Preliminary Studies On Detection Of Circulating Antigens From Urine Of Rabbit Infected With Schistosoma Japonicum By Western Blot Based On Igy

Schistosomiasis is a serious parasitical zoonosis with public health importance ThereaJ'e still many people in risk to be infected by Schistosomiasis in the south of the YangtzeRiver Basin and its 12 provinces,municipalities and autonomous regions,380 countiesEarly and correct diagnosis is the focus of schistosomiasis prevention and control work,and nowadays immunology methods are used on disease diagnosis and monitoring of theepidemic Immunological diagnosis of Schistosomiasis includes detection of circulatingantibodies(CAb)and circulating antigen(CAg)The technology of detecting circulatingantibody has been more mature and have a higher sensitivity and specificity,but it isdifficult to distinguish between past infection and current infection,can not assess efficacy,and also isn't conducive to the monitoring of schistosomiasis But the technology ofdetecting circulating antigen is preferable to give an indication ofthe activities of infectionand measure the infection degree,with a better value of assessing efficacy At the Sa2rletime it can also serve as the basis for current infection Moreover,circulating antigen levelsaJ'e relatively low,SO there aJ'e some issues as low sensitivity and specificity in the detectionof circulating antigen and the existence of circulating immune complexes(CIC)furtherreduce its detection rateTraditional immunological diagnostic methods aJ'e by detecting serum and not easilyaccepted by subjects．especially in large-scale epidemiological investigation with poor compliance in groups Moreover,it also may lead to irogenic infection Through thedetection of urine or saliva schistosomiasis diagnosis is a simple,practical and no injurymethod of immunization,with great prospects for developmentChicken egg yolk immunoglobulin(IgY)is a new polyclonal antibody,and it canimprove the sensitivity and specificity of immunological diagnosis Because of the highyield and nature of stability,in recent years,IgY has been widely used in diseases of theimmunological diagnosisIn this study,first of all,we used glycine-hydrochloride buffer solution to dissociate thecirculating immune complexes from urine of rabbit infected with Schistosoma japonicum,and then Western blot to detect circulating antigens after dissociation based on chicken eggyolk immunoglobulin(IgY)ofanti-soluble egg antigen of Schistosomajaponicum(SEA)For keeping the IgY antibody with a uniform titer,we first mixed the 30-60d IgYantibodies after immunization fully and package them,assayed the protein concentration byBCA method with 8 2ug/u1．and reappraisaled the potency by indirect ELISA method Theprotein concentration of SEA prepared for analyzing the titer of IgY antibody W'as 3 lug/ul,and the potency of SEA detected by indirect ELISA was as high as more than 1:12800The urine protein concentrations of rabbit infected with Schistosoma japonicum andnormal rabbit measured by BCA method were 9 9ug/ul and 2 8ug/u1．and we analyzed themby sodium dodecyl sulfate-polyacrylaanide gel electrophoresis(SDS-PAGE)The resultshowed that there was no obvious protein band in normal rabbit urine sample；infectedrabbit urine specimen had 2 main bands with the molecular weight of 146kDa and 60kDaand 3 secondary bands with the molecular weight of 108kDa,72kDa and 67kDa beforedissociating immune complexes and degenerating the protein But there were only 1 mainband with the molecular weight of 68kDa and 1 secondary band with the molecular weightof 86kDa after using glycine-hydrochloride buffer solution to dissociate the circulatingimmune complexes and degenerating the protein Using Western blot to analyze infectedrabbit urine after the treatment and the result showed the sa2ne protein band 68kDa had intnsive reaction with the serum from rabbit infected with Schistosoma japonicum and30-60d mixed IgY antibodies,but no protein band reacted with the normal rabbit serum andIgY from unimmunized egg yolkIn this study．we first used indirect ELISA method to identify the t"er of 30-60d mixedIgY antibodies,then SDS-PAGE to analyze the protein components of urine from rabbitinfected with Schistosoma japonicum and normal rabbit,and last Western blot method todetect the circulating antigens after dissociation in infected rabbit urine The conclusionpreliminary confirmed certain values of IgY in testing the circulating antigens of infectedrabbit urine,and was expected to further enrich and improve the immunological diagnosisof schistosomiasis.