| Schistosomiasis is one of zoonoses caused by worms of the genus Schistosoma, which is widely prevalent in tropical and subtropical regions, and more than 200million people are infected worldwide, with an estimated 600 million people at risk of schistosomiasis in 76 endemic countries. In China, Schistosomiasis was mainly endemic in 12 provinces, municipalities and autonomous regions along the Yangtze River Valley and the southern regions of the Yangtze. The transmission of schistosomiasis has been blocked in five provinces(Shanghai, Zhejiang, Guangdong, Guangxi and Fujian), but still remained in another seven provinces, so the prevention and control of schistosomiasis still faces serious challenges in China. Diagnosis plays an important role in the prevention and control of schistosomiasis. parasitological diagnosis as the gold standard for diagnosis of schistosomiasis, now becomes more and more difficult, for morbidity and infection intensity of the people with schistosoma japonicum being low,the patient's compliance is also becoming less and less, it calls for rapid, sensitive and accurate method of immunodiagnosis occurs.Detection of circulating antibodies couldn't distinguish between current and past infection, and evaluate the therapeutic effect, despite a good sensitivity, specificity, for their retention in the body are still a number of years after treatment. The detection of circulating antigens may reflect the existence of active infection of schistosome, and can be used on treatment assessment. but there are still problems of the detection techniques based on polyclonal antibody and monoclonal antibody of mammalian, such as low sensitivity, low specificity and cross-reactions with sera of patients with other parasitic diseases. Therefore, we need to find new methods of circulating antigen detection or alternative capture antibodies.Egg yolk immunoglobulin (IgY), a major immunoglobulin from birds, amphibians and reptiles, has similar functions and some different features to mammalian IgG. IgY has been widely used in the medical field, and proved potential application for diagnosis and treatment of disease. Therefore, this study was to explore potential applicational value of the IgY for detecting circulating antigen of schistosome.1.To prepare and identify preliminarily the specific IgY against soluble egg antigen (SEA) of Schistosoma japonicum, and observe the dynamic changes of the specific IgY obtained with two immunization routes in the chicken yolk.2.To establish a double antibodies sandwich enzyem-linked immunosorbent assay (S-ELISA) and colloidal gold immunochromatographic test strip method for detecting circulating antigens of Schistosoma japonicum in the sera of people infected with Schistosoma japonicum, to explore application value of the two methods in diagnosis of Schistosomiasis.Objective To prepare and identify preliminarily the specific IgY against soluble egg antigens (SEA) of Schistosoma japonicumMethods Seven New Zealand rabbits were infected with Schistosoma japonicum cercariae (1 500 per rabbit).After 42 days the rabbits were sacrificed to collect the eggs of schistosome from the livers and prepare the soluble eggs antigens(SEA). Six hens, being divided into two groups averagely, were intravenously and subcutaneously immunized with 50μg SEA of Schistosoma japonicum respectively All hens received five immunizations by the same dose of antigen, with 2-week interval for the first two doses, and 4-week interval for the rest doses. Hen eggs were collected before and after immunization. Egg yolk was diluted 1:6,1:8,1:10 by double distilled water respectively, freezing and thawing once or twice. then purified by using Saturated ammonium sulfate and method and EGG stract IgY Purifiction System. IgY concentration was determined by A260/A280 ratio. The titer of IgY in the egg yolk was evaluated by SEA-based ELISA, and the characteristics of IgY was analyzed by SDS-polyacrylamide gel electrophoretic (SDS-PAGE) and Western blotting.Results The best water dilution method was established:egg yolk was diluted 1:8 by double distilled water, freezing and thawing twice,and the concentration of crude IgY was about 6.5-9.0 mg/ml. SEA-based ELISA demonstrated that the titer of IgY was 1:256 00 and 1:128 OOby water dilution and by the kit,respectively.. SDS-PAGE demonstrated that the IgY contained two major protein bands with molecular weight of 68 000 and 25 000, western blotting demonstrated that only IgY purified from immunized egg yolk could be recognized by SEA.Conclusion Anti-SEA IgY with high specificity has been obtained and purified,It's a basis to further explore potential applicational value of the IgY for detecting circulating antigen of schistosome.Objective To observe the dynamic changes of the specific chicken egg yolk Antibodies (IgY) against soluble egg antigens (SEA) of Schistosoma japonicum in the yolk of two Immunization Routes.Methods Six hens, being divided into two groups averagely, were intravenously and subcutaneously immunized with 50μg SEA of Schistosoma japonicum respectively All hens received five immunizations by the same dose of antigen, with 2-week interval for the first two doses, and 4-week interval for the rest doses. The chicken eggs were collected pre-immunization and until eighteen weeks after immunization. Crude IgY of 2,4,6……18 week were extracted from egg yolks by water dilution.Agarose double diffusion method was used to evaluate the initial time of the antibody production, and the dynamic changes of the anti-SEA IgY were compared between the intravenous group and the subcutaneous group with SEA-based ELISA.Results The initial time of IgY in the intravenous group and the subcutaneous group were all come out the first week after immunization.The titer of IgY reached a peak at the 8th week in the intravenous group and at the 12th week in the subcutaneous group, respectively, and kept at a high level until the 18th week in the intravenous group after first immunization, and decreased gradually after the peak in the subcutaneous group.Conclusion Dynamic of chicken egg yolk antibody against soluble egg antigen of Schistosoma japonicum was received, provided a theoretical and experimental basis for the preparation of high titer of specific IgY.Objective To establish a double antibody sandwich ELISA (S-ELISA) to detect circulating antigen in serum of patients infected with Schistosoma japonicum, to explore application value of its diagnosis in Schistosomiasis.Methods Determination of the best concentration of coated antibody and optimal working concentration of IgM-HRP:each well of the ELISA plate was coated with 20μg/ml,40μg/ml,60μg/ml and 80μg/ml of the anti-SEA-IgY respectively, and stored at 4℃overnight. The plate was washed 3 times with PBS, and then blocked with Skim milk. One hundred microliters of SEA antigen (20μg/mL) was added to the wells, and the plate was incubated at 37℃for 1 hour. The plate was washed 3 times with PBS containing 0.05% Tween 20. One hundred microliters of peroxidase-conjugated Anti-SEA-IgM monoclonal antibody(HRP-IgM) was added with the dilution of 1:2000 ,1:1000 and 1:500 respectively, and the plate was incubated at 37℃for 1 hour. After the plate was washed, TMB substrate was added to each well, and the plate was incubated incubated for 10 minutes at 37℃. The reaction was stopped by adding 50μl of 2 Msulfuric acid to each well. The optical density (O.D.) was measured at 450nm.. The sera of patients with schistosomiasis and normal people were diluted to 1:10,1:5 and original liquid to be selected optimal dilution. The IgY-S-ELISA established was used to detect schistosome circulating antigens in the sera of patients with acute, chronic Schistosomasis japonica and other parasitoses, and the sensitivity, specificity and cross reactivity of the method were analyzed.Results The best working conditions of double-antibody sandwich ELISA was established:the optimal working concentration of IgM-HRP was 1:500; the best concentration of IgY antibody coated was 40μg/ml, Minimum detectable amount of SEA antigen was 300ng/ml. Among the sera from schistosomiasis patients, 100%(20/20) of acute and 91.3%(21/23) of chronic cases gave a positive reaction. 95%(38/40)of healthy persons gave a negative results, and only a few of Paragonimus westermani and Cysticercosis cases gave a false positive result. The patients suffering from other parasitic diseases gave negative results. Conclusion Double antibody sandwich ELISA based on IgY was established, gave higher specificity and sensitivity.Objective to develop a Colloidal gold immunochromatography rapid test strip for detecting circulating schistosome;to obtain the best pair of capture antibody and antibody labeled with gold particles.Methods The IgY-Au conjugate of anti-SEA IgY labeled with gold particles prepared with citrate reduction method, was sprayed on glass fiber membrane by two-dimensional spouted instrument, to obtain the gold conjugate pad. The nitrocellulose(NC) membrane was sprayed with another ant-SEA McAb and anti-mouse Polyclone antibody respectively, as test line (T line) and control line (C lines).(NC) membrane,the gold conjugate pad, sample pad, PVC plate were put together to make of colloidal gold immunochromatography rapid test strip.The McAb IgG1,IgG2b and IgM against SEA of Schistosoma japonicum matched with IgY-Au conjugate respectively,or the IgY matched with the McAb IgG1-Au,IgM-Au and IgG2b-Au conjugate respectively, tocompare their detection effect for testing schistosome circulating antigen.Results The pair of IgG1(1.5 mg/mL) and IgY-Au conjugate (30%) gave the best result.Among the sera from schistosomiasis patients, acute and chronic cases all gave positive reaction,healthy persons gave negative results.Conclusion colloidal gold immunochromatography rapid test strip for schistosomiasis japonica was preliminary established.The pair of IgG1(1.5 mg/mL) and IgY-Au conjugate (30%) gave the best results.1. Hailan chickens were immunized with SEA of Schistosoma japonicum intravenously and subcutaneously respectively, and the eggs were collected before and after immunization. IgY was extracted and purified from the egg yolk with water dilution method, then purified with saturated ammonium sulfate method and EGG stract IgY Purification System respectively. The method of water dilution was efficient for extracting the egg yolk IgY, and the concentration of crude IgY extracted by water dilution was up to 6.5-9.0 mg/ml. SDS-PAGE demonstrated that the IgY contained two major protein bands with molecular weight of 68 000 and 25 000, and western blotting demonstrated that SEA could be recognized by the IgY purified from immunized egg yolk.2. The chicken eggs were collected pre-immunization and 2-18weeks after immunization. The dynamic changes of the anti-SEA IgY extracted from egg yolks by water dilution were compared between the intravenous group and the subcutaneous group with SEA-based ELISA. The titer of IgY reached a peak at the 8th week in the intravenous group and at the 12th week in the subcutaneous group, respectively, and kept at a high level until the 18th week in the intravenous group after first immunization, and decreased gradually after the peak in the subcutaneous group.3. The double-antibody sandwich ELISA based on IgY (IgY-S-ELISA) was established with anti-SEA IgY as capture antibody, McAb IgM labeled with HRP as enzyme conjugate and used to detect schistosome circulating antigens in the sera of patients with acute, chronic Schistosomasis japonica and other parasitoses, and the sensitivity, specificity and cross reactivity of the method were analyzed. The best working conditions of IgY-S-ELISA was determined. The minimum detectable amount of SEA antigen was 300ng/ml. The results of detection showed the positive rates of 20 acute cases and 23 acute cases with schistosomiasis were 100%(20/20) and 91.3%(21/23), respectively, and 95% of healthy persons gave a negative results, and only few of Paragonimiasis westermani and Cysticercosis cases gave a false positive result. The patients suffering from other parasitic diseases gave negative results.4. Colloidal gold immunochromatographic strip was established by choosing the best one of pairs of capture antibodies and gold labeled antibodies(anti-SEA McAb IgM, IgG1, IgG2b, and Poly clone IgY), and used to detect the circulating antigens in the sera of patients with schistosomiasis.The pair of IgG1(1.5 mg/mL) and IgY-Au conjugate (30%) gave the best result. Among the sera from schistosomiasis patients, acute and chronic cases all gave positive reaction, healthy persons gave negative results.
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