| Echinococcus granulosus parasites in intestinal of dogs. Its infective eggs orsegments can be secreted with dog feces, excreting causing human and animalinfections. It is a serious threat to human health and the rapid development of animalhusbandry, which caused serious losses to the world economy. Therefore, preventionof disease is very important to human health and the development of animalhusbandry. All along, the researchers are trying to explore the effective methods forrapid diagnosis, the control and preventing from this disease. Although it had got a bigbreakthrough, it never stopped the prevalent of this disease. Dogs are the only finalhost, so the prevention of Canine E.granulosus is important for the control of thedisease. Currently, the fecal antigen detection methods of E.granulosus are mainlycontain arecoline cathartic, ELISA, PCR, and so on. But the reaction rate of arecolinecathartic method has some limitations; ELISA method often appears low sensitivityand some cross-reactivity; PCR method often appears false positive, and also needshigher equipment requirements. Also, the level of these methods detection is quitedifferent. The use of antigen also includes many types of adult worms antigen, cystfluid antigen, protoscolex parasite antigens and secretion antigens. So far there is nogold standard method and the antigen to detect Canine E. granulosus Therefore, it isnecessary to select a high specificity antigen, and establish a high sensitivity, highspecificity diagnostic method. This study screened E. granulosus immune-associatedantigen using the anti-E.granulosus hyperimmune serum. After the prokaryoticexpression, we performed the preparation of monoclonal antibody and polyclonalantibody to establish double-antibody sandwich ELISA method for detecting caninefecal antigen to search for an effective diagnostic antigen and to make a foundationfor an efficient and specific diagnostic method.Screening immune associated antigen from cDNA library of E.granulosus(adult worms) and expressed in Escherichia coli. In this study, SEREX was performed to screen related antigen from cDNA library of E. granulosus. First,λ-phage library was cultured on LB solid culture; Second, the expressed protein ofrelated gene was induced by IPTG; Third, related protein was screened by immunedserum of E. granulosus from infected rabit and HRP Anti-Rabbit IgG, and149positive results were acquired. The result of PCR and analysis by NCBI BLAST of positiveplaques insert was that10genes were acquired which were belong to Tapeworm genus. Avaluable gene was got with an864bp open reading frame, encoding288amino acids.The amino acids analysis by NCBI BLAST revealed the sequence has92%homologywith E. multilocularis diagnostic antigen gp50(CDJ06164.1). The sequence is maybean unknown new genes, named EdiagA864. Finally, EdiagA864recombinant proteinwas expressed in Escherichia coli. It was about51kDa and had reactionogenicity byWestern Blot analysis.The monoclonal antibody preparation and purification of EdiagA864Based on EdiagA864, we got4monoclonal antibodies by immunizing Balb/c mice,cell fusion and hybridoma screening and cloning, including2D12、3H4、4A6and6E5.After identification of chromosomal analysis and the analysis of antibody subtype, thechromosome number of2D12,3H4,4A6, and6E5were97,101,98, and96respectively.4monoclonal antibodies’ heavy chain subtypes were IgG1, the lightchain isoforms were Kappa.The antibody titer of2D12and3H4were higher than2×105. The antibody titer of4A6and6E5were higher than1×105. The purifiedmonoclonal antibodies got a light chain and a heavy chain by SDS-PAGE assay.The polyclonal antibody preparation of Echinococcus EdiagA864Based onEdiagA864, we got polyclonal antibody by immunizing rabits. The antibody couldspecifically recognize antigen EdiagA864, and had no cross-reaction with positivesera of Toxoplasma gondii, Neospora and canine heartworm.The establishing double antibody sandwich ELISA method of E. granulosusin dogs In this experiment, we established three double-antibody sandwich ELISAmethods by the monoclonal antibodies and rabbit polyclonal antibody: Method1,2D12monoclonal antibody as the capture antibody, HRP-labeled polyclonal antibodyas the detection antibody; Method2,2D12and3H4monoclonal antibodies mixed as the capture antibody, HRP-labeled polyclonal antibody labeled as a detection antibody;Method3,3H4monoclonal antibody as the capture antibody, HRP labeled2D12monoclonal antibody as the detection antibody. In the specificity tests, three methodshad no cross reaction with Giardia positive dog feces and roundworm positive dogfeces; In the sensitivity tests, the dilution of the positive samples of method1and2were both1:20. The dilution of the positive samples of method3was1:5. The threemethods were applied to detect E. granulosus positive dog feces: the positive rate ofmethod1and2were100%(8/8); The positive rate of method3was87.5%(7/8). Theresult of detecting64samples from Changchun was no posotive by the three kinds ofdouble-antibody sandwich ELISA. |
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