采用分子筛层析法分离纯化细粒棘球蚴囊液粗抗原

目的分离纯化细粒棘球蚴囊液抗原,去除其中的非特异性反应蛋白,探索优化囊液抗原制备方法。方法采用超滤法浓缩羊肝细粒棘球蚴囊液制备成粗抗原,并采用免疫印迹实验对囊液抗原进行分析,发现非特异性反应的主要蛋白条带。随后采用Superdex凝胶柱通过分子筛层析法对囊液抗原进行纯化。用44份细粒棘球蚴病患者、17份健康人和10份囊尾蚴患者血清进行酶联免疫实验,评估纯化后囊液抗原的检测能力。结果免疫印迹实验发现,非特异性反应的主要条带的相对分子质量(M_r)约为55 000,纯化后去除了该非特异性反应蛋白。纯化后的囊液抗原ELISA检测灵敏度为93.2%(41/44),特异性为94.1%(16/17),约登指数为0.87,与囊尾蚴病交叉反应性为30%(3/10)。未纯化前的灵敏度为90.9%(40/44),特异性为88.2%(15/17),约登指数0.79,与囊尾蚴病交叉反应性为60%(6/10)。纯化前后ELISA检测的灵敏度存在显著性差异。结论本研究采用分子筛层析法对羊源细粒棘球蚴的囊液抗原进行纯化,去除了引起非特异性反应的主要蛋白(M_r 55 000),为后续进行囊液抗原标准化、提高检测能力提供了依据。     

囊型棘球蚴和泡型棘球蚴不同组织抗原可溶性蛋白SDS—PAGE电泳分析

对囊型棘球蚴和泡型棘球蚴的不同组织及犬泡状带绦虫和豆状带绦虫、猪囊尾蚴，细颈囊尾蚴等四种异源绦虫组织共１４种抗原进行了ＳＤＳ－ＰＡＧＥ电泳分析。两种棘球蚴同种组织的主要蛋白质区带的电泳图谱差别不大，不同组织的电泳图谱差别较大。     

细粒棘球蚴Eg95重组蛋白表达及其血清学反应的研究

目的 诱导表达和纯化细粒棘球蚴Eg95抗原重组蛋白,对其与CE病人血清学反应情况进行研究.方法 IPTG诱导pET28a-Eg95原核表达质粒,表达和纯化Eg95重组蛋白,SDS-PAGE 电泳对其进行初步鉴定,Western blot法检测其与中间宿主CE病人血清学反应情况.结果 pET28a-Eg95重组蛋白得到成功表达,SDS-PAGE 电泳显示相对分子量约为15kDa.Western blot 结果 表明,pET28a-Eg95重组蛋白能被CE病人血清识别,11例CE病人血清中8例呈阳性反应.结论 细粒棘球蚴Eg95抗原存在于中间宿主CE病人体内,Eg95蛋白作为保护性抗原可能成为CE病人术后辅助性治疗的手段之一.     

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。     

检测包虫病患者血清特异IgG4诊断价值的研究

目的探讨检测患者血清特异IgG4诊断包虫病的效果.方法采用两种抗原(棘球蚴囊液粗抗原及其抗原B)通过ELISA法检测棘球蚴病病人、非棘球蚴病病人和健康对照血清特异IgG4.结果粗抗原和抗原B检测棘球蚴患者血清特异IgG4的阳性率分别为94.4%和89.8%;与部分猪囊尾蚴病患者血清出现交叉反应外,与肺吸虫病、旋毛虫病、血吸虫病、肝囊肿等患者的血清以及健康对照血清均未出现交叉反应.结论检测棘球蚴患者血清特异IgG4敏感性高,特异性强,具有较好的诊断价值.     

细粒棘球绦虫Eg18基因的克隆 表达和重组抗原免疫检测的初步评价

目的克隆、表达细粒棘球绦虫Eg18基因,评价重组蛋白的免疫反应性。方法根据已知Eg18基因序列,利用RT-PCR方法从青海绵羊肝棘球蚴原头节提取的总RNA中扩增目的基因,克隆到原核表达质粒pET-28a（＋）中,转化大肠埃希菌BL21（DE）,IPTG诱导表达。表达产物经亲和层析纯化,用免疫印迹试验（Western blot）和酶联免疫吸附试验（ELISA）初步评价其与棘球绦虫及其他蠕虫感染血清的反应性。结果 RT-PCR扩增产物编码的蛋白质序列与GenBank中的Eg18和Em18完全相同。重组蛋白与多种蠕虫病人血清中的IgG和IgG4均有交叉反应,但检测IgG4时,与其他蠕虫的交叉反应显著降低。结论 Eg18/Em18是细粒棘球绦虫和多房棘球绦虫的共同抗原,其特异性IgG4是泡型棘球蚴病较特异的诊断标志物。     

检测包虫病患者血清特异IgG4诊断价值的研究

目的 探讨检测患者血清特异IgG4诊断包虫病的效果。方法 采用两种抗原（棘球蚴囊液粗抗原及其抗原B）通过ELISA法检测棘球蚴病病人、非棘球蚴病病人和健康对照血清特异IgG4。结果 粗抗原和抗原B检测棘球蚴患者血清特异IgG4的阳性率分别为94．4％和89．8％;与部分猪囊尾蚴病患者血清出现交叉反应外,与肺吸虫病、旋毛虫病、血吸虫病、肝囊肿等患者的血清以及健康对照血清均未出现交叉反应。结论 检测棘球蚴患者血清特异IgG4敏感性高,特异性强,具有较好的诊断价值。     

包虫和猪囊虫抗原免疫化学性质及酶联免疫印斑技术临床应用研究

用 SDS 聚丙烯酰胺凝胶电泳和酶联免疫印斑技术分别对人源细粒棘球蚴囊液、棘球蚴砂、猪囊尾蚴囊液抗原组分进行分析研究,并对两种病血清学交叉反应进行了试验。结果显示包虫囊液抗原有29条蛋白区带,有两条糖蛋白带。猪囊虫囊液抗原显示36条蛋白带谱,两条糖蛋白带。棘球蚴砂显示23条蛋白带谱。包虫病人血清与印有包虫囊液抗原的硝酸纤维膜反应显示23条反应带,其中特异带为6条。包虫病人、猪囊虫病人血清与此膜反应显示4条共同反应带。包虫病人血清与棘球蚴砂抗原膜反应显示4条主要特异反应带,包虫病人血清、猪囊虫病人血清与此膜反应显示1条共同反应带。猪囊虫病人血清与猪囊虫囊液抗原膜反应显示22条反应带,其中有5条特异反应带;包虫病人和囊虫病人血清与此膜反应显示3条共同反应带。     

快速诊断多房棘球蚴病胶体金免疫层析试条方法的建立与评价

目的建立快速、简便诊断多房棘球蚴病的胶体金免疫层析试条方法。方法提取多房棘球蚴原头节总RNA,通过RT-PCR获得编码Em18基因片段并克隆入pGEX-3X表达载体,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达得到重组蛋白;采用柠檬酸三钠还原法制备胶体金,标记抗人IgG单克隆抗体;将重组Em18抗原包被于硝酸纤维素膜适当位置,制成检测特异抗体的胶体金免疫层析试条。用该试条检测多房棘球蚴病(56份)、细粒棘球蚴病(87份)、囊尾蚴病(30份)、日本血吸虫病(10份)和弓形虫病(10份)患者血清,以及健康人(50份)血清,以评价其检测的敏感性和特异性,同时用ELISA法进行平行检测,以评价该试条的诊断性能。结果以重组蛋白Em18为抗原胶体金免疫层析试条检测多房棘球蚴病患者血清,敏感性为92.9%(52/56)。与细粒棘球蚴病和囊尾蚴病患者血清分别存在9.2%(8/87)和3.3%(1/30)的交叉反应,与健康人血清存在8.0%(4/50)的假阳性率,与日本血吸虫病和弓形虫病患者血清则无交叉反应,特异性为93.0%(174/187)。ELISA法检测的敏感性和特异性分别为94.6%(54/56)和92%(172/187),与试条法比较两者差异均无统计学意义(P>0.05)。kappa分析结果显示,试条法与ELISA法检测多房棘球蚴病患者血清的结果高度一致(κ=0.98)。结论以重组Em18抗原建立的快速诊断胶体金免疫层析试条,检测多房棘球蚴病的敏感性和特异性均较高。     

组合抗原在人类包虫病与囊虫病快速鉴别诊断中的应用

为研究包虫病与囊虫病之间的鉴别方法,通过纯化羊肝细粒棘球蚴囊液抗原（EgB)、泡球蚴组织抗原（Em2)、猪囊虫囊液抗原（Tsc2)后,分别检测包虫病、囊虫病,其它寄生虫病及正常对照观察组的血清,结果发现它们对相应寄生虫病的敏感性都在91%以上,对除绦虫病以外寄生虫病患及正常健康对照组都有较好的特异性,但它们各自对细粒棘球蚴病,泡状棘球蚴病和囊虫病的诊断都有程度不同的交叉反应,然而将上述抗原进行组合后再对同一标本进行检测,表明EgB单项阳性可以诊断细粒棘球蚴病,Em2单项阳性及Em2 EgB阳性可以基本诊断泡状棘球蚴病,Tsc2单项阳性可以诊断囊虫病患,Em2 EgB Tsc2同时阳性则考虑囊虫病可能性为大,乘下极少数的患Tsc2 Em2同时阳性则需根据临床表现考虑对囊虫病和泡状棘球蚴病进行临床鉴别诊断。     

Development of Em18-immunoblot and Em18-ELISA for specific diagnosis of alveolar echinococcosis

Extensive experience has documented that Em2(plus)-ELISA, Em10-ELISA and Em18-immunoblot and Em18-ELISA are reliable serologic methods for detection of alveolar echinococcosis (AE) caused by the metacestodes of Echinococcus multilocularis. Among these, tests based on detection of antibodies to the specific Em18 antigen, either immunoblot or ELISA, appears to be the most specific for AE. Between 90 and 97% of AE cases with characteristic hepatic lesions detectable by image analysis have been positive in Em18-serology. In contrast Antigen B (8 kDa)-immunoblot is the most sensitive for all forms of echinococcosis, although it can not differentiate AE from cystic echinococcosis (CE). Primary serologic screening for echinococcosis, especially for CE using hydatid cyst fluid of Echinococcus granulosus appears to be highly sensitive in endemic areas. Glycoproteins (GPs) purified from cyst fluid of Taenia solium are highly specific for diagnosis of T. solium neuorcysticercosis (NCC). Using currently available antigens it is not difficult to differentiate these three larval cestodiases serologically. We recommend that (1) primary screening of CE in endemic areas should be carried out using hydatid cyst fluid of E. granulosus prepared from cysts in either sheep, human or mouse for immunoblot and from sheep or mouse for ELISA, (2) both primary screening and confirmation of AE in endemic areas should be carried out using Em18-ELISA, Em18-immunoblot or Em2(plus)-ELISA. Serodiagnosis in areas where both AE and CE are endemic, such as in China, should be carried out as a combination of (1) and (2), and (3) serology of NCC should be carried out using GP-ELISA or GP-immunoblot. All samples showing antibody to Em18 are exclusively from echinococcosis cases. There have been no false positive test reactions with sera from other diseases. Strongest Em18 responders are all from patients with AE but some weaker responses may be found in sera of persons with advanced complex lesions of CE. These highly reliable serodiagnostic methods using native, recombinant and synthetic antigens are briefly summarized and experiences with these methods in Japan is reviewed. We believe that use of these specific antigens in screening and confirmation programs for AE in Japan will improve specificity and reduce the confusion, anxiety and expense in persons whose sera give false positive reactions with crude echinococcal antigens.     

不同发育阶段广州管圆线虫的抗原分析

目的 分析不同发育阶段广州管圆线虫抗原差异 ,筛选优势诊断抗原分子。　方法　对不同发育阶段的广州管圆线虫的虫体蛋白进行十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和免疫印迹分析。结果　不同发育阶段的虫体蛋白质谱大致相同 ,SDS PAGE中出现的少数差异带在免疫印迹试验(Westernblotting)中无明显差异。各期虫体抗原相对分子质量为Mr 40 000、50 000、 66 000和 80 000的 ,与感染大鼠和正常大鼠血清均出现反应条带。 2～3周幼虫Mr 104 000与 2周感染血清出现明显反应条带。雌虫Mr 33 000及所有虫体的Mr 32 000抗原与感染后 2周血清出现反应条带 ,与感染 3周～5月的大鼠血清均出现明显反应 ,与正常大鼠血清均无明显反应。　结论　Mr 40 000、50 000、66 000和80 000抗原可能在以虫体粗抗原作探针的免疫诊断中引起非特异性反应。幼虫Mr 104 000、雌虫Mr 33 000和所有虫体的Mr 32 000可作为广州管圆线虫病早期诊断和流行病学调查的候选抗原分子。     

细粒棘球蚴原头节18 kU纯化抗原ELISA的建立及其对两型包虫病鉴别诊断的意义

目的用细粒棘球蚴原头节18ku纯化抗原建立ELISA诊断方法,用于两型包虫病的鉴别诊断.方法用18ku纯化抗原ELISA方法对44例泡型包虫病(AE)、70例囊型包虫病(CE)、29例囊虫病和30例健康人血清中特异性抗体进行检测,同时用羊包囊液抗原(Bu)常规ELISA法检测上述血清做比较.结果18ku纯化抗原检测不同血清的阳性率为AE90.91%、CE11.43%、囊虫病和健康人血清均为阴性;Bu抗原分别为AE97.73%、CE88.57%、囊虫病51.72%和健康人10.0%.18ku-ELISA对AE血清诊断敏感性为90.91%、特异性93.80%、阳性预示值为83.33%、阴性预示值96.80%.结论两型包虫病患者体内18ku抗体水平存在明显差异,纯化抗原18ku-ELISA可用于两型包虫病的鉴别诊断,较免疫印渍法简便快速.     

多房棘球蚴病特异性诊断抗原Em18的基因克隆、表达和血清学评价

目的　克隆从我国四川省分离的多房棘球绦虫Em18抗原基因片段,表达重组抗原,用于多房棘球蚴病(AE)的诊断,并用患者血清对其诊断价值进行评价。　方法　PCR扩增目的基因片段与质粒载体pET28a(+)连接构建表达质粒,转化大肠埃希菌BL21(DE3)菌株表达重组蛋白,用镍次氮基三乙酸琼脂糖树脂(NI NTAagarose)亲和层析纯化重组抗原,酶联免疫吸附测定(ELISA)评价重组抗原的血清学诊断效果。　结果　获得两个高效表达克隆ReEm181和ReEm182。其中ReEm181与预期序列一致,ReEm182为同一序列,但有27bp的核苷酸缺失。两个克隆表达的融合蛋白相对分子质量(Mr)分别为28000和26000。对101例AE、47例细粒棘球蚴病、30例囊尾蚴病、10例肝癌、9例血吸虫病和40名健康人共237份血清进行ELISA检测 ,结果显示,ReEm181和ReEm182重组抗原对AE血清诊断的敏感性为861%和901%、特异性为934%和941%、阳性预期值为906%和919%、阴性预期值为901%和928%、诊断效率分别为903%和924%;对58例AE患者肝脏病灶大小与重组抗原检测血清平均吸光度(A)的相关性比较分析结果表明,抗体反应水平在病程早期有较好的相关性。　结论　Em18重组抗原对AE具有特异性诊断价值     

Fertility of the cysts of Echinococcus granulosus in domestic herbivores from Benghazi, Libya, and the reactivity of antigens produced from them.

Unilocular cysts produced by Echinococcus granulosus were recovered from 110 domestic herbivores (60 sheep, 25 cattle, 20 goats and five camels) slaughtered in Benghazi. The proportion of the cysts from the sheep found to be fertile (75%) was higher than that of the cysts from the goats (55%), camels (40%), or cattle (0%). When tested in indirect haemagglutination assays (IHA) with eight sera from human cases of cystic echinococcosis, the fluid from the cattle cysts never gave a positive reaction. Antigens in the fluids collected from sheep or goat cysts did react with the sera, with antigens from each of the two sources giving similar titres with each serum. However, crude somatic antigens (prepared from protoscolices and brood capsules collected from sheep cysts) appeared to be more sensitive for the immunodiagnosis of human cystic echinococcosis than the cyst-fluid antigens.     

Immunoblot analysis of a 10 kDa antigen in cyst fluid of Taenia solium metacestodes

The diagnostic value of a 10 kDa subunit of 150 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was evaluated. Immunoblot analysis revealed that most sera from patients with neurocysticercosis recognized the 10 kDa subunit strongly (209/217 cases, 84.6%), while a few sera from individuals with other parasitic diseases including alveolar echinococcosis (AE, 2/20, 10%) and cystic echinococcosis (CE, 2/25, 8%) showed faint reactions. Sera of cases with other parasitic diseases, especially AE and CE, exhibited cross reactions against other bands in CF. Both differential immunoblot and immunoprecipitation analyses showed that the 10 kDa subunit was the most specific to cysticercosis and highly antigenic, whereas other components at 20–40, 64, 95 and 106 kDa in CF were cross-reactive. IgG subclass ELISA and immunoblot demonstrated that both IgG4 and IgG1 reactions were predominant in neurocysticercosis and recognized the 10 kDa .     

The immunodiagnostic potential of protoscolex antigens in human cystic echinococcosis and the possible influence of parasite strain

The results of ELISA, SDS-PAGE and western blotting indicated that the protoscolex antigens of Echinococcus granulosus (of 10-125 kDa) included antigens recognized by sera from human cases of cystic echinococcosis (CE). Some of the latter antigens (of approximately 79, 59, 45, 38, 31 and 29 kDa) exhibited cross-reactivity with sera from humans with other parasitic infections, including alveolar echinococcosis, cysticercosis and African trypanosomiasis. The 31-kDa antigen recognized by IgG antibodies in human CE sera only appeared to be present in the extracts of protoscoleces from sheep, and not in the corresponding extracts from horse or camel cysts. In contrast, the human CE sera recognized a 45-kDa protoscolex antigen only present in the horse cysts and a 125-kDa antigen present in the camel and horse (but not sheep) cysts. Extracts of protoscoleces from different species of hosts might therefore provide a source of strain-specific diagnostic antigens for human CE.     

细粒棘球绦虫抗原B重组蛋白的免疫反应性

目的 利用基因工程方法表达细粒棘球绦虫抗原B重组蛋白（rAgB）,并分析其免疫反应性。 方法 将rAgB基因片段插入原核表达载体pET41a（+）中,转化大肠埃希菌BL21（DE3）菌株,经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达,获得重组蛋白rAgB-GST。用谷胱甘肽琼脂糖树脂亲和层析柱（GST-sepharose 4B）纯化,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析重组蛋白的表达情况。用蛋白质印迹（Western blotting）分析其免疫反应性,并用免疫胶体金包虫诊断试剂盒作为对照。 结果 PCR、双酶切及DNA测序结果均显示重组质粒pET41a-rAgB构建成功。SDS-PAGE结果表明,重组蛋白rAgB-GST的相对分子质量（Mr）为40 800,纯化蛋白含量为78.4%。Western blotting分析结果显示,重组蛋白rAgB-GST检测细粒棘球蚴病和多房棘球蚴病患者血清的阳性率分别为79.2%（95/120）和51.1%（23/45）,但与卫氏并殖吸虫病患者、华支睾吸虫病患者血清以及健康人血清反应均为阴性。rAgB-GST的敏感性和特异性分别为79.2％（95/120）和81.0%（98/121）,均略高于免疫胶体金棘球蚴病诊断试剂盒的敏感性(72.8%,75/103）和特异性（76.9%,30/39）。 结论 重组rAgB-GST蛋白可被细粒棘球蚴病和多房棘球蚴病患者血清识别,有较好的免疫反应性。     

细粒棘球蚴粗抗原的免疫纯化方法及其在免疫诊断中的应用

采用免疫过筛法将细粒棘球蚴囊液粗抗原（ＥｇＣＦ）和头节抗原（ＥｇＰ）与正常人混合血清中杂抗体结合而除去产生非特异反应的粗抗原部分,得到部分纯化的抗原ＥｇＣＦ２,ＥｇＰ２,纯化前后的抗原在酶联免疫吸附试验（ＥＬＩＳＡ）及斑点免疫结合试验（ＤＩＢＡ）中对４５例细粒棘球蚴病（ＣＥ）的诊断阳性率分别为：ＥｇＣＦ９３．３％,ＥｇＰ９５．６％,ＥｇＣＦ２９１．１％,ＥｇＰ２９３．３％,它们的敏感性无显差异。     

Dot immunogold filtration assay (DIGFA) with multiple native antigens for rapid serodiagnosis of human cystic and alveolar echinococcosis

A new 3-min rapid dot immunogold filtration assay (DIGFA) for serodiagnosis of human cystic and alveolar echinococcosis was developed using four native antigen preparations: crude and partially purified hydatid cyst fluid extracts from Echinococcus granulosus (EgCF and AgB), E. granulosus protoscolex extract (EgP) and Echinococcus multilocularis metacestode antigen (Em2). The overall sensitivity of DIGFA in a hospital diagnostic setting was 80.7% for human cystic echinococcosis (CE) (n = 857) and 92.9% for human alveolar echinococcosis (AE) (n = 42). Highest specificity was 93.4% with AgB extract for CE, and 90.3% with Em2 antigen for AE when CE versus AE cross-reactivity was excluded. Anti-AgB antibodies were present in 35.5% of AE cases and anti-Em2 in 7.4% of CE cases. In endemic communities in northwest China screened for echinococcosis, the sensitivity of DIGFA ranged from 71.8% to 90.7% in comparison to abdominal ultrasound; specificity for CE using AgB was 94.6% and for AE using Em2 was 97.1%. This simple eye-read rapid test can be used for both clinical diagnostic support, as well as in conjunction with ultrasound for mass screening in endemic CE and AE areas.