高强度聚焦超声波辐照细粒棘球蚴包囊的热效应

目的通过检测高强度聚焦超声波(HIFU)照射棘球蚴包囊后囊液、囊壁、包囊周围肝组织温度和原头蚴死亡率,了解HIFU处理后不同部位的升温效应,探索HIFU杀伤棘球蚴的方法和效果。方法采集感染细粒棘球绦虫的新鲜羊肝,选取囊壁较薄,触摸弹性较好,直径介于10mm到45mm之间的包囊42个。采用随机区组设计的方法分组,对照组用普通超声照射,实验组用200W声功率HIFU直线扫描的方法照射,照射时间分别为2min,4min,6min,8min,10min。照射后立即测定囊液、囊壁、距包囊5mm部位肝组织的温度。抽取囊液、涂片,经台盼兰染色后计数原头蚴的死亡率。结果HIFU照射后,棘球蚴囊壁、囊液、包囊周围肝组织的温度均有升高,其中囊壁的温度升高最为明显,且与囊液、包囊周围肝组织之间有显著差异;囊液中的原头蚴死亡率增加。结论HIFU照射使棘球蚴包囊的囊壁产生明显的升温效应,HIFU照射对原头蚴有杀伤作用。     

抗细粒棘球绦虫成虫单克隆抗体的制备及鉴定

目的 建立能分泌与细粒棘球绦虫(简称包虫)成虫特异结合的抗包虫成虫单克隆抗体的杂交瘤细胞株。方法 用包虫成虫抗原免疫BALB／c小鼠后，获取免疫的脾细胞，与小鼠骨髓瘤细胞SP2／0融合。结果 ELISA方法筛选出1株能持续稳定分泌抗包虫成虫单克隆抗体的杂交瘤细胞株，1F5E3C9E9D5杂交瘤细胞株分泌的单克隆抗体可以与包虫成虫、棘球蚴结合，与囊液抗原呈边缘性阳性反应，而与泡球蚴EM2抗原呈阴性反应。冻存1个月后复苏，仍能稳定分泌。经免疫组织化学检测发现，该单克隆抗体与棘球蚴的生发层，棘球蚴原头节虫体呈阳性反应。结论 1F5E3C9E9D5是一株稳定的杂交瘤细胞株，所分泌的抗细粒棘球绦虫成虫单克隆抗体在粪抗原的检测方面有应用前景。     

高强度聚焦超声辐照后细粒棘球蚴囊壁的病理变化

目的 观察细粒棘球蚴囊壁在高强度聚焦超声（HIFU）辐照后的病理改变。方法 采集感染细粒棘球蚴的新鲜羊肝，选取囊壁较薄、触摸弹性较好的细粒棘球蚴30个。采用随机抽样方法等分为3组，每组10个包囊。对照组，用普通诊断超声照射2 min。处理组1和处理组2分别用150 W和250 W声功率对细粒棘球蚴包囊进行沿囊壁多层面的环形扫描，层面间距为5 mm，扫描速度为3 mm/s，照射时间2～10 min（根据包囊大小）。取出照射后先肉眼观察细粒棘球蚴包囊大体改变，后取囊壁组织分别制作病理切片和透射电镜切片，观察其病理改变。 结果 HIFU（250 W）辐照后，细粒棘球蚴包囊剪开处内囊壁立即发生卷曲，剥离出的内囊颜色变白、变硬、透光度降低。病理切片显示，HIFU辐照后细粒棘球蚴的内囊壁上角皮层与生发层大部分发生分离。电镜观察结果显示，HIFU辐照后细粒棘球蚴的角皮层纤维纹理明显改变，生发层细胞发生裂解性破坏。 结论 HIFU沿细粒棘球蚴囊壁的多层面的环形照射可明显损害细粒棘球蚴囊壁。     

青海省果洛州人与动物棘球蚴病调查

目的通过对青海省果洛州人群与动物棘球蚴病的调查与分析,了解当地棘球蚴病在人群和动物中的流行与分布状况。方法应用B超对人群进行患病率调查;囊液抗原间接血凝试验(IHA)法筛查人群棘球蚴抗体的阳性率;并分别用重组Em18(rEm18)、重组抗原B(rAgB)和Eg囊液抗原(EgcF),对随机选取的B超检查和IHA检测阳性者进行ELISA试验,与B超诊断结果进行符合率比较,评价泡型棘球蚴病(AE)、囊型棘球蚴病(CE)的分布情况;应用ELISA检测家犬和藏狐粪抗原阳性率;现场检查牛、羊胸腹腔,确定牛、羊感染率。结果人群调查:①B超检查2826人,总患病率为9.45%。血清IHA法检查1113人,阳性率25.79%。女性人群的患病率和血清阳性率均显著高于男性;②血清囊液抗原IHA检测阳性者(108)与ELISA法检测EgcF,阳性符合率为66.94%;3种重组抗原检测,ELISA法与IHA法阳性符合率为75.00%。③B超法诊断棘球蚴病(88人)与ELISA法检测,符合率EgcF为94.32%,rAgB为68.18%,rEm18为5l-4%。EgcF阳性率符合率为69.44%;3种抗原检测与IHA检测总体阳性符合率为75.00%。61例CE患者中,血清ELISA法检测,EgcF阳性率为96.72%,rAgB为68.85%,rEm18为33.33%;26例AE患者中,EgcF和rEm18阳性率均为92.31%,rAgB为65.39%。动物调查:①高原鼠兔棘球蚴感染率21.74%。经cytb基因分析,证实有多房棘球绦虫(E.multilocularis)和石渠棘球绦虫(E.shiquicus)感染。绵羊棘球蚴感染率82.61%,牦牛棘球蚴感染率78.52%。②野犬、藏狐、猞猁均有不同程度的棘球绦虫感染。结论青海省果洛州人群与动物中存在棘球蚴病的高度流行。E.shiquicus种的幼虫和成虫分别在高原鼠兔和藏狐体内寄生已得到证实,提示果洛州为3种棘球蚴的混合流行区。     

细粒棘球蚴病患者血清特异性IgG亚型抗体的检测

目的用细粒棘球蚴囊液抗原检测甘肃省环县地区细粒棘球蚴病患者血清中特异性Ig G及其亚型抗体。方法采集甘肃省环县地区经B超确诊的37例细粒棘球蚴病患者和29例健康人血清,ELISA法检测血清中抗棘球蚴囊液抗原的特异性Ig G抗体及其亚型Ig G1、Ig G2和Ig G4。应用Med Calc软件,以B超检测结果为金标准,根据ELISA结果绘制受试者工作特征(ROC)曲线,通过曲线下面积的配对z检验,比较这4种抗体的诊断性能差异,确定最佳诊断阈值。用卡方检验分析并比较棘球蚴囊液抗原用于检测4种抗体的灵敏性和特异性。结果用囊液抗原检测Ig G、Ig G1、Ig G2和Ig G4抗体的ROC曲线下面积分别为0.722、0.919、0.712和0.835,其中Ig G1的曲线下面积显著大于Ig G和Ig G2的面积(P0.05)。棘球蚴囊液抗原检测这4种抗体的灵敏性分别为54.1%、91.9%、67.6%和75.7%,其中Ig G1的灵敏性高于Ig G、Ig G2和Ig G4(P0.05);特异性分别为89.7%、82.8%、72.4%和89.7%,Ig G1的特异性与Ig G、Ig G2和Ig G4相比,差异无统计学意义(P0.05)。检测Ig G4抗体在CEⅠ-Ⅲ型病例中的灵敏性高于CEⅣ-Ⅴ型病例(P0.05)。结论棘球蚴囊液抗原检测血清中Ig G1抗体的灵敏性优于其他3种抗体,但特异性与其他3种抗体间差异无统计学意义。     

分泌抗细粒棘球绦虫单克隆抗体杂交瘤细胞株的建立

为了对包虫病的免疫诊断和免疫预防提供有价值的单克隆抗体,通过对骨髓瘤细胞SP2/0与用细粒棘球蚴囊液免疫的BALB/c小鼠脾细胞融合试验及多次筛选、克隆,建立了分泌单克降抗体的细胞株NIA_2。用ELISA及免疫电镜等方法检测证实该细胞株有特异性。其培养上清分泌抗体滴度为1:2560,注入同系小鼠腹腔,诱生肿瘤和滴度为1:163840的腹水。免疫球蛋白鉴定表明是IgG_1,未见对囊虫的阳性反应。该杂交瘤细胞株经组织培养传代10个月,分泌抗细粒棘球绦虫抗体性能稳定。     

棘球蚴感染中的免疫逃避相关分子

棘球蚴可以依赖有效的免疫逃避机制在宿主体内长期生存,并造成慢性感染。包囊囊壁、原头节和囊液等所含抗原物质多而复杂,在逃避宿主的免疫应答中起重要作用。棘球蚴逃避宿主免疫反应机制分为主动免疫逃避与免疫调节两种:棘球蚴通过抑制补体的激活,耗竭特异性抗体,影响T细胞活性,下调B细胞功能等有效地避开宿主的免疫反应。棘球蚴的EgTeg蛋白和囊液中的抗原B等能明显抑制中性粒细胞的趋化作用;刺激宿主产生TH2型细胞因子,激发非保护性的免疫应答;干扰单核细胞分化和调节树突状细胞的表型,逃逸宿主免疫监视。     

细粒棘球绦虫水通道蛋白9的多肽特征及免疫定位研究

目的 研制细粒棘球绦虫水通道蛋白9 (EgAQP9)的特异性多肽抗体,并用于检测其在棘球蚴囊壁、生发细胞及原头蚴中的组织分布情况。 方法 根据EgAQP9特异氨基酸序列设计合成B细胞抗原多肽,再用合成的多肽偶联KLH作为免疫原注射免疫新西兰兔以制备多克隆抗体。酶联免疫吸附试验(ELISA)方法检测多肽抗体滴度,蛋白免疫印迹法(Western blot)测定多肽抗体免疫活性,免疫荧光实验分析EgAQP9的亚细胞定位,免疫组化实验分析EgAQP9组织定位。 结果 免疫新西兰兔成功制备出多肽抗体;间接ELISA检测其多肽抗体效价高达1∶256 000;Western blot结果显示多肽抗体能够特异识别细粒棘球绦虫中36 kDa处的条带,与EgAQP9预测的相对分子量大小相符;免疫荧光实验结果显示EgAQP9分布于棘球蚴生发细胞的胞质、胞膜中;免疫组化实验显示该蛋白主要分布在原头蚴表面及棘球蚴囊泡生发层。结论 本研究以制备的EgAQP9兔源多克隆抗体定位了AQP9在棘球蚴生发细胞、原头蚴及棘球蚴囊壁中的分布状况。     

检测棘球蚴病患者血清内循环抗原的免疫层析试条的制备和评价

目的 以棘球蚴囊液纯化抗原特异性单克隆抗体为基础建立一种快速、简便诊断棘球蚴病的胶体金免疫层析试条方法,并对其进行评价。方法 纯化棘球蚴囊液抗原,并以此免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体,对所制备的单克隆抗体确定其亚类和效价。筛选基于棘球蚴囊液纯化抗原制备的单克隆抗体对,采用柠檬酸三钠还原法制备胶体金颗粒,标记筛选到的单克隆抗体,并将其吸附于交联垫;将另一筛选到的单克隆抗体划线包被于同一硝酸纤维素膜适当位置,制成免疫层析试条。用该试条检测手术确诊的细粒棘球蚴病(87例)、多房棘球蚴病(40例)、囊尾蚴病(25例)、日本血吸虫病(10例)、弓形虫病(5例)、并殖吸虫病(5例)、华支睾吸虫病(5例)患者血清,以及60例健康者血清,以评价其检测的敏感性和特异性。结果 以棘球蚴囊液纯化抗原为免疫源制备单克隆抗体,共筛选了11株能高效分泌效价在1∶25 600～1∶102 400特异抗体的细胞株,抗体亚类为IgG₁或IgG_2a。筛选到的单克隆抗体F₃B₆作为标记抗体,单克隆抗体C_4<...     

分泌抗细粒棘球蚴单克隆抗体杂交瘤细胞株的建立

本文采用棘球蚴囊壁生发展制备抗原免疫ＢＡＬＢ／ｃ小鼠脾细胞与骨髓瘤ＳＰ２／Ｏ进行细胞融合，经多次筛选克隆后建立了分泌抗细粒棘球蚴单克隆抗体细胞株Ｃ５Ｆ２Ｃ１１Ａ１１，Ｃ１２Ｃ１１Ｆ６Ｇ３，Ｃ１２Ｈ１２Ｆ８Ｄ５三株。应用ＥＬＩＳＡ及免疫组化法检测该三株具有特异性，培养上清分泌抗体滴度分别为１：２５６０，１：５１２０，１：５１２０。     

包虫病人循环免疫复合物的初步研究

用包虫病人血清与亲和层析纯化的包虫病抗原制成人工免疫复合物,证实了可溶性免疫复合物主要为抗原轻度过剩的复合物成分。提示其具有致病的作用。用PEG沉淀法检测49例包虫病人血清免疫复合物的阳性率为22.45％。用Pernice法检测特异性免疫复合物的阳性率仅为8.16％。PEG沉淀物经8M尿素解离后,包虫抗原的检出率为20.4％。36例肝包虫病人pEG沉淀法阳性者10例(27.77％)尿素解离物中检出抗原者9例(25％)。8例肝包虫病人用pEG沉淀法未检出阳性,而解离物中检出抗原者1例(12.5％)。经IHA和间接ELISA法未检出血清抗体的包虫病人中,在pEG沉淀的解离物中检出抗原者各为18.18％(4/22)和10％(1/10)认为在血清pEG沉淀的解离物中检测包虫抗原是测定特异性免疫复合物的较好方法。     

分泌抗包虫囊液抗原McAb杂交瘤细胞株的建立

应用Oriel法制备的包虫囊液抗原经腹腔注入供实验的小鼠,作为脾细胞供体。此脾细胞与不能分泌的骨髓瘤细胞株SP2/0和P3u_1相融合,以生成杂交瘤细胞株。用ELISA法从这些混合细胞培养基中筛选出上清液,用稀释法将上清液克隆,再克隆化之后,生成能分泌单克隆抗体、抗包虫囊液活性的杂交瘤,其染色体数目在80～100范围之内。单克隆抗体类及亚类13Fs和31G_(12)分别是IgG_1和IgA。DD和ELISA用于鉴定它们的敏感性和特异性。应用ELISA法比较杂交瘤和绦虫囊尾蚴液等其它寄生虫抗原制备物,包虫囊液和Pf抗原,表明单克隆抗体对包虫囊寄生虫有很高的特异性。     

Comparison of serologic tests for the diagnosis and follow-up of alveolar hydatid disease

Alveolar hydatid disease is a serious and often fatal condition caused by infection with the metacestode form of Echinococcus multilocularis. Sera of 21 patients with histologically confirmed disease were tested by an enzyme-linked immunosorbent assay (ELISA) using a semi-purified E. multilocularis antigen fraction (Em2) and by indirect hemagglutination (IHA) and double diffusion (DD5) tests using antigens prepared from E. granulosus cyst fluid. At diagnosis, sera from all 21 patients were positive by Em2 ELISA, 18 (86%) by IHA, and 5 (24%) by DD5. Em2 ELISA detected an antibody response earlier than IHA in 4 of 9 patients from whom sera were available before diagnosis. Following complete surgical resection, Em2 ELISA converted from positive to negative in serum of 2 of 3 patients, while IHA results did not change. Following incomplete resection, 14 of 15 patients tested remained positive by Em2 ELISA, while 12 remained positive by IHA. Of sera from 361 healthy persons from regions free of E. multilocularis, none were positive by Em2 ELISA, while 8% were positive by IHA. Of sera from 59 patients with non-echinococcal parasitic infections, none were positive by Em2 ELISA, while 31% were positive by IHA. Thus, in comparison with tests using E. granulosus antigens, Em2 ELISA appears to be more sensitive and specific for diagnosing AHD, useful on follow-up of resected patients, and positive earlier in the course of disease.     

Echinococcus granulosus: diagnosis of hydatid disease in man

Several antigen fractions were prepared from sheep hydatid fluid and scolices of Echinococcus granulosus by salting out with ammonium sulphate. Sera from subjects with hydatid disease and from uninfected controls were assayed by the IHA, Latex, ELISA and Complement Fixation tests. The greatest sensitivity was given by the hydatid fluid 0.8 M fraction in the IHA test. This antigen also gave good results with the ELISA technique. Antigens from sheep fertile fluid were diagnostically superior to those from scolices. The specificity was excellent for all antigens examined.     

Detection of specific circulating antigen, immune complexes and antibodies in human hydatidosis from Turkana (Kenya) and Great Britain, by enzyme-immunoassay

Circulating antigen, specific immune complexes (IgG and IgM) and specific antibodies (IgG, IgM, IgE and IgA) were detected by enzyme-linked immunosorbent assay (ELISA) in the sera of hydatid (Echinococcus granulosus) patients from Turkana (Kenya) and the UK. Specific IgG and IgM antibodies predominated in current UK hydatid infections, while all classes of specific antibodies were lower in the Turkana patients. Circulating antigen, detected in 3% polyethylene glycol (PEG) precipitated complexes, using peroxidase conjugated hyperimmune human hydatid IgG (Fab) was more specific in ELISA than either antibody or immune complex assays where peroxidase conjugated anti-human IgG was used. Anti-human immunoglobulin ('rheumatoid' factor) was not detected in hydatid sera. Serum antigen, specific IgM immune complexes and specific IgM antibodies were associated with UK cases of current hydatid infection in contrast to patients with previous histories of hydatidosis. In 3 hydatid patients (from UK) levels of circulating antigen and specific IgM immune complexes rapidly declined within 1-4 months after surgical cyst removal. The detection of specific IgG and antigen in PEG precipitated immune complexes from false-negative/low responder Turkana hydatid sera, suggests that antibody 'mopping' by specific antigen may be occurring. After SDS-PAGE/immunoblotting analysis, antigen of mol. wt 67 000, present in hydatid cyst fluid and protoscoleces, was identified as putative circulating antigen in 3% PEG precipitates of sera from albendazole treated hydatid patients.     

Comparison of three immunological tests for seroepidemiological purposes in human echinococcosis

The comparative sensitivity and specificity of three immunodiagnostic tests for hydatid disease—the latex agglutination (LA) test, the indirect haemag-glutination (IHA) test and the counterimmunoelectrophoresis (CIEP) test—were studied. The results of the IHA test were evaluated using three positivity criteria. Antigen from the same source—hydatid cyst fluid pool of ovine origin—was utilized for the three tests. A total of 123 sera were studied; 30 preoperative sera from patients with other parasitic and infectious diseases, 30 sera from patients with non-parasitic diseases and 30 sera from normal blood donors. The results of the three tests were correlated to the immunoelectrophoresis (IEP) test based on the presence of arc 5. The same sensitivity (86.7%) and specificity (99%) was obtained in LA, CIEP and IHA tests when the minimum cross-reactivity titre criterion was employed for the latter. Non-specificity was relatively high (13%) in the IHA test when only serological reactivity was considered. The sensitivity of the IHA test decreased (83.3%) using the diagnostic titre criterion and non-specific reactions were eliminated. All three tests correlated well with the IEP test.     

细粒棘球蚴囊液抗原纯化方法的比较评价

对于绵羊肝脏细粒棘球蚴囊液用常规粗制法,通过抗绵羊全血清抗体免疫吸附柱的亲和层析法,Oriol氏纯化法,Burstein氏纯化法,以及将纯化抗原再经酚提取法制备的六种抗原,以SDS-PAGE,免疫电泳,ELISA,IHA等方法进行了抗原得率、纯度、组份、免疫活性和特异性等方面的比较。结果表明:亲和层析抗原得率最高(l6.25％～22.64％),Oriol氏法纯化抗原最低(3.35％)。与囊液粗抗原相比,纯化抗原中Burstein抗原组份最多。经酚提取的纯化抗原组份最少。各种抗原组份数目显然相差明显,但在免疫电泳中均出现弧5沉淀线。酚提取法不能达到将抗原5和抗原B分离的目的。在ELISA和IHA中,Burstein法纯化抗原的活性最高。作者分析了亲和层析抗原活性低于Burstein和Oriol两种抗原的原因可能是亲和层析抗原缺少68kDa和36.5kDa两种宿主组份。因而认为这两个组份可能同时具有寄生虫抗原的性质。在ELISA试验中,各种纯化抗原均与囊虫病人血清产生部分交叉反应,但比粗抗原低。在IHA试验中,纯化抗原均不与囊虫病人血清产生交叉反应。作者推荐由于Burstein法纯化抗原得率高,提纯程序较简单,抗原活性高而且在IHA中没有与囊虫病人血清的交叉反应,因而可作为常规诊断抗原使用。     

Immunological responses to antigen B from Echinococcus granulosus cyst fluid in hydatid patients

The immunological reactivity of Echinococcus granulosus antigen B was evaluated in 30 hydatid patients. Antigen B was purified from sheep hydatid cyst fluid by electroelution from a non-reducing SDS-PAGE gel (AgB). In ELISA and immunoblotting (IB), determining antibody production in sera from patients with hydatid disease and with other parasitic infections, purified AgB showed higher specificity than a partially purified antigen named pH₅PPT (100% vs 83% in pH5PPT-ELISA and 58% in pH₅PPT-IB). AgB-IB achieved higher sensitivity than AgB-ELISA (80% vs 63%). All AgB-IB positive sera recognized the 12 kDa subunit. Qualitative AgB-IB assessment of IgG isotype responses identified IgG4 as the predominant isotype (87%). The other isotypes showed a lower percentage of positive reactions: IgG1, 33%; IgG2, 21%; and IgG3, 17%. PBMC proliferative assay revealed a cellular response to AgB in 100% of patients' PBMC. These findings confirm antigen B, especially its smallest subunit, as a good diagnostic molecule.     

Echinococcus granulosus human infection stimulates low avidity anticarbohydrate IgG2 and high avidity antipeptide IgG4 antibodies

Total IgG and IgG subclasses recognizing carbohydrate and peptidic epitopes from native and periodate treated partially purified hydatid cyst fluid (ppHCFA) and protoscolex somatic antigens (PSA) were tested by ELISA in hydatid patients sera. Binding of the major cross-reactive antiphosphorylcholine antibodies was inhibited with free reagent. A predominant anticarbohydrate antibody response against ppHCFA and PSA is shown. Although the main contributing IgG subclass to the antipeptide response against both antigens was IgG4, IgG1 also significantly contributed to the anti-PSA peptidic epitopes response. Western blot showed that IgG1 antibodies strongly recognized in ppHCFA a periodate susceptible 38 kDa antigen. The IgG4 antibodies mainly recognized the periodate-resistant 12, 16 and 24 kDa antigens. In addition, IgG2 antibodies recognized three strongly periodate-susceptible broad bands (116, 55 and 24 kDa antigens). PSA-specific IgG1 and IgG4 antibodies showed similar patterns of antigen recognition as well as no significant reduction of reactivity after periodate treatment while the IgG2 antibody recognition was strongly affected by this treatment. Furthermore, IgG2 showed significantly lower avidities than IgG1 and IgG4 antibodies recognizing both antigens. In conclusion, hydatid patients showed an enhanced production of low avidity anticarbohydrate IgG2 as well as high avidity antipeptide IgG4 antibodies.     

绵羊细粒棘球蚴囊液抗原诱发小鼠免疫反应及保护性免疫的研究

本文用绵羊细粒棘球蚴囊液抗原制剂,给小鼠3次腹腔注射诱发初次免疫,然后腹腔移植泡球蚴组织作攻击感染,观察保护性免疫。实验提示,注射免疫原制剂后1月能诱发小鼠免疫反应,间接血凝试验(IHA)可测出血清抗体;攻击感染后23周,IHA阳性率达94.4％,而未诱发初次免疫的对照鼠IHA阳性率为90.0％,表明无论是初次免疫或攻击感染,均能诱发小鼠产生抗体,二者无显著差别(P>0.05);但IHA血清滴度1:256百分率分别为94.1％和55.5％,有显著差别(P<0.01),表明攻击感染有强化初次免疫的作用。攻击感染后23周,对比免疫鼠和对照鼠的腹腔泡球蚴湿重均值和病理分级率均无显著差别(P>0.05),提示本文小鼠初次免疫未显保护性免疫之效。