Analysis Of Target Antigen In Fecal Samples Of Dogs Infected With Echinococcus Granulosus

Hydatidosis,also known as Echinococcosis,is a serious parasitic zoonosis caused by Echinococcus granulosus,which seriously damages the health of humans and livestock,and brings harm and obstacles to animal husbandry.The feces of dogs may contain metabolites,nodules,eggs,secretions and other tissues generated during the growth of Echinococcus granulosus as those canines have been the final host.In this paper,the screening and identification of target antigens in dog feces provide a basis for the preparation of standard samples in the ELISA detection method.And the accuracy of detection of Echinococcus granulosus in the final host in endemic areas can also provide technical support for the formulation of comprehensive measures and the control of infection.The method in this study can be an important reference method for screening and identification of target antigens and the result of it may provide significant experience for improving the sensitivity and specificity of the ELISA kit for fecal antigen detection.Method:After artificially infecting dogs with Echinococcus granulosus,collect their feces samples with high load of parasite,and select a suitable buffer solution to initially extract the antigens in the fecal samples.Then use the polyclonal serum which can be combined with fecal antigen to form an antigen-antibody complex,followed by purifcation and separation with ammonium sulfate precipitation and chromatography respectively.Finally the antigens are identified by a 2-D electrophoresis.Result:(1)PBST buffer solution is superior in the precipitation of soluble protein to borate borax buffer solution and Tris-HCl buffer solution.(2)Under the prerequisition that the volume of the solution is unchanged,the optimal concentration of polyclonal serum would be 60%～70%for the binding of fecal antigen-antibody;yet after the precipitation with saturated ammonium sulfate,the required concentration has reduced to 5%～20%.(3)The properties of the antigen-antibody complex hardly change after chromatography,and in approximately 45.5 to 50.5 minutes the peak would appear,which suggests the samples of pure positive antigen-antibody complex required are to be collected.(4)The IPG strip of the two-dimensional electrophoresis is a 7 cm map in pH 3～10 and the 7 cm IPG strips in pH 4～7 shows that some dot-like spots are formed.And the western blot result in a 7 cm IPG strip in pH 4～7 shows specific protein sites.Result:Compared with Tris-HCl buffer solution and boric acid borax buffer solution,PBST buffer solution is superior in the precipitation of soluble protein in fecal samples.And the optimal concentration of polyclonal serum could be 20%for the binding of fecal antigen-antibody under the prerequisition that the volume of the solution is unchanged.The antigen-antibody complex can be received in the time period from 45.5 min to 50.5 min after passing through the gel chromatography column.Exploring the immunological binding sites of Echinococcus granulosus antigens and antibodies in the feces of final hosts through these methods can provide reference experience for the prevention and detection of echinococcus in future.