230503092606.0 m o j ta 060808s2000 xx r |||| u|eng d (iLib)UPMNL-00000056076 UPM-CPH eng LG996 2000 P94 P33 Padilla, Mildred A. Analysis of antigens of Philippine fasciola gigantica cobbold, 1885 towards the development of a monoclonal antibody-based antigen capture assay Mildred A. Padilla. 2000 162 leaves. Thesis (DrPH)--University of the Philippines Manila. This dissertation aims to develop and evaluate a sandwich ELISA to detect antigen in serum of naturally infected carabaos (Bubalus bubalis). Excretory-secretory products were recovered from Philippine Fasciola gigantica adult (FgES) and juvenile (FgJES) worms cultured in vitro. By SDS-PAGE analysis, FgES and FgJES shared molecules with molecular mass of 12-14, 15, 17, 26, 30, 43 and 63 kDa. FgES was chosen over FgJES as the source of diagnostic antigen because the adult worms were abundant compared to the juveniles. Moreover, the use of adult ES products was justified based on the demonstration of shared molecules by SDS-PAGE analysis and the finding of a 12-14 kDa antigen that was shown in several studies to be present in early Fasciola infection. By immunoblotting and indirect ELISA, cross reactivity was observed among F. gigantica, Schistosoma japonicum and Fischoederius spp. Eleven molecules in FgES were recognized by sera from rabbits immunized with FgES. These were in the approximate moleculas maa of 12-14, 17, 23, 25, 26, 28, 33, 36, 38, 45 and 55 kDa. All except the 38 kDa molecule were also recognized by sera from rabbits immunized against the adult worm antigen of S. japonicum. Sera from rabbits immunized against somatic extracts of Fischoederius spp. recognized ES antigens in the estimated molecular mass of 15, 23 and 33 kDa. The cross reactions observed when assaying for Fasciola antibodies point to the need for a specific immunodiagnostic test which discriminates an infection due to Fasciola from other parasites since multiple helminth infection is the rule rather than the exception. FgES was used to immunize BALB/c mice to produce monoclonal antibodies specific for FgES. Five MAbs were generated of IgM and IgG1 isotypes, kappa chain and all recognized the 12-14 kDa molecule in FgES. Compared to others, the target epitope recognized by MAb1H3F7 was species-specific, showing no cross reactions with Schistosoma japonicum or Fischoederius spp. The target epitopes of the remaining MAbs namely, 2C10E3, 1H3F7, 1H8C11 and 2D11B4 cross reacted. The 12-14 kDa target antigen of these MAbs seems to be a glycoprotein as suggested by smeary appearance in Western blots and it could be the fatty acid binding protein (FABP) reported in literature. The MAbs were subsequently used in an indirect sandwich ELISA as capture antibodies (15 ?g ml-1), along with a rabbit anti-FgES serum (1: 2000 dilution) as detecting antibody. This MAb-rabbit antiserum sandwich ELISA was initially used to detect varying concentrations of FgES and FgSom somatic antigens. Under optimal assay condictions, all the MAbs captured small amounts of both antigen preparations. MAb2C10E3 and MAb1H3F7, and MAb1H8C11 seemed to be the most promising MAbs when assessed for capturing the smallest amount of the target antigen (0.032 ?g ml-1) in FgES and FgSom, respectively. Purified rabbit polyclonal anti-FgES (PAb) as capture and MAb as detection antibodies were tried to detect varying amounts of FgES but was successful. Hence, the MAb-rabbit antiserum was adopted to detect antigen in sera from naturally infected carabaos that represent different levels of FEC and ELISA titer. Sera from these animals were first pretreated with trichloroacetic acid to dissociate immune complexes that might interfere with test results. This test system failed to detect antigen in the carabao sera. The most likely explanation for the failure to demonstrate the antigen is the absence or low levels of target antigen in the sera. If indeed the target antigen of the MAbs was a FABP, then the strong antibody response of the host would cause the immediate clearing of antigen in the circulation. FABP has been reported to be highly immunogenic antigen. In addition to the above findings, a glutathione-S-transferases (GSTs) with an apparent molecular mass of 26 kDa, and 26 and 26.5 kDa were purified from FgES and FgSom, respectively. This is the first report of GSTs from ES products of F. gigantica. GSTs are candidate vaccine molecules against fasciolosis. Protease activity was also demonstrated from both antigen preparations by gelatin substrate SDS-PAGE. Proteases like cysteine proteases are currently vaccine and diagnostic molecules for fasciolosis. Since the MAbs produced in this study recognized only a single target antigen that was very immunogenic, more MAbs should be generated that are directed against ES antigens of F. gigantica that could be obtained from laboratory infections. These MAbs could then be used to develop antigen capture assays. Fascioliasis. Fasciola. Monoclonal antibody. Antigen analysis. UP UPMNL CPH LG996 2000 P94 P33 Thesis