细粒棘球绦虫成虫表膜抗原特异性基因的筛选及克隆

为筛选细粒棘球绦虫(Echinococcus granulosus,Eg)表膜抗原基因或具有诊断性的特异性基因,提取Eg成虫表膜抗原,经ELISA、Western blot对该抗原的免疫学特性进行初步研究.以Eg成虫表膜抗原高免鼠血清为探针,筛选Eg成虫cDNA文库,将阳性噬菌斑的PCR产物和pGEM-T载体连接,转染到DH5α,对得到的重组子进行测序,并进行同源性分析.ELISA检测显示,表膜抗原免疫鼠诱导产生了特异性抗体,Westem blot鉴定该抗体能识别Eg成虫表膜抗原、原头蚴、Eg发育不成熟、Eg发育成熟抗原.筛选出6个阳性克隆,DNA片段大小在1～2kb之间.对阳性克隆进行同源性分析,结果与EgP-29mRNA同源性为99%,与Eg14-3-3蛋白mRNA同源性80%-99%.筛选Eg成虫cDNA文库所获得的基因,证明了Eg虫体表膜抗原的存在,有望成为犬细粒棘球绦虫的诊断抗原以及犬抗细粒棘球绦虫的候选基因.     

犬细粒棘球绦虫粪抗原夹心ELISA检测方法的建立

为建立特异性和敏感性高的检验犬细粒棘球绦虫感染的方法。用细粒棘球绦虫(简称,Eg)成虫抗原分别免疫兔和绵羊,收集高免血清,纯化的高免抗体。依据抗体夹心ELISA工作原理,以兔抗体包被,检测感染Eg、不同犬带科绦虫的实验犬和空白犬粪样,绵羊抗体扑捉抗原,HRP标记兔抗绵羊IgG(1∶8 000)催化显色,用酶标仪测定OD 405nm吸光度,用以确定其特异性和敏感性。试验结果表明,敏感性为82.69%(43/52),特异性为85.88%(140/163);粪抗原在感染细粒棘球绦虫16d后可检出,最低抗原浓度为9.7ng/mL即犬感染5条成虫时可检测出阳性。该检测方法具有较好的特异性和灵敏性,为进一步研制检测细粒棘球绦虫虫体抗原ELISA检测试剂盒奠定了基础。     

大豆抗原蛋白血清抗体间接ELISA检测方法的建立

旨在建立检测血清大豆抗原蛋白抗体的间接ELISA方法。经琼脂糖凝胶层析纯化大豆抗原蛋白,以不同剂量皮下注射免疫小鼠,采用方阵滴定法确定最佳抗原包被浓度及血清稀释度,并对其他条件进行优化,最终建立检测血清大豆抗原蛋白抗体的间接ELISA方法,利用该方法检测小鼠免疫后血清抗体水平。通过方阵滴定法确定11S蛋白最佳包被浓度为5.0μg/mL,血清稀释倍数为1∶800;7S蛋白抗原最佳包被浓度为2.5μg/mL,血清稀释倍数为1∶1 600;两者的批内、批间系数均小于10%,重复性较好,通过ELISA法确定11S和7S蛋白的最佳免疫次数为2次,免疫剂量为1 000μg/kg。结果表明本试验初步建立大豆抗原蛋白抗体检测间接ELISA方法,具有很强的特异性、敏感性和重复性,可用于大豆抗原蛋白过敏反应的临床检测。     

间接ELISA检测禽霍乱血清抗体的研究

比较不同ELISA包被抗原对禽霍乱免疫血清的检测结果,选择最佳的包被抗原建立禽霍乱血清抗体的间接ELISA检测方法.分别 使用超声波裂解抗原、脂多糖蛋白抗原和全菌抗原作为包被抗原,应用间接ELISA方法检测 禽霍乱免疫血清,并对不同抗原建立的ELISA方法进行重复性试验.根据三种抗原对 免疫血清的检测结果以及重复性试验,发现全菌抗原的敏感度、特异性、稳定性均优于其他 两种抗原.全菌抗原作为ELISA包被抗原用于禽霍乱血清学检测具有敏感度高、特异性强、稳定性好的特点.     

羊伪结核棒状杆菌的分离鉴定及ELISA检测方法的建立

从疑似山羊干酪性淋巴结炎羊的肩前淋巴结脓肿内分离到2株菌,经鉴定均为羊伪结核棒状杆菌。利用羊伪结核棒状杆菌标准菌株(ATCC19410株)制成外毒素作为检测抗原,通过酶标抗体、阳性血清、外毒素抗原最适浓度的选择试验,确定适当的抗原、抗体和酶标抗体稀释浓度,建立了间接ELISA方法用于检测抗体。用建立的ELISA方法检测100份待检羊血清,其中阳性血清4份,阳性检出率为4％。     

抗伊氏锥虫表膜单克隆抗体的研究 Ⅱ.用抗伊氏锥虫表膜抗原McAb检测循环抗原

利用抗伊氏锥虫表膜抗原的McAb双抗体夹心法,检测了48匹健康马(骡)、5匹人工接种伊氏锥虫马(骡),9匹先用锥虫弱毒株免疫后用强毒株攻毒马(骡)共278份血清的循环抗原(CA),同时用常规间接ELISA检测了相应的循环抗体(CAb)。结果: 278份血清的CA、CAb阳性率无显著差异,但其阳性检出时机不完全吻合;人工接种马(骡)的CA阳性检出率高于CAb,于接虫后经1-2个CA高峰而死,濒死前4/5动物的CA呈现阳性,免疫马(骡)在攻虫前CA即可呈现阳性,多经1-3个CA高峰后逐渐转阴;人工接种马(骡)的CA水玉高于免疫马(骡),二次攻虫后的CA水平高于首次。     

双抗体夹心ABC-ELISA检测弓形虫循环抗原方法的建立

为检测弓形虫循环抗原,以实现弓形虫急性感染的早期诊断,本研究建立了基于ABC(avidin biotin-peroxidase complex)放大系统的双抗体夹心ELISA方法。通过制备弓形虫排泄分泌抗原(ESA)并免疫动物,经3种抗原的筛选以获得抗循环抗原(CAg)的单抗,以方阵滴定法确定最佳多抗包被浓度和单抗工作浓度,利用夹心法和ABC放大系统建立检测弓形虫循环抗原的夹心ABC-ELISA方法,用该方法对人工感染犬血清和其他阳性血清样本进行检测以确定该方法的检出时间和准确性,并应用于临床样品的检测。结果:获得了3株针对不同抗原表位的单克隆抗体,并对CAg有较高特异性。双抗体夹心ABC-ELISA反应条件:兔多抗包被浓度为3.7μg/mL,生物素标记3A5、3E5和10F5-3单抗在混合工作液中的浓度分别为0.1、0.13和0.12μg/mL。该ELISA方法对ESA最低检测限为11.9 ng/mL,并与隐孢子虫早期感染牛血清、血吸虫尾蚴早期感染牛血清、艾美耳球虫早期感染鸡血清、犬瘟热急性感染早期血清、犬细小病毒急性感染血清无交叉反应。用该ELISA方法检测人工感染的犬,于感染后2 d血清即显示阳性,该方法能明显区分标准阳性血清和阴性血清。用该方法检测68份猪临床血样(其中包括2份标准阳性血清),检测结果与Nest-PCR结果一致。表明该双抗体夹心ABC-ELISA方法特异性强、敏感性高,可用于弓形虫急性感染的早期或活动期的诊断。     

SIVp27抗原双抗体夹心ELISA检测方法的研究

目的建立检测SIVp27抗原的双抗体夹心ELISA方法并进行初步应用。方法使用ProteinA亲和层析柱纯化腹水中的SIVp27单抗,用过碘酸钠法对单抗进行辣根过氧化物酶标记。经抗体配对实验确定包被抗体和检测抗体,再用棋盘滴定法确定抗体工作浓度后,探索构建检测SIVp27抗原的双抗体夹心ELISA法。应用建立的双抗体夹心ELISA法,对SIV模型猴血浆标本和SHIV模型猴病毒分离培养上清标本进行了检测,并将其结果与Coulter公司试剂盒检测结果进行比较。结果以稀释至20ug/mL的2E12为包被抗体、1:400稀释的酶标3G3为检测抗体为ELISA最优体系。模型猴血浆标本和病毒分离培养上清标本的检测结果表明自建体系可以用于SIVp27抗原的检测,特异性良好,灵敏度略低于Coulter公司试剂盒。结论检测SIVp27抗原的双抗体夹心ELISA方法初步建成,为研制有独立知识产权的检测试剂奠定了一定的基础。     

用完整菌体作抗原包被进行ELISA检测激光照射前后血清抗体含量的初步研究

本研究用改良ELISA法检测激光照射前后抗大肠杆菌血清抗体的含量。结果表明:利用完整菌体作抗原包被进行ELISA试验,完全符合一般可溶性抗原的ELISA规律;并与传统血清学方法凝集反应进行了比较。激光照射穴位后使血清中抗体的含量增加,提示激光照射后能增强机体的免疫功能。     

多房棘球绦虫粪抗原双抗体夹心ELISA检测方法的建立

建立多房棘球绦虫粪抗原双抗体夹心ELISA检测方法,为犬感染多房棘球绦虫的早期诊断提供技术支撑。以5E10H5杂交瘤细胞株腹腔接种Balb/c鼠制备的腹水作为包被抗体,多房棘球绦虫成虫可溶性抗原免疫新西兰大白兔制备的多克隆抗体血清作为检测抗体,HRP标记的驴抗兔IgG作为二抗,建立双抗体夹心ELISA方法,检测试验犬(感染多房棘球绦虫)和阴性对照犬犬粪。结果显示,多房棘球绦虫成虫可溶性抗原具有良好的抗原性并能产生高效价抗体;对该方法的灵敏度进行检测,阳性粪样稀释至1∶10 000时仍显示为阳性;在感染动态分析中,该方法最早可在犬感染72 h后,最迟6 d后检测到多房棘球绦虫粪抗原。表明建立的方法可用于诊断犬多房棘球绦虫感染,为后期研制犬多房棘球绦虫粪抗原双抗体夹心ELISA检测试剂盒奠定了基础。     

Use of Echinococcus granulosus worm antigens for immunodiagnosis of E. granulosus infection in dogs.

Echinococcus granulosus worm excretory/secretory antigens (WES) were used in ELISA for diagnosis of E. granulosus infection in dogs and compared with protoscolex somatic antigens (PSM). Sera from 224 dogs were tested. There was no correlation between ELISA absorbance values and E. granulosus worm burdens using either antigen. There was a significant linear relationship between absorbance values of sera tested in the ELISA using WES (W-ELISA) and the ELISA using PSM (P-ELISA). However, there was a small but significant difference between the absorbance values of the sera tested against the two antigens. Western blot analysis of WES using sera from E. granulosus-infected and uninfected dogs revealed antigenic components of relative molecular mass (Mr) larger than 94,000, Mr 94,000-68,000 and Mr 43,000-39,000 in worms, and these were specific for E. granulosus and not identified in PSM; these antigenic differences may be responsible for differences in reactivity in ELISA. The sensitivities of W-ELISA and P-ELISA were 80.8% and 75.6%, respectively. The specificities of W-ELISA and P-ELISA were 93.7% and 97.9%, respectively. The reduced specificity in W-ELISA was mainly attributable to increased background reactivity of sera from Taenia hydatigena-infected dogs. Despite the reduction in specificity, both ELISAs are valuable epidemiological tools to determine the prevalence of antibody to E. granulosus in dog populations and to monitor the success of hydatid control campaigns.     

Specific antibody responses to Taenia hydatigena, Taenia pisiformis and Echinococcus granulosus infection in dogs

Groups of dogs reared free of both nematodes and cestodes were infected with Taenia hydatigena, Taenia pisiformis or Echinococcus granulosus. After infections with the Taenia spp became patent, dogs were purged to remove the worms. They were later reinfected and the second infections again removed by purging after patency. A group of 3 uninfected worm free dogs was kept as age-matched controls. The dogs were bled at intervals of 5 days and their serums tested for antibodies using the enzyme-linked immunosorbent assay (ELISA) with excretory/secretory (ES) antigens collected during in vitro incubation of evaginated scoleces (scolex ES antigen) and oncosphere antigens. Antibodies to scolex ES antigen were detected by 3 weeks after infection with each cestode species whereas antibodies to oncosphere antigen were not detected until about one week after eggs were found in the faeces of the infected dogs. Antibody responses to both oncosphere and scolex ES antigens decreased rapidly following removal of the worms by purging. Uninfected control dogs were invariably negative to both oncospheral and scolex ES antigens. There were cross-reactions between the serums from dogs infected with T. pisiformis and T. hydatigena when tested with scolex ES antigens, but oncospheral antigens showed a high degree of species specificity. Scolex ES antigens from E. granulosus were compared with those prepared from T. hydatigena and T. pisiformis for their ability to discriminate between antibodies in serums collected from dogs 31 and 32 days after infection with 100,000 protoscoleces of E. granulosus or dogs infected with Taenia spp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative immunoelectrophoretic analysis of Echinococcus granulosus, Taenia hydatigena and Taenia pisiformis cyst fluid antigens by hyperimmune rabbit sera.

Cyst fluid antigens of Echinococcus granulosus, Taenia hydatigena and T pisiformis were examined by electrophoresis using homologous and heterologous hyperimmune rabbit sera to these antigens. While arc 5 forming antibodies were identified in sera from rabbits immunised with E granulosus and T hydatigena cyst fluids, antibodies responsible for forming precipitating antigen B band were detected in rabbit antisera to E granulosus, T hydatigena and T pisiformis antigens. T hydatigena cyst fluid appears to contain antigen similar to E granulosus antigen 5 and probably antigen B while T pisiformis cyst fluid has mainly an antigen close to hydatid antigen B.     

Comparative antigenic characterisation of Echinococcus granulosus and Taenia hydatigena cyst fluids by immunoelectrophoresis.

Hydatid cyst fluid from Echinococcus granulosus (HCF) and cyst fluid from Taenia hydatigena (TCF) cysts were compared in reciprocal immunoelectrophoresis (IEP) tests using homologous and heterologous antisera which were free of antibodies to host serum contaminants. The antigens for the E granulosus arc 5 were demonstrated in TCF. Antibody activity to these and other antigens common to HCF and TCF was removed from homologous antisera by absorptions with the heterologous antigenic preparation. Antigens not shared by the two metacestodes fluids were then demonstrated by IEP tests. These findings are discussed in terms of their significance to phylogenetic and immunodiagnostic studies of these parasites in their immediate hosts.     

Use of the enzyme-linked immunosorbent assay in the diagnosis of Dictyocaulus filaria infection

The enzyme-linked immunosorbent assay (ELISA) was used to detect and quantify antibodies to whole worm extract antigens of Dictyocaulus filaria. The results provided evidence that the test was highly sensitive and could be used as a tool for epidemiological studies. No cross reactions were observed with sera from lambs infected with the hookworm Gaigeria pachyscelis.     

Use of two polysaccharide antigens in ELISA for the detection of antibodies to Echinococcus granulosus in sheep sera

Polysaccharide antigens were obtained from either the secretions produced during in vitro cultivation of Echinococcus granulosus protoscoleces or from mouse hydatid cyst membranes by phenol extraction. When either of these antigens was used in an enzyme-linked immunosorbent assay antibody activities were detected in sera from sheep infected 27 or more weeks earlier with at least 100 E granulosus eggs. These antibody responses were significantly higher (P less than 0.05) than those of sheep infected with Taenia hydatigena or T ovis and tested with the E granulosus antigens. Very high cross-reacting antibody responses in sera from sheep recently infected with T hydatigena were only detected with the protoscoleces secretions antigen. Neither antigen was sufficiently sensitive or specific for serodiagnostic use. However, when sera were first tested with one antigen and then with the other, and only sera that were positive in both tests were regarded as positive, the overall sensitivity and specificity of this two antigen method increased to about 80 per cent.     

Antigens of Hyostrongylus rubidus, the red stomach worm of pigs. Analyses by means of passive haemagglutination and immediate hypersensitivity tests.

Antigens were prepared from different stages of worm development and adult worm antigens were fractionated by ion exchange and exclusion chromatography. The antigens, or fractions of them, were assessed for their activity in passive haemagglutination reactions or intradermal tests. The results of the passive haemagglutination reactions indicated that circulating agglutinins were more readily detected when adult stage specific antigens were used. Antigen prepared from worms isolated when the population was declining was less sensitive. Fractions of adult worm antigen did not confer greater sensitivity than the whole worm extract.     

Efficacy of divided doses of fospirate against immature Echinococcus granulosus infections in dogs.

Seventy dogs were used to evaluate efficacy of divided doses of fospirate against immature Echinococcus granulosus infections in dogs. Dose rates of 10 to 80 mg/kg given on 1 or 2 occasions resulted in the clearance of 70.6 to 94.5% of expected worm numbers. At least 3 treatments may be required before dogs can be free from E granulosus. Vomiting, which occurred in dogs given doses of 40 mg or more/kg, seemed to interfere with anthelmintic efficacy in some dogs.     

Immunodiagnosis of experimental Parelaphostrongylus tenuis infection in elk

Elk infected with the meningeal worm, Parelaphostrongylus tenuis (Protostrongylidae), do not consistently excrete larvae in feces, making the current method of diagnosing live animals using the Baermann fecal technique unreliable. Serological diagnosis could prove more useful in diagnosing field-infected animals but depends on the identification and availability of good quality antigen. To mimic field infections, 2 elk were inoculated with 6 infective L3 larvae of P. tenuis, and another 2 with 20 L3 larvae. Fecal samples were examined for nematode larvae using the Baermann technique and serum samples taken were tested for anti-P. tenuis antibody with ELISAs by using the excretory-secretory (ES) products of L3, and sonicated adult worms as antigens. One animal passed first-stage larvae in its feces 202 days postinoculation, but passed none thereafter. The remaining 3 inoculated animals did not pass larvae. In contrast to parasite detection, antibodies against larval ES products were detected in all animals starting from 14 to 28 days postinoculation and persisted until the termination of the experiment on day 243 in 2 animals that harbored adult worms. Antibodies against somatic antigens of the adult worm were not detected until day 56 but also persisted until the end of the experiment in the animals with adult worms. In 2 elk that had no adult worms at necropsy, anti-ES antibodies were detected transiently in both, while anti-adult worm antibodies were present transiently in one. These findings confirm the superiority of P. tenuis larval ES products over somatic adult worm antigens as serodiagnostic antigens, as previously observed in studies of infected white-tailed deer, and extend the application of the newly developed ELISA test in diagnosing and monitoring cervids experimentally infected with P. tenuis.