血吸虫病免疫诊断的研究——放射免疫检测循环抗原初报

为了提高血吸虫病免疫诊断的敏感性与特异性,试以同位素(~(125)I或~(131)I)标记抗循环原抗体(抗CSA IgG),进行检测血吸虫病循环抗原试验,现分三部分小结如下。 一、抗循环抗原体IgG制备,同位素标记及标记物的鉴定: 以日本血吸虫尾蚴5,000条感染家兔,感染后30天心脏取血,分离血清,用三氯醋酸沉淀血清蛋白。离心后取上清液透析。再经超滤取样,冰冻干燥获取循环抗原。再将此循环抗原加甲基化兔白蛋     

单克隆抗体识别日本血吸虫循环虫卵抗原表位的初步研究

本文采用免疫印迹试验对抗日本血吸虫卵可溶性糖蛋白单克隆抗体SM_(21-2)识别感染兔血清中循环虫卵抗原表位作了初步的分析。将Dot-ELISA检测呈阳性反应的感染兔血清进行SDS凝胶电泳,再转移至NC膜上,采用间接ELISA染色。结果发现SM_(21-3)能识别感染10、100、1000条尾蚴兔血清中27kDa的循环虫卵抗原表位,而正常血清无此区带。同时发现在46kDa处存在非特异性反应。不同的封闭剂对印迹效果有一定的影响,用0.3％Tween-PBS获得了较好的结果。     

日本血吸虫未成熟虫卵可溶性抗原诊断血吸虫病的初步探讨

用日本血吸虫未成熟虫卵可溶性抗原(SIEA)作探针，采用ELISA和ELIB检测血吸虫病人血清抗体，对急慢性血吸虫病人的控出率分别为100％和98．2％，未出现假阳性及交叉阳性反应；治疗后3、6和12个月血清抗体的阴转率分别为32．3％、50．9％和64％。其中抗82，73，67，52，42，38，32，31，28，26．21，18kDa分子的血清抗体消失较快。结果表明，SIEA在血吸虫病的诊断中具有较高的敏感性、特异性和潜在的疗效考核价值。     

甲状腺未分化癌人源单链抗体制备及初步鉴定

目的从大容量甲状腺未分化癌(anaplastic thyroid carcinoma,ATC)人源单链抗体(single chain variable fragments,sc Fv)库中筛选特异性抗ATC单链抗体并进行生物特性鉴定。方法对该抗体库进行4轮筛选后,挑取抗ATC阳性克隆感染E.coli HB2151;并用异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导该sc Fv抗体可溶性表达,ELISA法检测其可溶性表达;经HiTrap~(TM) Anti E-tag亲和柱层析纯化抗体;细胞ELISA鉴定可溶性抗体与细胞结合的特异性,分别设置TT细胞、ARO细胞、人肝癌Hep G2细胞及PBS空白对照组,用酶标仪检测各组在450 nm处的吸光度。SDS-PAGE法及Western blot检测抗体的表达和相对分子质量;流式细胞术观察细胞凋亡情况。结果经4轮筛选后抗体实现了显著富集,并得到了纯化抗体。ELISA结果表明筛选出的抗体与甲状腺未分化癌细胞能够特异性结合。SDS-PAGE和Western blot检测结果显示ATC抗体的相对分子质量约为2.9×10~4。流式细胞术结果显示ARO细胞经sc Fv特异性作用后细胞明显凋亡。结论成功获得抗ATC特异性人源单链抗体,鉴定结果显示抗体活性较好。     

抗日本血吸虫SEA特异性IgY应用于循环抗原检测的研究

本研究应用抗日本血吸虫可溶性虫卵抗原(SEA)的鸡卵黄免疫球蛋白(IgY)建立敏感、特异的检测循环抗原的双抗体夹心酶联免疫吸附试验( ELISA).用SEA皮下多点注射法免疫海蓝鸡,水稀释法制备IgY抗体,以辣根过氧化物酶标记纯化的IgY抗体(IgY-E)和兔抗IgG抗体(IgG-E)分别作为检测抗体,IgY抗体和兔抗...     

循环免疫复合物检测的影响因素——特异性与敏感性

如所周知,循环免疫复合物(CIC)的检测方法一般可分为抗原特异性法和抗原非特异性法两大类。前者适用于检测一种已知抗原及其相应抗体组成的CIO,如DNA-抗DNA、HBsAg-抗HBs、甲状腺球蛋白     

东方田鼠免疫抑制模型对日本血吸虫感染性的研究

目的建立东方田鼠免疫抑制模型,初步探讨淋巴细胞在东方田鼠抗血吸虫病机制中的作用。方法成年健康的东方田鼠40只,分成4组（每组10只）：免疫抑制＋血吸虫感染组、免疫抑制组、血吸虫感染组和空白对照组。免疫抑制剂每周注射3次,连续使用6周,建立东方田鼠免疫抑制的动物模型,用脾淋巴细胞转化试验和ELISA抗体检测进行免疫抑制模型的初步鉴定。在尾蚴攻击感染各组动物后42d,麻醉处死各组动物后进行解剖,灌注冲虫,收集虫体和肝脏虫卵镜检。结果免疫抑制模型淋巴细胞增殖受到显著抑制;免疫抑制组血吸虫抗体水平显著低于未抑制组和空白对照组;免疫抑制和未抑制的东方田鼠均未检出成虫和虫卵。结论成功建立东方田鼠免疫抑制模型,淋巴细胞在东方田鼠抗血吸虫病机制中可能不发挥直接作用。     

甲型肝炎患者循环免疫复合物的检测及其临床意义

本文对20例甲肝病人血清用聚乙二醇组分分析法进行了循环免疫复合物(CIC)的检测,发现:(1)甲肝急性期IgM抗体和IgM型CIC明显增高,且IgM型CIC与抗HAV-IgM效价存在着明显的正相关,间接提示抗体IgM以甲肝病毒作为抗原形成CIC(循环免疫复合物),参与病毒的排除。(2)病人血清中CIC水平变化与病情有一定的关系。     

先天性感染日本血吸虫家兔攻击感染后血清抗体动态的观察

日本血吸虫抗原系统相对比较复杂，汤益等对先天性感染日本血吸虫仔兔于出生后第55天攻击感染，以ELISA法检测仔兔血清特异性针对可溶性虫卵抗原(SEA)抗体，结果仔兔对攻击性感染呈低反应状态。本研究采用ELISA法检测先天性感染仔兔攻击感染后血清针对日本血吸虫抗原(AWA)IgC、IgM抗体并观察其动态变化，同时重复SEA-ELISA法检测结果，进一步探讨日本血吸虫病先天性感染仔兔的体液免疫应答。     

包虫病患者血清中循环免疫复合物测定及其临床意义的探讨

<正> 包虫病患者血清中循环免疫复合物(Circulating Immune Complexes,CIC)升高已为一些学者的研究所证实。但其临床意义尚不能肯定。本文测定了62例确诊为包虫病的患者血清中CIC浓度,同时检测了其血清中抗细粒棘球蚴(Echinococcus granulosus,E.g)抗体滴度,旨在了解包虫病人血清中CIC水平,探讨CIC与包虫囊肿临床表现及血清中抗E.g.特异性抗体水平之间的关系。     

Circulating antigens and immune complexes in Schistosoma mansoni-infected rats. Characterization by radioimmunoprecipitation-PEG assay (RIPEGA)

Circulating schistosome antigens (CSA) and circulating immune complexes (CLC) were investigated in rats infected with Schistosoma mansoni. The radioimmunoprecipitation-polyethylene glycol (PEG) assay (RIPEGA), with 125I-labelled anti-S. mansoni anti-serum, detected CSA during two distinct periods of the infection; the first between the 11th and the 14th week of infection and the second between the 11th and 14th week after infection. The CH50 deviation test revealed the presence of CIC in sera from infected rats, approximately at the two periods when CSA were detected. At 6 weeks of infection, the levels of CIC in infected rats were not different from those in control rats. However, a more sensitive method characterized IgG2a in C1q-binding C1C from infected rats. At weeks 5 and 6, IgE immune complexes were also detected in the serum from infected rats. In fact, the use of RIPEGA on the material eluted from infected rat serum after passage through an anti-IgE immunosorbent showed the presence of schistosome antigen at week 4, and at higher levels at week 6. Levels of 50% haemolytic complement in infected rat serum were lowered between the 2nd and the 4th week, the 5th and the 8th week and after the 12th week of infection. The possible role played by CIC in the protective mechanisms to a S. mansoni challenge infection in rats is discussed.     

Experimental study for early diagnosis of prepatent schistosomiasis mansoni by detection of free circulating DNA in serum

Sensitive and specific diagnostic methods of schistosomiasis at an early stage of infection are crucial to avoid irreversible pathological reactions induced by eggs. This study aimed to evaluate the PCR technique for detection of free circulating Schistosoma mansoni DNA in serum in the early prepatent period in experimentally infected mice, in comparison to the commonly used indirect hemagglutination assay (IHA) for the detection of bilharzial antibody and stool examination. Sixty-four mice were experimentally infected with S. mansoni, and every 3 or 4 days through the 8 weeks postinfection (p.i.), serum samples were collected from randomly chosen four infected mice, then pooled and examined for circulating DNA and bilharzial antibody. The results showed that the earliest deposition of eggs in the small intestine was observed at the fifth week p.i., and the eggs were detected in feces in the seventh week p.i. PCR detected free circulating DNA of S. mansoni starting from the third day p.i., while IHA failed to detect infection up to the eighth week p.i. It is concluded that detection of free circulating DNA by PCR can be used as a valuable test for early diagnosis of prepatent S. mansoni infection.     

Demonstration of Toxoplasma antigen containing complexes in active toxoplasmosis.

With an enzyme-linked immunosorbent assay antigen, specific circulating immune complexes (CIC) were demonstrated in experimental and human toxoplasmosis. In experimentally infected mice, CIC became demonstrable as soon as antibodies appeared after fatal infection. When a nonvirulent strain of Toxoplasma was used CIC remained detectable for several weeks. This period was characterized by clinically healthy animals with increasing antibody titers and by cysts growing in the brains of the animals, indicating a subacute stage of the toxoplasma infection. In the human sera, a surprisingly high percentage of CIC was demonstrated. Both immunoglobulin G (IgG) and IgM were found in the CIC; however, IgG was seen in the majority. If the humans were grouped according to other serological results, such as a combination with IgM antibodies, circulating antigens, or both, or a positive complement fixation test, increasingly more CIC were observed. When sera were selected from patients with clinical symptoms generally associated with toxoplasmosis, more CIC were also again demonstrated. On the contrary, in healthy individuals (blood donors), CIC were also regularly observed, suggesting that exacerbations of latent infections or reinfections may regularly occur without leading to clinical signs. In conclusion, we propose that the interpretation of a positive CIC test requires great care but may provide useful information about the activity of a toxoplasma infection.     

Circulating antigens, immune complexes and C3d levels in human schistosomiasis: relationship with Schistosoma mansoni egg output.

Circulating schistosome antigens (CSA), circulating immune complexes (CIC) and C3 breakdown product - C3d - were investigated in human schistosomiasis in comparison to the S. mansoni egg count. A close relationship was observed between the mean number of eggs/g of stool and the detection of CSA (evaluated by the radioimmunoprecipitation-PEG assay - Ripega), CIC (Clq-binding test) and C3d levels (quantitated by radial immunodiffusion). All the patients with more than 500 S. mansoni eggs/g of stool also presented antigen '4', specific of the genus Schistosoma, in the serum. A significant correlation was noticed between levels of CSA and CIC. This suggests the involvement of several schistosome antigens in the detected CIC. No relationship was noted between CIC and C3d levels. In contrast, there was a highly significant correlation between levels of CSA and C3d. The interaction between certain schistosome antigens and the complement system is discussed.     

Experimental infection of pigs with aTaenia species from Korea: parasitological and serological aspects

Belgian Landrace piglets were experimentally infected with eggs of aTaenia sp. of Korean origin. At autopsy, metacestodes were present only in the livers. The proportion of degenerated metacestodes increased from 12%–39% at 5 weeks to 94%–100% at 10 weeks after infection. A sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies raised against the excretory-secretory products ofT. saginata metacestodes detected circulating antigen in the sera of the pigs at 1 week post-infection. A good correlation was found between the presence of viable metacestodes and the detection of circulating antigen; the latter disappeared as the metacestodes died off. However, the antibodies were detected only after 3 weeks of infection and onwards until the necropsy of the pigs.Financial support for this study was provided by the Institute for Scientific Research in Industries and Agriculture (IWONL, Brussels)     

Evaluation of murine cytomegalovirus antibody detection by serological techniques.

Naturally acquired murine cytomegalovirus (MCMV) infection in laboratory strains of mice induces antibody levels which are generally undetectable by standard techniques; therefore, MCMV has not been included routinely in mouse viral antibody screening programs. The relative sensitivity of three assay systems, the nuclear anticomplement immunofluorescence (NACIF), the enzyme-linked immunosorbent assay (ELISA), and the complement fixation (CF) test, was evaluated for the detection of MCMV antibodies. Sera were harvested from CD1 male mice (33 days old) infected intraperitoneally with salivary gland-passaged MCMV (Smith strain). The sera were assayed separately at weeks 1 through 8, and at week 11, 16, and 25 post-inoculation; a total of 167 mice in 11 groups were tested. The animals tested at 1 week post-inoculation had low levels of antibodies to MCMV as measured by the NACIF test (1:10), whereas only 25% were positive by ELISA, and none was positive by CF until 5 weeks post-inoculation. A higher titer of MCMV antibodies was measured by CF (1:640) than by NACIF (1:40) at 6 months post-inoculation; yet, a titer of 1:3,200 was detected by ELISA from the same serum. The ELISA technique was more sensitive for detecting persistent infection with MCMV, and NACIF was more useful for detecting acute MCMV infection. Since MCMV can have significant long-term effects on the immune system, it is recommended that testing for antibodies to MCMV be included in mouse viral antibody screening protocols.     

Disturbances in humoral and cell-mediated immunity in rats with experimental depressive syndrome induced by systemic administration of 1-methyl-4-phenyl-1,2,3,6—tetrahydropyridine

Changes in humoral and cell-mediated immunity are studied in rats with 1-methyl-4-phenyl-1,2,3,6—tetrahydropyridine (MPTP)-induced depressive syndrome. A decrease in the lymphocyte count and in absolute and relative T cell counts and absolute B cell counts in peripheral blood and an increase in serum concentration of circulating immune complexes (CIC) are demonstrated. Serum CIC content increases, while the relative count of peripheral blood T cells remains decreased two weeks after discontinuation of MPTP and normalization of rats' behavior. Serum CIC content decreases and T cell count normalizes one month after discontinuation of MPTP. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 3, pp. 308–311, March, 1997     

Comparative evaluation of the diagnostic significance of circulating immune complexes and antibodies to phosphatidylinositomannosides in pulmonary tuberculosis by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay was developed to detect circulating immune complexes (CIC) and antibodies to phosphatidylinositomannosides (PIM) of mycobacteria for the diagnosis of pulmonary tuberculosis. Identification of PIM in CIC of smear and culture positive (SPCP) cases revealed 88 % sensitivity and antibodies to PIM were detected in circulation in 94% cases. The smear negative and culture positive (SNCP) group had 67% positivity with regards to PIM in CIC and 83% for antibodies to PIM in circulation. In the smear negative and culture negative (SNCN) group, 40% samples were found to have PIM in CIC and 20% exhibited antibodies to PIM. The high degree of sensitivity was observed with both antigen detection in CIC and antibodies to PIM in circulation.     

Adaptation of a protein a binding assay for measurement of immune complexes in sera of rats

We describe a simple and sensitive method for the detection of circulating immune complexes (CIC) in rats and have applied the method to test sera from tumor-bearing rats. CIC were precipitated preferentially with 6% polyethylene glycol, dissolved, and then bound to Staphylococcus aureus that are Protein A (PA) positive. Rabbit-anti-rat IgG (RARG) antibodies were then added and followed by 125I-PA. The RARG sandwich antibody enhanced the sensitivity of detection of CIC in rats at least 5 fold compared to that observed using 125I-PA without RARG.