Released August 2007 - The Indian Society for Parasitology

24 Prasad et al.blocked with blocking solution. After washing with contortus: Dot-ELISA was performed with adultphosphate buffered soline-tween (PBST), these stripsst, nd rdsomatic antigen and 1 2 and 3 week experimentalwere incubated with primary antibody (1:100) for 2 h sera, as well as with positive, negative and controland washed with PBST. The strips were then animal sera. No dot formation was observed with 1 stincubated with donkey anti-sheep horse-reddishndand 2 week sera, as well as with negative control andperoxidase conjugate. After washing, finally diamino control animal sera. A solid dot formation wasrdbenzidine (DAB) substrate was added and dot observed with positive control sera. When 3 weekformation was observed.sera was allowed to react with adult somatic antigen, asolid dot was formed which indicated the recognitionSDS-PAGE: The adult somatic antigen andof antibodies at this stage (Fig. 1 A, B, C).immunoaffinity purified adult somatic antigen wereanalyzed through SDS-PAGE (Laemmli, 1970) byAusing mini vertical slab gel electrophoresis system.1 2 3 4Tris glycine gel (12%) under non-reducing conditionwas used at constant voltage (100 V). The proteinsamples were mixed with the sample buffercontaining (0.05M Tris pH 6.8, 5% SDS, 20% glyceroland 0.02% bromophenol blue) in the ratio of 1:1, and10–20 µg protein/lane was loaded. The gel was runapproximately for 90 min. After complete run, the gelwas stained with 0.1% Coomassie Brilliant Blue G-B250, and destained with destaining solution (30%methanol and 10% acetic acid). Molecular weights ofthe fractionated polypeptides were determined byusing molecular weight markers.1 2 3 4RESULTSCoproculture of H. contortus larvae: L larvae of H.3contortus were obtained from coproculture and wereidentified by the presence of kinky tail and tubercleson the body surface as described by Levine (1980). Allthe larvae were confirmed to be H. contortus.Positive control sera: Hyperimmune sera raised inrabbits were used as positive controls in dot-ELISA,previously tested in DID which showed precipitin linewith adult somatic antigen.Experimental sera: Experimental sera werecollected after infection with H. contortus larvae fromstrd1 week to 3 week post-infection. Coprologicalexaminations of faeces, when conducted up to threeweek post-infection, were found negative whichindicated prepatency.Dot-ELISA with adult somatic antigen and sera ofsheep naturally-infected with H. contortus: Dot-ELISA performed with adult somatic antigen and seraof sheep naturally-infected with H. contortus revealedsolid dot formation with five out of ten serum samplesat 1:100 serum dilution, whereas solid dot formationwas observed with positive control and no dotformation occurred with control animal sera (Fig. 2 A,B, C).Protein estimation: In the adult somatic antigen,protein concentration was estimated to be 6.3 mg/ml,whereas in the pooled immunoaffinity purifiedfraction of adult somatic antigen, the proteinconcentration was estimated to be 0.2 mg/ml.Dot-ELISA with adult somatic antigen andexperimental sera of sheep infected with H.C1 2 3 4Fig. 1: Dot-ELISA with the 1st, 2nd and 3rd week experimentalsera and somatic antigen of H. contortus.A. 1: Somatic antigen and positive control; 2: Somatic antigenand negative control; 3: Somatic antigen antigen and controlanimal sera; 4: Somatic antigen and 1st week sera.B. 1: Somatic antigen and positive control: 2: Somatic antigenand negative control: 3:Somatic antigen and control animalsera;4: Somatic antigen and 2nd week sera.C. 1: Somatic antigen and positive control:2: Somatic antigenand negative control:3:Somatic antigen and control animalssera; 4: Somatic antigen and 3rd week sera.