摘要
血吸虫病是一种严重危害人类健康并阻碍疫区社会经济发展的人畜共患病，目前全球约有7．79亿人受威胁，感染者达2．07亿人。我国日本血吸虫病主要分布于长江流域及其以南的12个省市自治区，至2006年底仍有日本血吸虫病人67．13万。血吸虫病仍然是我国乃至世界范围内的严重危害人类健康的公共卫生问题之一。因此，我国血防工作所面临的形势仍然十分严峻。
由于吡喹酮仍然是杀灭血吸虫的有效药物，因此诊断在血吸虫病防治中处于重要位置。早期正确的诊断和及时有效的治疗是防止慢性和晚期血吸虫病发生的关键。目前血吸虫病诊断主要依靠粪检发现虫卵或血清学试验检测特异性抗体，但由于粪检易受感染度和感染时间的影响，且工作量大、受检者的依从性差，因此单纯依靠粪检诊断血吸虫病已不能适应血防工作的需要。随着免疫学技术的发展，国内外逐渐利用检测人群血清中特异性抗体的方法来弥补粪检方法的不足。检测血清中特异性抗体的方法具有方法简便、快速和人群依从性较好的优点，但由于血吸虫特异性抗体能在感染者体内存在很长时间，甚至在治愈后数年仍可从血清中测出，因此此法不能区分现症感染和既往感染，也无考核疗效的价值。为了克服抗体检测技术的不足，从上世纪80年代开始，人们转而研究检测血吸虫循环抗原的技术。由于循环抗原是由活虫排放的，虫体死亡后患者体内的循环抗原也会很快消失，因此检测血吸虫循环抗原的优点在于它能区分现症感染和既往感染，且具有考核疗效的价值。已报道的检测血吸虫循环抗原的方法有间接红细胞凝集试验、时间分辨免疫荧光分析方法、磁珠抗原捕获酶联免疫试验、杂交瘤细胞凝集试验、试纸条法和酶联免疫吸附试验等，但迄今为止，尚无敏感性高、特异性强的检测循环抗原的技术问世，至此，血吸虫病的免疫诊断成为制约血吸虫病防治工作发展的瓶颈问题。利用免疫学的新技术和新方法，开发具有敏感性高、特异性强、适于现场应用的检测血吸虫循环抗原的新技术，以适应新形式下血防工作的需要是当前亟待解决的问题。
IgY是鸡的卵黄免疫球蛋白的简称(Immunoglobulin of egg yolk，IgY)，通过皮下注射特异性抗原，鸡可产生相应的特异性抗体并可从母鸡的血清传递到蛋黄中。这种从蛋黄中提取的抗体称为IgY。由于IgY可与抗原上的更多表位发生反应，起到信号放大作用，因此可提高免疫学诊断的敏感性。Ohnishi等发现，IgY可使ELISA检测的灵敏度提高10倍。此外，IgY不与补体系统、类风湿因子、哺乳动物抗体以及人或细菌Fc受体等结合，因此可减少免疫检测中哺乳动物血清样本中无关因子的干扰从而避免假阳性或假阴性结果的产生。IgY的独特优势使其被欧洲实验方法替换确认中心(European Centre for the Validation of Alternative Method，ECVAM)推荐为哺乳动物IgG的替代免疫球蛋白，并被应用于多种疾病的免疫诊断中。但迄今为止，尚未见到将其应用于血吸虫病免疫学诊断的报道。
免疫磁珠酶联免疫法(Immunomagnetic bead ELISA，IMB-ELISA)是20世纪80年代末发明的一种新的检测技术，该技术以高均一的纳米磁性微球为固相支持物，由于纳米磁珠颗粒小、表面积大，可结合更多的诊断分子，因此可提高Ig分子的Fab与抗原结合的机会，使蛋白吸附能力超出酶标板载体的1000倍以上，克服了普通酶标板酶联免疫技术的缺点。
本课题结合上述两项新技术的优点，制备了抗日本血吸虫可溶性虫卵抗原(Soluble egg antigen，SEA)的IgY(anti-SEA IgY)，并将anti-SEA IgY耦联在纳米磁珠上，建立了基于IgY的免疫磁珠夹心ELISA法(Immunomagnetic bead ELISA based onIgY，IgY-IMB-ELISA)。用IgY-IMB-ELISA法检测血吸虫感染小鼠血清和尿液中的循环可溶性虫卵抗原(Circulating soluble egg antigen CSEA)，并将其用于检测血吸虫病患者血清中CSEA，从而为检测日本血吸虫循环抗原提供一种新的技术。
本课题分为以下四个部分：
一、IgY-IMB-ELISA法的建立
本实验用日本血吸虫SEA免疫莱杭母鸡，用EGGstract IgY Purification System提取和纯化了anti-SEA IgY，并对其进行了SDS-PAGE和Western-blotting鉴定，成功制备了分子量为130 kDa的特异性IgY多克隆抗体。该多克隆抗体可特异性地识别日本血吸虫SEA中140 kDa、100 kDa和69 kDa三种抗原成分。将IgY与免疫磁珠偶联并将其作为捕获抗体，以辣根过氧化物酶标记的小鼠抗SEA单克隆抗体NP28-5B(NP28-5B labeled horseradish peroxidase，HRP-NP28-5B)作为检测抗体，建立了IgY-IMB-ELISA法，此法最低可检测出5ng／ml的SEA。
二、IgY-IMB-ELISA法检测日本血吸虫感染小鼠血清中循环抗原的研究
用IgY-IMB-ELISA法检测日本血吸虫不同感染度的小鼠血清中CSEA的动态变化，结果发现，重度感染组(30只尾蚴／每鼠)和轻度感染组(10只尾蚴／每鼠)小鼠血清中CSEA分别在感染后4周和5周出现阳性，之后血清中CSEA水平迅速升高，至感染后第8周到达高峰，然后维持至少6周的平台期，直至实验结束(感染后14周)。研究结果表明宿主体内的CSEA水平与感染度呈正相关。
此外，本研究观察了吡喹酮治疗对重度感染组小鼠血清中CSEA的影响。感染小鼠血清中CSEA水平在吡喹酮治疗后6周降至阴性对照组水平，说明本研究所建立的检测方法可用于评估化疗的效果。
三、IgY-IMB-ELISA法检测日本血吸虫病患者血清中循环抗原的研究
本研究在利用IgY-IMB-ELISA法成功检测出动物血清中CSEA的基础上，进而将其用于检测血吸虫病患者血清中的CSEA。研究发现，IgY-IMB-ELISA法对急性和慢性血吸虫病患者的检出率分别是100％(40／40)和91．5％(107／1 17)。与正常人血清未出现阳性反应(0／49)。与肺吸虫病人血清无交叉反应(0／20)，与肝吸虫病人的交叉反应为3．3％(1／30)。IgY-IMB-ELISA法检测血吸虫病的特异性为99．0％，该法检测急性和慢性血吸虫病的约登指数分别为0．99和0．91。说明IgY-IMB-ELISA法是一种敏感性和特异性均较高的检测血吸虫循环抗原的方法。
四、IgY-IMB-ELISA法检测日本吸虫感染小鼠尿液中循环抗原的研究
本研究用IgY-IMB-ELISA法检测血吸虫感染小鼠尿液中CSEA并观察其动态变化，结果发现重度和轻度感染组分别于感染后8周和10周尿液中CSEA水平达到高峰，之后一直维持平台期直至实验结束。在感染后8周至14周中，重度感染组小鼠尿液中CSEA水平明显高于轻度感染组。
重度感染组小鼠在吡喹酮治疗4周后尿液中CSEA水平明显升高，然后下降，治疗后8周降至阴性对照组的水平。与同期血清中CSEA水平相比，尿液中CSEA出现的时间较晚、水平较低。尿液中CSEA水平受吡喹酮治疗影响明显滞后于其对血清中CSEA水平的影响，这可能与肾脏对循环抗原的浓缩和延迟排出作用有关。
本研究探讨了血吸虫感染小鼠尿液中CSEA的动态变化及吡喹酮化疗对其的影响，为下一步检测血吸虫病患者尿液循环抗原奠定了良好的基础。
综上所述，本课题研究结果为：
1．首次成功地制备了分子量为130 kDa的抗SEA的特异性IgY多克隆抗体，该抗体可特异性地识别日本血吸虫SEA中140 kDa、100 kDa和69 kDa三种抗原成分。
2．结合IgY和IMB-ELISA的优点，首次建立了IgY-IMB-ELISA法，该法可检出低至5ng／ml的SEA。
3．利用IgY-IMB-ELISA法检测不同感染度小鼠血清和尿液中的CSEA，初步阐明了CSEA在感染小鼠血清和尿液中的动态变化规律，为血吸虫循环抗原的适宜检测时间提供了实验依据。
4．动物和人体实验证实，IgY-IMB-ELISA法可有效地检测出不同感染度小鼠和感染后不同时期患者血清中CSEA，提示IgY-IMB-ELISA法是一种敏感性和特异性均较高、操作简便的血吸虫循环抗原的检测方法。
5．动物实验证明，经吡喹酮有效治疗后，感染小鼠血清和尿液中CSEA分别于治疗后6周和8周消失，提示IgY-IMB-ELISA法不仅可区分现症感染和既往感染，还可用于疗效考核。
6．动物实验证明，IgY-IMB-ELISA法可检测出不同感染度小鼠尿液中的CSEA，为无创伤性诊断人体血吸虫病奠定了实验基础。
7．将IgY-IMB-ELISA法用于检测血吸虫病人尿液中的循环抗原，从而建立一种简便、快速且对人体无任何损害的诊断技术是本课题的下一步目标。
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-ELISA
Leghorn 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-ELISA
IgY-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-ELISA
The 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.
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