Preparation Of Anti-SEA Chicken Immunoglobulin Y And Detection Of Circulating Schistosoma Japonica Antigen

Objective:Immunodiagnosis of schistosomiasis japonicum mainly consists of detection of antibodies and circulating antigens.Generally, detection of circulating antibodies is cost-efficient and less affected by the immunity of host.But it is hard to distinguish the present infections from the past,as antibodies can exist a relatively longer time after the treatment.It means it is of no significance in evaluation of chemotherapeutic efficacy.While detection of circulating antigens reflects active infection,which means higher detection significance and a better method to evaluate chemotherapeutic efficacy.However,current assay to detect circulating antigens is of low specificity and of low sensitivity for chronic infectors.So a hot field in basic schistosomiasis research is to explore a method with better sensitivity,specificity and value on evaluating chemotherapeutic efficacy.Chicken egg yolk antibody(IgY),the main immunoglobulin in chicken egg yolk,produced by hens immunized by some specific antigens,and accumulated in the yolk,also referred to as chicken IgG,has recently attracted considerable attention as an inexpensive source of antibodies.Compared with mammalian IgG;IgY is economical and easily prepared with good stability.It neither binds Fc receptors,nor activates the complement system,or reacts to rheumatoid factor.In addition,far from the mammal species IgY is less likely to show cross-reactivity to mammalian IgG,which can decrease false positive rate and increase specificity.Our study aims at the development of a sandwich ELISA by using purified anti-SEA IgY as capture antibody and a HRP conjugated mAb(NP28-5B)as detection antibody to detect circulating antigens in schistosomiasis.Methods:1.IgY preparation and identification:25-week-old Hailan hens were immunized 4 times with soluble egg antigen of schistosome japonicum intravenously and subcutaneously.Antigen dose for initial immunization was 60μg.Three boost injections were given at ten-day intervals with 30μg SEA.IgY was extracted from eggs 35d after the initial immunization by WD(water-dilution)method.Simultaneously,eggs of non-immunized hens were used as negative control.Concentration of IgY was measured by BCA,its relative molecule weight and purity were determined by SDS-PAGE,and specificity by ELISA.2.Production and labeling of Monoclonal antibody NP28-5B:The anti-SEA hybridoma cell line was obtained from a fusion of SP2/0 and spleen cells of a BALB/c mouse chronically infected with schistosoma japonicum for 4 months and identified by screening with soluble egg antigen(SEA)of S.japonicum,indicating that the NP28-5B was an anti-SEA antibody.The supernatant of cultured hybridoma cells was harvested after sub-culturing,precipitated with 50%ammonium sulfate and re-dissolved in 10mM PBS(pH7.4),then reserved at -70℃.HRP was bound to the monoclonal antibody by simplified labeling method.3.Sandwich ELISA:by using purified anti-SEA chicken IgY as the capture antibody and anti-SEA mouse monoclonal antibody NP28-5B conjugated with HRP as the detection antibody,a sandwich ELISA was set to examine the sera of acute and chronic schistosomiasis patients and healthy people along with the SEA-ELISA,which is the classical method for detecting antibodies.The established S-ELISA was further improved by using orthogonal test to determine the appropriate test factors(coating concentration of the capture and detection antibody,incubating time of sera and detection antibody).4.Source of infected sera:9 acute infected sera and 45 chronic infected sera were respectively collected from Duchang County of JiangXi Province and WeiShan County of Yunnan Province.50 healthy people sera collected from BingZhou city of ShanDong province,where is the non-epidemic field of schistosomiasis japonicum.Results:35d after initial immunization,approximately 61mg IgY can be extracted from each egg yolk.Purified IgY,with relative molecular weight of 180 000,shows a main protein marker by SDS-PAGE. Non-denatured SDS-PAGE results showed 7 bands of the IgY,with 2 major bands,containing 66000 and 35000 of Mw.The supernatant of cultured hybridoma cells was further purified by 50%ammonium sulfate precipitation and completely dialyzed against PBS,and the final concentration of monoclonal antibody was 61.75mg/ml.Before conjugated with HRP,its activity should be determined by indirect ELISA.The optimal work concentration of HRP-NP28-5B is 1:600～1:1000.There exists a significantly positive correlation between SEA concentrations and A450 values.The regression equation was deduced (r=0.9481,y=459.22x-108.14).The positive rates of acute and chronic schistosomiasis by S-ELISA were 100%(9/9)and 84%(38/45) respectively.Its specificity for healthy people was 96%.The statistical analysis showed no difference in the detection rate between the two methods.Conclusions:1.The anti-SEA IgY shows high sensitivity and specificity after purification.2.The established sandwich ELISA also showed high sensitivity and specificity and is available for immunodiagnosis of schistosomiasis.