抗日本血吸虫抗独特型抗体的制备及初步鉴定和应用

本研究用1株针对日本血吸虫尾蚴和童虫表膜抗原的单克隆抗体(N15D9)免疫新西兰兔制备多克隆抗N15D9独特型抗体。结果表明,多克隆抗N15D9独特型抗体具有较高的抗体滴度,经用间接免疫荧光法和ELISA分析,该抗体显示较高的结合特异性,与具有相同靶抗原结合特异性的单克隆抗体有中度的交叉反应。当用抗N15D9独特型抗体作为诊断抗原检测日本血吸虫感染的病人血清时,其阳性检出率为39％。本研究结果初步揭示,抗N15D9独特型抗体具有一定的分子模拟作帮和诊断应用的潜能。     

减弱的血吸虫童虫不同途径接种小鼠的转归与诱导抗曼氏血吸虫免疫力的关系

作者将照射尾蚴,用人工转化法获得3小时龄(3h)减弱童虫。另自接种10000条照射尾蚴的小鼠肺部收集8日龄(D8)减弱童虫。分别经血管(i.v)、气管(i.t)、腹膜(i.p)和皮内(i.d)4种途径接种于小鼠。免疫剂量为300条减弱童虫。每次试验用一组小鼠腹部皮肤(p.c)感染500条照射尾蚴,以估价每批虫的免疫原性。接种照射尾蚴或3h减弱童虫后5周及接种D8减弱童虫后4周测定抵抗力。即用尾浸没法以200条尾蚴攻击,5周后门静脉灌注,计数成虫。以下列公式计算抵抗力(R)。B=(C-T)/C×100％。公式中C,T分别为灌注试验小鼠及对照小鼠     

抗狂犬病病毒N蛋白单克隆抗体的制备与鉴定

目的制备抗狂犬病病毒N蛋白单克隆抗体,为建立快速准确的狂犬病病毒抗原检测方法奠定基础。方法将狂犬病病毒Flury LEP株N基因克隆至pGEX-6P-1表达载体中,将纯化后的pGEX-6P-1-RV-N蛋白免疫BALB/c小鼠3次。取小鼠脾脏细胞和SP2/0细胞融合后,用pET-32a-RV-N重组蛋白作为筛选抗原,经3次亚克隆筛选后得到1D9、2B6、3C5、6B5及5F2 5株单克隆抗体细胞株。亚类鉴定结果表明,其中1D9、2B6、6B5 3株亚类为IgG1,3C5、5F2的亚类为IgM。间接ELISA方法检测2B6单抗细胞腹水效价可达1∶106。经Protein G亲和层析柱纯化和脱盐后可得到浓度为2.5mg/mL的高纯度的单克隆抗体。其免疫荧光试验结果表明5株单克隆抗体均能特异地与狂犬病病毒结合。结论制备的单抗具有良好的特异性和敏感性,为后期诊断方法的建立奠定了基础。     

抗日本血吸虫小鼠IgE单克隆抗体的产生与性质

本文描述一种对Sj的小鼠IgE单克隆抗体,在感染早期被动转移该抗体对小鼠有部分的而明显的保护作用。材料与方法:用机械方法制备童虫,然后在5％CO_2、37℃下孵育3小时。按Chaffee法略加改良制备Sj、Sm及肝片形吸虫,卫氏并殖吸虫,旋毛虫抗原。在制备Sj成虫抗原时,单性雄虫与配对的成虫分开制备。转化的Sj童虫抗原亦分别制备。慢性感染血清为感染20～30条Sj尾蚴8～12周后的小鼠血清。McAb的制备:经Sj成虫抗原免疫对Sj产生高水平IgE抗体的C3H/He小鼠的脾细胞与NS-1骨髓瘤细胞融合,以被动皮肤过敏反应(PCA)筛选阳性杂交瘤,经克隆化获得IgE单克隆抗体。     

抗日本血吸虫蛋白质“靶抗原”单克隆抗体的研究

在成功地抽提出国际公认在诱导血吸虫保护性免疫力上起重要作用的24-26kD和90kD蛋白质“靶抗原”的基础上,进一步应用上述抗原免疫小鼠,经融合试验及克隆化获得分泌抗日本血吸虫蛋白质“靶抗原”单克隆抗体(McAb)的杂交瘤细胞29株,其中IgG类16株(IgG_1 7株,IgG_(2a) 5株,IgG_(2b) 1株,IgG_3 3株),IgM 13株。以后,又从McAb的定位研究、McAb体外ADCC对血吸虫童虫的杀伤效应、McAb在不同血吸虫抗原中的识别位点(抗原结合位)等方面,对报获的McAb进行了研究。     

死疫苗诱导的抗曼氏血吸虫保护性免疫Ⅰ.用可溶性血吸虫提取物免疫的小鼠血清抗体所识别的抗原的部分特征

本实验用可溶性虫体提取物成功地诱导抗虫免疫,并用抗血清作抗原分析,提示用这些异质性抗原和BCG,有可能制备有效的抗曼氏血吸虫疫苗。 C57BL/6J小鼠每10只一组,抗原包括经冻融灭活的尾蚴、童虫(机械断尾制备)及冻融童虫经12,000g离心5分钟后的可溶性(上清)及颗粒(沉淀)组分;另冻融童虫经超声处理及10万g离心后制成可溶性童虫抗     

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。     

抗-MSA_1单克隆抗体与血吸虫和非血吸虫抗原提取物的反应

所用抗原除MSA_1外,(?)有下列(?)融的可溶性抗原:曼氏血吸虫各期包括尾蚴、童虫、成虫和虫卵抗原,亚氏双脐螺(SBaHA)和光滑双脐螺(SBgHA)血淋巴抗原以及肝吸虫成虫抗原。并用磷酸缓冲液作为抗原对照。抗牛血清白蛋白单克隆抗体作为抗MSA_1单克隆抗体对照。应用免疫扩散及酶联免疫吸附试验的观察结果表明:抗MSA_1单克隆抗体与所有上述抗原制剂均产生不同程度的反应。免疫扩散试验可见抗MSA_1单克隆抗体与MSA_1出现清晰单一的沉淀线。与童虫抗原和SBgHA     

用纯化的表面抗原作抗曼氏血吸虫的预防接种

本文报道了以单克隆抗体亲和层析分离的两种血吸虫表面抗原免疫接种小鼠实验的初步结果。在被动转移实验中查见有一抗童虫表面抗原的保护性单克隆抗体IgM,以免疫电转移技术可识别成虫洗提液中分子量为155K的抗原,在童虫蛋白中虽未检出IgM相应的抗原,但用亲和层析法却在表面活性剂提取的童虫浸液中得到了极微量的155K抗原。用pH7.0凝胶过滤部分纯化的IgM和相同的pH经交联活化的Sepharose 4B可反复用于从表面活性剂提取成虫浸液中分离到纯     

用单克隆抗体证实一种主要的曼氏血吸虫表面膜抗原(28KDa)

作者取波多黎各株曼氏血吸虫尾蚴,分别用机械法获得童虫、尾静脉注射法获得肺内的虫体或感染法获得成虫。从感染8周小鼠肝脏分离虫卵,经孵化收集毛蚴。将童虫经脱胆酸盐提取后的产物,免疫BALB/c鼠,取脾脏,进行细胞融合和克隆化培养,上清液用于实验。童虫培养于不含蛋氨酸的培养液中,加入[~(35)S]蛋氨酸于37℃、7％CO_2培养16小时,然后用脱胆酸盐处理。经ELISA和间接免疫荧光试验分析鉴定。将所有毛蚴、童虫、肺内的虫体和成虫抗原用去垢剂处理,以~(135)I     

Schistosoma mansoni: characterization of two protective monoclonal antibodies

Two monoclonal antibodies against the surface of S. mansoni schistosomula were found to confer significant passive protection to mice (M7B3A, range 28-70%; M22H12C, range 14-58%). No additive effect was observed when both were transferred together. Neither McAb bound to the cercarial surface but both bound to the surface of in vitro derived schistosomula and schistosomula recovered from mouse skin up to 3 days after infection. The McAbs were species specific, but not S. mansoni strain specific. M22H12C immunoprecipitated an 125I-labelled surface antigen of relative molecular weight (mol. wt) 32 000. In Western blotting of an NP40 schistosomular extract, M7B3A recognized an antigen smear of 13 000-18 000 with a dominant band at 16 000. This 16 000 antigen was recognized by serum from demonstrably immune mice and rats vaccinated with highly irradiated carcariae but not by sera from mice with chronic single sex or bisexual infections.     

A preliminary analysis of Wuchereria bancrofti microfilarial antigens for potential use in diagnosis

Several antigens from the microfilarial stage of Wuchereria bancrofti have been identified using immunoblots of microfilarial antigens and screening with immune sera and tropical pulmonary eosinophilia (TPE) sera. This analysis revealed an array of antigens with apparent molecular weights of 14kDa, 35kDa, 42kDa, 63kDa, 88kDa, 97kDa and 200kDa. Among these only the 14kDa and 42kDa antigens were consistently recognized by most of the immune sera. A 132kDa antigen was recognized only by TPE sera. Analysis of rabbit immune sera revealed that the 42kDa antigen was shared by two developmental stages of W. bancrofti, namely L3 and mF. This antigen could become a potential vaccine candidate. The 14kDa antigen seems specific for the microfilarial stage and therefore could be a diagnostic marker for active infection. The 132kDa antigen could aid in the diagnosis of TPE.     

Protection of mice against Schistosoma mansoni infection by passive transfer of sera from infected rabbits

Sera from rabbits infected with unattenuated Schistosoma mansoni cercariae conferred significant levels of protection against S. mansoni challenge ( P  < 0.001) after passive transfer to mice. Infected rabbit sera were only effective in conferring protection when transferred during the first week of infection, and were not effective when administered against liver-stage worms. Immunoglobulins isolated from the infected rabbit sera with Protein A-Sepharose were shown to be responsible for the transfer of protection to mice. Immunofluorescence studies demonstrated that the sera were more reactive against the surface of three hour-old mechanically transformed schistosomula than against the surfaces of lung-stage schistosomula. The sera from infected rabbits reacted polyspecifically against antigens in cercaria, schistosomula, and the worm and egg stages of the S. mansoni life-cycle. The host parasite relationship of S. mansoni in the rabbit is discussed.     

Fractionated sera from Schistosoma mansoni infected patients confers passive protection in mice

Sera from humans with chronic Schistosoma mansoni infections (CHS) were chromatographed with CNBr-activated Sepharose 4 B conjugated with NP-40 extracts obtained from live 3 hr schistosomula. Both unbound (CHSUB) and bound (CHSB) fractions which contained IgG and IgM isotypes were characterized by ELISA, immunofluorescence, and complement-mediated in vitro killing assays. ELISA data showed that the CHSB fraction recognized schistosomula NP-40 extracts, whereas the CHSUB fraction did not. However, both CHSUB and CHSB fractions recognized 8 M urea adult worm extracts and 8 M urea egg extracts. By indirect immunofluorescence assay, the CHSB fraction recognized epitopes on the surface of live schistosomula 3 hr-29 days of age, whereas the CHSUB fraction showed surface fluorescence only on 24- and 29-day-old worms. The CHSB fraction mediated 95% killing of schistosomula in a complement dependent in vitro assay, the CHSUB fraction and the unfractionated CHS did not exhibit killing ability. The CHSUB fraction was able to titrate out the killing ability of the CHSB fraction in in vitro cytotoxic assays when mixed with the CHSB fraction at increasing concentrations. In passive immunization experiments, the CHSB fraction provided approximately 30% passive protection in mice when injected 1 day or 6 days after challenge and 20% protection when injected at 15 days, but failed to provide protection when administered greater than or equal to 24 days after challenge. Unfractionated CHS failed to mediate passive protection.     

Schistosoma japonicum myosin: cloning, expression and vaccination studies with the homologue of the S. mansoni myosin fragment IrV-5

The Schistosoma japonicum homologue of the 62 kDa fragment of S. mansoni myosin (SmIrV-5), which has proved highly protective against S. mansoni infection in mice and rats, has been cloned and expressed as the full length 62 kDa equivalent, Sj62, and a truncated 44 kDa version, Sj44. DNA sequencing showed the Sj62 sequence to be 88.4% identical at the nucleic acid level and 96% identical in deduced amino acid sequence to that of SmIrV-5. The recombinant proteins (rSj44 and rSj62) were strongly recognized in Western blotting by sera from mice multiply vaccinated with UV-irradiated S. japonicum cercariae and weakly recognized by S. japonicum chronic infection mouse sera. Unlike SmIrV-5, mouse antisera against the recombinant S. japonicum proteins did not give positive recognition in immunofluorescence assay with the surface of newly transformed schistosomula of the homologous species, S. japonicum, nor did they react with S. mansoni schistosomula. However, the anti-rSj62 sera clearly localized the native antigen to the subtegumental muscle layers in male adult worm sections by immunoelectron microscopy. Vaccination of several groups of mice and/or rats with rSj44 and rSj62 incorporated into different adjuvants induced high titres of specific IgG but in only one experimental group was there a significant reduction in worm burden (27%, P < 0.05). The possible reasons for the disparity between the vaccination results presented here and those demonstrated in experiments using rSm62 (IrV-5) are discussed .     

弓形虫速殖子表膜蛋白的组分和抗原性分析

目的试验分析弓形虫速殖子表膜蛋白的组分及其抗原性。方法用聚丙烯酰胺凝胶电泳分析弓形虫速殖子表膜蛋白的组分和用弓形虫病人阳性血清及兔抗弓形虫表膜抗原血清识别硝酸纤维膜载体上的速殖子表膜抗原的免疫反应靶位。结果弓形虫速殖子表膜抗原的独特蛋白区带分子为16和17kDa,弓形虫病人特异性抗血清识别的速殖子表膜抗原的免疫反应位点为16kDa,免抗血清识别出64,35,32,26,16kDa等8个位点。结论弓形虫速殖子表膜蛋白存在特异的抗原决定簇和可被特异性抗体识别的受体,证明速殖子表膜蛋白具有较好的抗原性,此对于弓形虫病的免疫学诊断具有重要的意义。     

日本血吸虫尾蚴成虫及虫卵的早期诊断组分抗原分析

目的寻找具有血吸虫病早期诊断价值的组分抗原.方法以感染后不同时间的兔血清,用SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹试验（Western blotting）方法,对日本血吸虫尾蚴、成虫和虫卵中的可溶性抗原的成分进行免疫分析.结果可溶性尾蚴抗原（SCA）94、48、41、40 kDa和38 kDa组分抗原最早出现免疫印迹反应,能被感染后2周兔血清所识别,可被感染后3周兔血清识别的SCA 71、23 kDa组分抗原反应最强.可溶性成虫抗原（AWA）最早出现反应的组分抗原是71、58 kDa,能被感染后3周兔血清所识别.可溶性虫卵抗原（SEA）最早出现反应的组分抗原是270、151、73、69、50 kDa和24 kDa,能被感染后4周兔血清所识别.结论 SCA 94、71、48、41、40、38、23 kDa,AWA 71、58 kDa和SEA 270、151、73、69、50、24 kDa能被急性感染兔血清所识别,是具有潜在早期诊断价值的组分抗原.     

旋毛虫部分抗原表位的识别与分析

[目的 ]筛选旋毛虫肌幼虫可溶性抗原中具有免疫显性的表位。 [方法 ]采用杂交瘤技术 ,获得 15株特异性单克隆抗体 ,随后用酶联免疫吸附试验 (ELISA)、免疫印迹法 (Westernblotting)和间接免疫荧光试验(IFA)对部分免疫显性抗原进行分析。 [结果 ]Westernblotting试验显示 ,6株单抗与旋毛虫肌幼虫可溶性抗原反应显示有特异条带 ,分子量为 40～ 70kDa ;而多抗血清则可识别 2 0～ 2 0 0kDa之间 10条条带。IFA可观察到 ,6株单抗中有 4株单抗的靶抗原定位在旋毛虫肌幼虫表皮层上 ,另 2株定位于杆状体 (stichosome)及表皮层。 [结论 ]识别与分析部分旋毛虫肌幼虫可溶性抗原中具有免疫显性的表位 ,为纯化旋毛虫的抗原及疫苗靶抗原的研制提供了有价值的实验依据。     

SD大鼠抗日本血吸虫感染机理的初步研究

目的　对SD大鼠天然抗日本血吸虫感染机理进行初步研究。　方法　用免疫印迹技术分析正常SD大鼠血清(NRS)、感染SD大鼠血清 (IRS)对日本血吸虫成虫可溶性抗原 (AWA)的识别 ;并用间接ELISA方法检测NRS抗AWA、日本血吸虫可溶性虫卵抗原 (SEA)、日本血吸虫可溶性肺期童虫抗原 (SSA)的IgG抗体及亚类 ;并观察了NRS、去补体血清对体外培养的童虫的杀伤作用。　结果　NRS可识别AWA中的 75、47、3 4.5和 2 3ku的抗原分子 ,IRS则主要识别分子质量为 62～ 86、5 4.7、47、3 4.5、3 0 .3和 2 3ku的特异性条带 ;NRS抗AWA、SEA、SSA特异性IgG1 抗体水平明显高于正常昆明鼠血清中相应的抗体水平 ,而IgG2b则无明显增高 ;SD大鼠去补体血清在体外对童虫的杀伤作用低于正常血清 ,培养 48h童虫的死亡率分别为 41.0 %和 76.2 %。　结论　SD大鼠血清中存在天然抗日本血吸虫感染的抗体 ,此抗体在SD大鼠血清的杀伤机制中起重要作用。     

Schistosoma japonicum (Chinese strain): characterization of two monoclonal antibodies which recognized common epitopes in the tegument of adult worms and schistosomula

Two monoclonal antibodies have been produced that bind to the common epitopes on adult and schistosomula of S. japonicum (Chinese strain). The isotypes of these monoclonal antibodies were determined to be IgM (designated 8G9-5) and IgG2a (designed 9E7) respectively. The target epitopes of 8G9-5 and 9E7, as analysed by immunoblotting assay, are at Mr of 64 kDa and 54 kDa respectively. Indirect immunofluorescence assay using frozen sections of adult worms, intact mechanically transformed schistosomula and 5-day-old lung schistosomula indicated that both epitopes are located in the tegument of both stages of parasite. These epitopes appear to be two of several major immunogenic proteins on the tegument of both young and adult worms.