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。     

猪囊尾蚴表面蛋白及其抗原性的初步测定分析

本文初步分析了猪囊尾蚴囊壁表面蛋白的特性。根据囊尾蚴漂洗液PAGE和SDS-PAGE考马斯亮蓝染色带分别显示了9条及14条蛋白区带,其中大部分与宿主骨骼肌组织的可溶性蛋白有一致的迁移率,但其中第5、7、10条蛋白带为其独有。漂洗液与囊液兔高免血清IE可出现1条沉淀弧;用漂洗液蛋白抗原对35例囊虫病人血清做ELISA,阳性率为60％。此外,对囊虫表面宿主蛋白的可能作用,做了初步探讨。     

抗囊虫卵黄免疫球蛋白的提取

猪囊虫病是严重危害人体健康和养殖业发展的重要人兽共患病。医学和兽医科技工作者对检测猪囊尾蚴病进行了大量的研究 ,已取得了一定的进展。但在以卵黄IgY作为抗体方面尚未有人尝试。本试验旨在探索一个提取抗囊虫抗体的新方法。为囊虫病的防治及检测开辟一条新的途径。1　猪囊虫抗原的纯化1 1　材料　猪囊虫含生理盐水 15 0ml。1 2　方法1 2 1　囊液的收集　从新鲜囊虫猪肉摘取虫囊 ,然后将虫体取出置灭菌平皿中 ,用生理盐水洗涤 4次 ,吸干多余液体。用剪刀剪破囊壁使囊液自然流出 ,收集囊液于灭菌瓶内 ,置4℃过夜。次日将囊液做 30 0 0…     

猪囊虫病的免疫学诊断研究.一、猪囊尾蚴蛋白质及其抗原制剂的电泳分析

本文报道猪囊尾蚴头节和囊壁混合蛋白质与可溶性抗原及囊液蛋白质和可溶性抗原的电泳区带。头节和囊壁混合抗原有13条(PAGE)和22条(SDS-PAGE)考马斯亮蓝染色带,免疫学抗原11种;囊液抗原有10条(PAGE)和16条(SDS-PAGE)考马斯亮蓝染色带、PAS和苏丹黑B染色带各1条,免疫学抗原14种。囊液富含蛋白质、糖蛋白和脂蛋白,且有一种抗原活性较强的抗原组分。两种抗原有共同抗原3种,均含耐热抗原组分.     

猪囊虫体表抗原用于 ELISA 诊断囊虫病的研究

应用猪囊尾蚴体表抗原对66例临床确诊的囊虫病人用 ELISA 进行检测,同时以囊液粗抗原作比较观察。阳性率分别为95.5%和68.2%,两者差异非常显著(P<0.005)。两种抗原对同批血清的反应强度也呈现明显差异(P<0.05)。用囊虫体表抗原和囊液粗抗原同时检测50名健康人血清,假阳性率分别为2%与12%;两种抗原与10例包虫病人血清分别有8例和4例出现交叉反应。     

猪囊尾蚴液、固相多种抗原对囊虫病免疫诊断效果评价

目的　筛选适合多种寄生虫病流行区使用的特异性较高的囊虫病免疫诊断抗原。　方法　制备猪囊尾蚴各类型液相抗原和固相抗原 ,用ELISA或IEST检测囊虫病流行区患者阳性血清抗体 ,比较各种抗原的敏感性和特异性。　结果　猪囊尾蚴全囊及囊液尿素溶性抗原与水溶性纯化抗原ELISA检测囊虫抗体的特异性差异无显著性 (P >0 .0 5 )。固相抗原IEST有敏感性好、操作简易的特点。　结论　猪囊尾蚴全囊尿素溶性纯化抗原是较理想的囊虫病ELISA诊断抗原 ,有诊断应用价值。固相石腊切片抗原IEST较为适合流行区现场或基层应用。     

间接血凝试验在囊虫病诊断中的应用

囊虫病是人畜共患疾病,严重损害人民健康。本文报道用间接血凝试验检测人血清中囊虫抗体,具有较高的特异性和敏感性。 1 囊虫抗原致敏血球的制备 1·1 囊虫抗原的制备,选取新鲜的含猪囊蚴的猪肉,用消毒空针抽取囊液,经3000rpm/分离心10min,上清即为囊液抗原。用分光光度计测量蛋白     

猪肉和人皮下猪囊虫囊液抗原的免疫印迹分析

应用SPA-ELISA、SDS-PAGE和IB(免疫印迹)技术对猪肉及人皮下结节内猪囊虫囊液的抗原成份进行了分析比较。结果表明,凝胶中考马氏亮蓝和氨基黑染色所示的蛋白条带以及血清抗体所识别的特异性抗原成份中,取自猪肉中的囊虫囊液较取自人体的囊液含量高,免疫反应性强。两种抗原用于SPA-ELISA诊断未见有明显差异。     

猪囊虫囊液游离氨基酸组分的研究

应用氨基酸自动分析仪测定猪囊虫囊液的游离氨基酸组分及含量。从14只猪的皮下肌肉囊虫及10只猪的脑囊虫的囊液进行测定。在两者囊虫囊液所测定的18种氨基酸中,均以内氨酸含量最高;在皮下肌肉囊虫囊液,其次为甘氨酸和脯氨酸,在脑囊虫囊液,则以苏氨酸为次。用配对T检验方法比较7只猪的皮下肌肉和脑囊虫囊液中各种氨基酸含量表明,在不同部位囊虫囊液所含的15种氨基酸中,有12种含量存在显著差异,特别是苏氨酸、丝氨酸、丙氨酸、锁氨酸和酪氨酸出现差异非常显著。本研究结果提示上述氨基酸代谢差异,可能与猪囊虫在宿主的寄生部位不同有关.     

猪囊尾蚴蛋白质聚丙烯酰胺凝胶电泳和等电聚焦分析

猪囊尾蚴粗抗原在进行人体囊虫病的免疫诊断中有比较好的效果。但猪囊尾蚴抗原成分复杂,为了进一步提高阳性率,减少交叉反应,需要纯化抗原,分离出特异的抗原组分。我们用聚丙烯酰胺凝胶电泳(PAGE)和薄层水平等电聚焦(IEF)对猪囊尾蚴的囊液、头节囊壁和全囊抗原蛋白质组分进行了比较分析。     

Chromatographic and antigenic properties of Echinococcus granulosus hydatid cyst-derived glycolipids

The neutral and acidic fraction glycolipids of Echinococcus granulosus metacestode tissue compartments were isolated, defined by their chromatographic and antigenic properties, and assessed as to their efficacy as antigens in the serodiagnosis of human hepatic cystic and alveolar echinococcosis, and other helminthiases. Analyses were accomplished by thin-layer chromatography immunostaining and ELISA. The neutral glycolipid fraction's major carbohydrate epitope was the same as or very similar to that of Taenia crassiceps neutral glyco(sphingo)lipids, as represented by the 'neogala'-series core structure. The blood group-active, carbohydrate epitope P1 was expressed by a number of neutral fraction glycolipid component bands. The reverse-phase, thin-layer chromatography-isolated neutral fraction glycolipid component, designated Ag1, was efficient in the serological discrimination of cystic echinococcosis medium to high-titred sera. Ag1 did not specifically discriminate low-titred sera, i.e., other human helminthiases. The detected sialic acid residues of the acidic fraction glycolipids, on enzymatic cleavage, were identified as N-acylneuraminic acid and terminal. The acidic fraction glycolipids exhibited the paradox of only chemically minor components being antigenic towards cystic and alveolar echinococcosis infection sera. The combined acidic fraction glycolipid components Ra and Rx were capable of serological discrimination between cystic echinococcosis, alveolar echinococcosis and other helminthiases.     

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。     

棘球蚴特异性抗原的蛋白质印迹分析

目的　寻找细粒棘球蚴 (Eg)和多房棘球蚴 (Em)特异性抗原组分用于免疫诊断。　方法　对Eg、Em的囊液、原头节、囊壁角质层、囊壁生发层及犬泡状带绦虫、犬豆状带绦虫、羊细颈囊尾蚴、猪囊尾蚴等 4种异源性绦虫 ,共14种粗抗原进行免疫学分析。采用蛋白质印迹试验 (Western blotting)、Genesnap和Genetool分析软件比较分析不同抗原蛋白条带分别与囊型棘球蚴病 (CE)及泡型棘球蚴病 (AE)患者血清反应的差异。　结果　 14种粗抗原中与CE、AE患者血清发生交叉反应的 11条蛋白条带相对分子质量 (Mr)为 130000、100000、94000、80000、75000、66000、62000、52000、38000、32000及 24000 ;与CE患者血清有特异性反应的 5条蛋白条带Mr为 41000、40000、22000、160 00和12000 ;与AE患者血清具有特异性反应的 8条蛋白条带Mr为 120000、109000、86000、59000、43000、28000、20000及 18000。　结论　确定了Eg和Em共有的交叉反应性抗原和具有进一步研究价值的特异性抗原组分 ,为进一步分离和鉴定具有诊断价值的特异性抗原提供了基础资料     

绵羊牦牛棘球蚴囊液及囊壁抗原的SDS—PAGE分析

本文应用十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)对绵羊、耗牛棘球蚴囊液及绵羊棘球蚴囊壁可溶性粗抗原的蛋白组分进行了分析。使用10％凝胶,采用垂直板型电泳、考马斯亮兰R—250染色进行SDS-PAGE的结果表明,绵羊及耗牛棘球蚴囊液分别含有16及11种蛋白组分,分子量范围分别为270～16.5KD及280～17KD;主带均为8条。绵羊棘球蚴囊壁可溶性粗抗原共显示17条蛋白带,分子量范围245～17KD,主带8条。本项研究为进一步分析分离特异性抗原奠定了基础。     

RAPD-PCR characterization of Pseudomonas aeruginosa strains obtained from cystic fibrosis patients

OBJECTIVE: To characterize P. aeruginosa strains isolated from bronchoalveolar lavage fluid of cystic fibrosis (CF) patients over a 3 year period. MATERIAL AND METHODS: A prospective follow-up study was carried out in a population of cystic fibrosis patients. The random amplified polymorphic DNA (RAP.D) technique was used to amplify DNA of P. aeruginosa strains isolated from bronchoalveolar lavage fluid samples of five CF patients from the Servicio de Neumología y Cirugía del Tórax del Instituto Nacional de Pediatría (Mexico City Chest Clinic of the National Pediatrics Institute) in Mexico City, between June 1996 and June 2002. Amplification patterns were established for each isolate to accurately identify all strains and to carry out an epidemiological analysis of P. aeruginosa among the selected CF patients. RESULTS: Eighteen different DNA amplification patterns were defined and used to identify each P. aeruginosa strain isolated from the different bronchoalveolar lavage samples. No correlation was observed between the different P. aeruginosa strain genotypes and mucoid or non-mucoid phenotypes, as strains with different phenotypes showed similar amplification patterns. Several strains with different amplification patterns were identified in samples obtained from the same patient, suggesting coinfection with ore than one P. aeruginosa strain. Two siblings with CF shared similargenotypes, suggesting the occurrence of cross- contamination. Similar genotypes of P. aeruginosa strains were isolated throughout the study period. CONCLUSION: Genotypic characterization of P. aeruginosa strains in CF patients allows more accurate epidemiological analyses of this important host-agent relationship.     

三裂叶豚草花粉致敏蛋白组分的分离、纯化及鉴定

目的分离、纯化及鉴定三裂叶豚草花粉致敏蛋白组分。方法提取三裂叶豚草花粉粗提液,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离粗提液蛋白组分并测定其分子量。收集存在变态反应患者血清,通过免疫印迹法(Western-blotting)对花粉致敏蛋白组分进行鉴定,用离子交换层析初步纯化其致敏蛋白组分,并通过Western-blotting鉴定。结果三裂叶豚草花粉有30余条蛋白条带,其中有14条主要条带,其特异性致敏蛋白组分的相对分子质量为63 000、56 000、40 000和36 500,主要为63 000、40 000和36 500。三裂叶豚草花粉通过离子交换层析纯化出相对分子质量分别为63 000、40 000和36 500的致敏蛋白组分主要集中在Ⅳ、Ⅲ、Ⅱ、Ⅰ峰。结论初步分离、纯化及鉴定了三裂叶豚草花粉致敏蛋白组分。     

Serologic Reactivity to the Emerging Pathogen Granulibacter bethesdensis

Background.?Granulibacter bethesdensis is a recently described member of the Acetobacteraceae family that has been isolated from patients with chronic granulomatous disease (CGD). Its pathogenesis, environmental reservoir(s), and incidence of infection among CGD patients and the general population are unknown. Methods.?Detected antigens were identified by mass spectroscopy after 2-dimensional electrophoresis and immunoaffinity chromatography. The prevalence of Granulibacter immunoreactivity was assessed through immunoblotting and enzyme-linked immunosorbent assay (ELISA). Results.?Methanol dehydrogenase (MDH) and formaldehyde-activating enzyme were recognized during analysis of sera from infected patients. Unique patterns of immunoreactive bands were identified in Granulibacter extracts, compared with extracts of other Acetobacteraceae species. By use of criteria based on these specific bands, specimens from 79 of 175 CGD patients (45.1%) and 23 of 93 healthy donors (24.7%) reacted to all 11 bands. An ELISA that used native MDH to capture and detect immunoglobulin G was developed and revealed high-titer MDH seroreactivity in culture-confirmed cases and 5 additional CGD patients. Testing of samples collected prior to culture-confirmed infection demonstrated instances of recent seroconversion, as well as sustained seropositivity. Infection of CGD mice with G. bethesdensis confirmed acquisition of high-titer antibody-recognizing MDH. Conclusions.?These serologic tests suggest that Granulibacter immunoreactivity is more common among CGD patients and, perhaps, among healthy donors than was previously suspected. This finding raises the possibility that clinical presentations of Granulibacter infection may be underappreciated.     

犬弓首线虫幼虫培养,排泄分泌抗原的制备和分析

本文报道犬弓首线虫幼虫体外培养、排泄分泌抗原(TES抗原)的制备和分析结果。采用自行研制的幼虫分离器分离人工孵化的犬弓首线虫第二期幼虫,用Eagle's基础培养基培养幼虫,并用饱和硫酸铵盐析法从培养液中提取TES抗原均取得满意结果。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示此抗原,可见到7条蛋白质区带。免疫双扩散表明本抗原与本虫感染的免血清(1:32)作用呈阳性反应。免疫电泳显示至少含有四条沉淀线。转移电泳免疫识别显示本抗原含有5～7个血清学抗原成份。     

Differences in excretory-secretory products and surface antigens among 19 isolates of Giardia

The excretory-secretory (E-S) products and surface antigens of 19 isolates of Giardia were compared by reactivity of E-S products with antisera to homologous and heterologous organisms and by acrylamide gel electrophoresis of surface-labeled Giardia. Isolates could be divided into three broad groups on the basis of the previously reported DNA studies and the present studies. Group 1 consisted of five isolates with similar or highly cross-reactive E-S. These showed identical DNA banding patterns after endonuclease restriction analysis; four of five had identical surface antigens, and the remaining isolate showed a similar but different major surface antigen. Group 2 consisted of 11 isolates with moderate reactivity amongst themselves. DNA patterns showed some bands in common with group 1 organisms and themselves, but the surface-antigen molecular weight patterns were different. Group 3 consisted of three isolates with reactivity only amongst themselves. There were no DNA bands in common with group 1, and the molecular weights of the surface antigens were diverse. Surface-antigen differences are common among isolates of Giardia lamblia. These differences correlated to some degree with the DNA banding patterns observed after endonuclease restriction analysis and may result in altered virulence and host response.     

华支睾吸虫未脱脂和脱脂成虫抗原的免疫印迹分析

目的 寻找华支睾吸虫成虫抗原中具有特异性诊断价值的抗原组分。方法 应用SDS—PAGE和Westernblot分析华支睾吸虫成虫未脱脂、脱脂的可溶性抗原蛋白组分的血清学反应特征。结果 未脱脂成虫抗原有14条蛋白带可与华支睾吸虫病病人血清反应，其中分子质量单位为180、170、100、67、41、37、35、32、26ku是主带，180、170、37、35ku条带还可与日本血吸虫病、卫氏并殖吸虫病病人血清发生交叉反应。脱脂成虫抗原有10条蛋白带可与华支睾吸虫病病人血清反应，其中分子质量单位为180、100、67、37、35、24、20ku是主带，此外，180ku带可与日本血吸虫病和卫氏并殖吸虫病病人血清发生交叉反应，78ku带可以与血吸虫病病人血清发生交叉反应。结论 华支睾吸虫成虫抗原经脱脂处理后可减少与日本血吸虫病和卫氏并殖吸虫病病人血清交叉反应的蛋白组分数量。华支睾吸虫未脱脂和脱脂成虫抗原中主要的特异性抗原组分分别是100、67、41、32、26ku和100、67、37、35、24、20ku，这些组分抗原可用于华支睾吸虫病的免疫诊断。