Detection Of Circulating Antigen Of Schistosoma Japonicum By Immunomagnetic Bead Sandwich ELISA Based On IgY

Schistosomiasis is a serious zoonosis which affects human health and hinders theeconomy development in endemic areas.There are 779 million people at risk of infectionwith schistosome and among them 207 million people are suffering from schistosomiasisall over the world.It is estimated that there are 671.3 thousand cases of schistosomiasisjaponica in our country in 2006.Schistomiasis still remains one of major public healthproblems in China as well as in the world.Diagnosis of schistomiasis plays a key role in the control programs.So far,diagnosis of schistomiasis is usually performed by parasitological or immunologicalmethods.Definite diagnosis still relies on parasitological detection,mainly through thedetection of eggs in feces or miracidium hatching method.Nowadays,both the prevalenceand infection intensity have been reduced drastically due to repeated chemotherapy inendemic areas.As a result,repeated stool examination is usually required to increase thesensitivity,especially in the case of light infection and for efficacy evaluation.However,repeat stool examination is difficult to be operated because people are reluctant to providefeces samples repeatedly.In addition,stool examination is influenced by unevendistribution of eggs in solid matrix and day-to-day variation of egg excretion.In this condition,the prevalence of schistomiasis is surely to be underestimated especially inendemic areas with low intensity by parasitological detection.Thus,parasitologicalexamination can not meet the needs of schistosomiasis control.Immunological diagnosesare used most widely to detect the antibodies.However,antibody detection is impossible todistinguish acute case from past infection since antibodies can still be detected in the hosteven a long time after successful chemotherapy.The detection of schistosome circulatingantigens seems an effective way to discriminate between previous exposure and currentinfection.In this respect,the detection of schistosome circulating antigen takes a prominentplace.Various methods have been developed for detecting schistosome circulating antigen,including ELISA,indirect haemagglutination,time-resolved immunofluorometric assay,magnetic bead antigen immunoassay,hybridoma cell agglutination and reagent strips.Unfortunately,the sensitivity and specificity of current techniques to detect circulationantigen are unsatisfied.Therefore,exploring a method with better sensitivity and specificitybecomes a hot research field.Chicken egg yolk immunoglobulin (IgY),produced by hens immunized byspecific antigens and transferred from serum to egg yolk,has been recognized as anexcellent source of polyclonal antibodies for over decades.Specific antibodies produced inchickens offer several important advantages over producing antibodies in mammals.Due tothe evolutionary difference between mammals and birds,chicken IgY reacts with moreepitopes on a mammalian antigen,which will give an amplification of the signal.It wasreported that the sensitivity of ELISA could be improved ten fold by the application of IgY.In addition,the interference in immunological assays could be reduced because IgY doesn'treact with the complement system,rheumatoid factors,anti-mammalian antibodies,orhuman and bacterial Fc receptors.Because of the above advantages,IgY has been widelyused to diagnosis in different diseases.However,to our knowledge,there is no report on theuse of IgY in the detection of schistosomiasis.The magnetic bead antigen immunoassay combines the use of magnetic beads with a high binding capacity as a solid phase and the rapid reaction kinetics of solutionswith the simple separation of bound and unbound material on the solid phase,whichprovides the chance of enhancing the sensitivity of antigen detection.The immunomagneticbead ELISA is found to provide higher sensitivity compared to a microplate-based ELISAtechnique.In the present study,we produced and identified IgY polyclonal antibody againstsoluble egg antigen (SEA) of Schistosoma japonicum and further developed animmunomagnetic bead ELISA based on IgY (IgY-IMB-ELISA) for detection ofschistosome circulating antigen.The assay involved the use of anti-SEA IgY coupled tomagnetic bead as a capture antibody and anti-SEA mouse monoclonal antibody NP28-5Blabeled horseradish peroxidase (HRP-NP28-5B) as a detecting antibody.In addition,IgY-IMB-ELISA was used to detect circulating soluble egg antigen (CSEA) in serum andurine of mice infected with S.japonicum.The effect of praziquantel on the CSEA levels inserum and urine from murine schistosomiasis was also investigated.Furthermore,the novelassay was used to diagnose human schistosmiasis japonica.The main contents and results of this study are follows:1.Development of IgY-IMB-ELISALeghorn hens were immunized subcutaneously with SEA of S.japonicum andthen IgY polyclonal antibody was extracted from immunized yolk according to the protocolof EGGstract^TM IgY Purification System.Protein content of IgY was checked by BCAProtein Assay Kit.The purified IgY was analyzed by non-reducing SDS-PAGE andidentified by Western-blotting.The result showed that,ant-SEA IgY with MW of 130 kDawas produced from immunized leghorn hens and it could recognize three protein bandswith MW of 140 kDa,100 kDa and 69 kDa of SEA.Moreover,we developedIgY-IMB-ELISA for detection of schistosome circulating antigen.The assay involved theuse of ant-SEA IgY coupled with magnetic bead as a capture antibody and HRP-NP28-5Bas a detecting antibody.IgY-IMB-ELISA demonstrated the high sensitivity since its lower detection limit of SEA was 5ng/ml.2.Detection of CSEA in serum of mice infected with Schistosoma japonicum byIgY-IMB-ELISAIgY-IMB-ELISA was used to detect CSEA in serum from murine schistosomiasis.Two groups of BALB/c mice infected with S.japonicum cercariae were used:lightlyinfected mice (infected with 10 S.japonicum cercariae) and heavily infected mice (infectedwith 30 S.japonicum cercariae).The CSEA was detectable as early as 4 and 5 weeks afterinfection in the sera of heavily and lightly infected mice,respectively.The CSEA levelsrose rapidly and reached a peak in 8 weeks after infection and then remained a plateau forat least another 6 weeks in both groups.Moreover,the effect of praziquantel on the CSEAlevels was also investigated.The heavily infected mice were treated with praziquantel andthe CSEA levels in sera increased dramatically in the first week post treatment and thendecreased to the control level by 6 weeks after treatment.The novel assay appears to besensitive for detection of schistosomal antigenemia and valuable to assess the efficacy ofchemotherapy in murine schistosomiasis.3.Detection of CSEA in serum of human schistosomiasis japonica byIgY-IMB-ELISAThe sensitivity of IgY-IMB-ELISA was 100% (40/40) in 40 cases of acuteinfection and 91.5% (107/117) in 117 chronic schistosomiasis cases,and no positivereaction (0/49) was found in healthy individuals.The cross-reaction with clonorchiasis was3.3% (1/30) and 0 (0/20) with paragonimiasis.The specificity of the novel assay was 99.0%for detection of human schistosomiasis.Youden's indexes of IgY-IMB-ELISA for detectionof acute and chronic cases were 0.99 and 0.91,respectively.The results indicated thatIgY-IMB-ELISA is sensitive and specific for the detection of human schistosomiasisjaponica.4.Detection of CSEA in urine of mice infected with Schistosoma japonicum byIgY-IMB-ELISA To investigate the possibility of IgY-IMB-ELISA in application of urine diagnosisof schistosomiasis,the novel assay was used to detect CSEA in urine of murineschistosomiasis with different infection intensity.The results showed that the CSEA levelsin urine of lightly and heavily infected mice reached a peak in 8 and 10 weeks afterinfection,respectively.And then it remained a plateau in both groups by the end of theexperiment (14 weeks after infection).The CSEA level in urine of heavily infected micewas much higher than that of lightly infected mice during 8 to 14 weeks after infection.Inaddition,the effect of praziquantel treatment on the CSEA level in urine of heavily infectedmice was also investigated.It was found that the CSEA level in urine of heavily infectedmice increased dramatically in 4 weeks after treatment and then decreased to the controllevel by 8 weeks after treatment.Compared to that in sera,the CSEA in urine appeared laterand its corresponding level was much lower.Additionally,the effect time of praziquanteltreatment on the CSEA level in urine was also later than that in sera.The results indicatedthat IgY-IMB-ELISA is valuable to detect CSEA in urine of infected mice and monitor theefficacy of chemotherapy,which provides scientific basis for its further application todetect circulating antigen in urine of human schistosomiasis.In conclusion,these data demonstrated:1.It was firstly reported that anti-SEA IgY was produced from immunized leghorn henswith SEA immunization and the specific IgY could recognize three protein bands withMW of 140 kDa,100 kDa and 69 kDa of SEA.2.It was firstly reported that IgY-IMB-ELISA was developed for detection of circulatingantigen of schistosomiasis and its lower detection limit of SEA was 5 ng/ml.3.The kinetics of CSEA in sera and urine of infected mice with different intensity weredemonstrated clearly by IgY-IMB-ELISA,which provided scientific basis for thedetection time of circulating antigen of schistosomiasis japonica.4.The serum CSEA of infected mice with different infection intensity and of humanschistosomiasis with different stage could be detectable by IgY-IMB-ELISA,which showed that the novel assay is a valuable method to diagnose schistosomiasis with goodsensitivity and specificity.5.CSEA in sera and urine of infected mice disappeared in 6 weeks and 8 weeks afterpraziquantel treatment,respectively.The resulted showed that IgY-IMB-ELISA couldbe used not only to distinguish acute case from past infection,but also to assess theefficacy of chemotherapy in murine schistosomiasis.6.IgY-IMB-ELISA was valuable to detect CSEA in urine of infected mice with differentintensity,which laid a good foundation to the application of this noninvasive diagnosismethod to human schistosomiasis.7.We will investigate the application of IgY-IMB-ELISA for detection of circulatingantigen in urine of human schistsomiasis in the further study.