斑点免疫金渗滤法同时检测猪瘟和猪繁殖与呼吸综合征抗体的研究

在猪瘟、猪繁殖与呼吸综合征单重斑点免疫金渗滤法的基础上,建立能同时检测HC和PRRS的双重斑点免疫金渗滤法。将提纯的HC和PRRS抗原同时包被在硝酸纤维膜上,利用胶体金标记SPA显色,当猪血清中含有HC、PRRS抗体时,则相应的点会显现红色(呈红色斑点),结果易于判断。且操作简单,耗时短,与猪细小病毒、口蹄疫和猪伪狂犬病毒阳性血清不发生交叉反应。     

胶体金法与TPPA法检测梅毒特异性抗体的对比研究

目的 :探讨梅毒螺旋体特异性抗体胶体金法 (TPAb)在临床工作中的应用。方法 :以梅毒累旋体明胶凝集试验 (TPPA)为“金标准” ,同时用TPAb法检测梅毒高危人群的血清。结果 :两种方法经 χ2 检验P >0 0 5 ,差异没有显著性 ,Kappa值为 0 86 ,TPAb法其敏感性 88 9% ,特异性 97 8% ,准确性 93%。结论 :TPAb法可推荐用于血清学梅毒特异性抗体检查 ,有助于临床梅毒的快速诊断     

胶体金渗滤方法检测乙肝患者血清中乙肝核心抗体的研究

目的:建立胶体金免疫渗滤法检测乙肝患者血清中乙肝核心抗体(anti-HBc Ig M)抗体的方法,评价其灵敏度、特异性和重复性。方法:预先在硝酸纤维素膜上包被乙肝核心抗原HBc Ag,将血清或者血浆加在硝酸纤维素膜上,血清或者血浆中的anti-HBc Ig M与HBc Ag结合。洗涤去掉非特异结合,加入胶体金标记的抗人-Ig M,洗涤去掉没有结合的抗人Ig M抗体。硝酸纤维素膜上的抗原-Ig M-抗人Ig M形成的\"三明治\"复合物呈现红色圆点,证实实验有效。结果:102份乙肝患者血清,anti-HBc Ig M阳性标本99份。检出率97%。50份正常血清中,有一份检测结果弱阳性,特异性98%。结论:胶体金免疫渗滤法检测anti-HBc Ig M敏感性高、特异性强、重复性好,且方便快捷,不需要特殊的仪器设备,在anti-HBc Ig M快速检测上有广阔的应用前景。     

番木瓜乳管结构及木瓜蛋白酶的免疫电镜研究

采用免疫细胞化学方法, 以透射电镜观察, 对番木瓜（Ｃａｒｉｃａ ｐａｐａｙａ Ｌ．）乳管分化及木瓜蛋白酶生成的超微结构环境进行了研究。实验结果表明：１．正在分化的乳管细胞内质网分泌旺盛, 线粒体和聚核糖体非常丰富; 之后细胞器逐渐解体, 内质网断裂、膨胀, 细胞壁多处穿孔; 经过内膜系统的重新组合, 成熟乳管被小泡充满, 小泡内有无定形物质凝聚, 已经没有任何细胞器残留, 但原生质膜一直存在。２．经过纯化的兔抗ｃｈｙｍ ｏｐａｐａｉｎ ＩｇＧ 为第一抗体, 羊抗兔ＩｇＧ－金复合物（１０ ｎｍ 直径）为间接抗体, 进行适当的免疫标记反应, 发现金颗粒主要在乳管细胞内, 附近的薄壁细胞及导管只是偶尔出现金标, 说明免疫标记的特异性较强, 通过各种对照试验, 证明了非特异性吸附相当微弱。显示木瓜蛋白酶的原初生成部位是正在分化的乳管细胞的内质网, 它暂时贮存于分泌泡, 随乳管的发育和乳汁其它成分一起被“组装”为乳汁小泡而充满成熟乳管     

Nitric oxide (NO) expression during annual reproductive activity in buffalo epididymis: a histochemical and immunocytochemical study

The buffalo is one of the few domestic animals that has a seasonal mating cycle, influenced by the photoperiod. It is known that the photoperiod regulates gonadal function probably via the pineal and/or hypothalamus-pituitary axis. Moreover, the hypothalamus (melatonin) and gonads influence the production of the signaling transmitter nitric oxide (NO), suggesting that the NO may have an important role in the regulation of gonadotropin-releasing hormone secretion. This further suggests the hypothesis that NO in the epididymis has an important role in the maturation of spermatozoa and their motility and posterior fertilization capacity. The aim of the present study is to investigate the seasonal variations in the morphology of the epididymis by means histochemical and immunocytochemical techniques. We used the NADPH-d, nitric oxide synthase (NOS) I and NOS III to clarify the relationship between epididymis function and NO signaling activity. The results of this work show that NO is present in the caput of epididymis during short photoperiods, i.e., periods of maximum gonadal activity (winter) and absent during long photoperiods, i.e., periods of gonadal regression according to the previously described role of NO in spermatozoa capacitation and motility in the caput epididymis.     

Localization of profilin- and actin-like immunoreactivity in in vitro-germinated tobacco pollen tubes by electron microscopy after special water-free fixation techniques

Double immunogold labeling of profilin and actin was performed on ultrathin sections of in vitro germinated tobacco pollen using different anti-profilin and anti-actin antibodies. Since profilin, besides its role as an actin-binding protein, is known as an allergen, water-free fixation in p-formaldehyde vapor was used. Profilin labeling occurs throughout the cytoplasm of the pollen tube. There is no profilin in the pollen tube wall. Actin reactivity is found in the cytoplasm and extracellularly in the pollen tube wall where three out of four different anti-actin antibodies give a positive signal. This labeling of the pollen tube wall may result from a wall-bound actin, an isoform of actin not yet described or from the presence of a molecule immunologically indistinguishable from actin.     

免疫胶体金标记显示波形纤维与核孔复合体的结合

中间纤维与细胞核的关系是一个亟待解决的重要问题。本文采用火鸡红细胞作为研究材料,首先用细胞分级抽提结合免疫印迹反应显示火鸡红细胞中间纤维蛋白为波形纤维蛋白。然后,我们采用细胞分级抽提结合包埋前免疫胶体金标记的方法显示胞质中间纤维被抗波形纤维蛋白抗体-蛋白A-胶体金特异标记。同时,我们显示结合于核孔复合体上的胞质纤维被抗波形纤维蛋白抗体-蛋白A-胶体金所特异标记。本文结果表明,结合于核孔复合体上的胞质纤维是波形纤维,并且提示波形纤维可能通过与Nup 180的结合附着在核孔复合体上,为进一步探讨中间纤维与核孔运输的关系提供了初步实验证据。     

Assessment of antibody responses to antigens of Mycobacterium tuberculosis and Cysticercus cellulosae in cerebrospinal fluid of chronic meningitis patients for definitive diagnosis as TBM/NCC by passive hemagglutination and immunoblot assays

Tanned sheep erythrocytes stabilized with pyruvic aldehyde and glutaraldehyde, called double-aldehyde-stabilized cells, were used to standardize passive hemagglutination assay (PHA) for detection of antibody responses to sonicate extract of Mycobacterium tuberculosis and Cysticercus cellulosae soluble antigens. PHA was performed in the following groups of cerebrospinal fluid (CSF) samples: group I - chronic infections of the central nervous system with the possible diagnosis of tuberculous meningitis (TBM), tuberculoma and neurocysticercosis (NCC) (n=88), and group II - controls which included (a) non-infectious non-neurological conditions (n=30), (b) infectious neurological conditions (n=21) and (c) non-infectious neurological conditions (n=133). PHA could detect anti-mycobacterial antibodies at the sensitivity level of 80.76% with a specificity of 92.4% and anti-cysticercal antibodies with a sensitivity of 100% and specificity of 92.94%. However, in 6.33% (i.e. 14/221) of group I and group II (c) CSFs both anti-mycobacterial and anti-cysticercal antibodies were detected. Immunoblot analysis of CSFs derived from TBM patients reacted predominantly to 120-kDa, 96-kDa, 65-kDa, 38-kDa, 26-kDa, 23-kDa, 19-kDa and 12-14-kDa and 4-6-kDa antigens of M. tuberculosis sonicate extract (MTSE), whilst CSFs of proven NCC reacted to >110-kDa, 96-kDa, 80-kDa, 66-68-kDa, 52-kDa and 26-28-kDa antigens of porcine whole cyst sonicate extract (PCSE). On immunoblot analysis, some of the CSFs of TBM patients were PHA positive for both MTSE and PCSE showed antibody reactivity to 70-kDa and 10-kDa antigens of C. cellulosae. Similarly CSF antibody of some Guillain Barre syndrome and myeloradiculopathy patients reacted with cysticercal antigens. But per se no cross-reactivity between MTSE and anti-cysticercal antibodies and vice-versa were observed. However, findings of this study should alert laboratory personnel especially in endemic areas to be extra careful in interpretation of antibody detection results.     

斑点免疫测定法在植物病毒研究中的应用及技术要点

斑点免疫测定法（ＤＩＡ）能快速、有效地检测出植物组织及种子中的病毒,且灵敏度高,不需特殊设备。本文介绍了ＤＩＡ的基本方法及步骤,并重点论述了进行ＤＩＡ测定的技术要点。     

Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth,Plodia interpunctella

We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium.     

Intracellular localization of protein C inhibitor (PCI) and urinary plasminogen activator in renal tubular epithelial cells from humans and human PCI gene transgenic mice

Urinary plasminogen activator (uPA) is a serine protease that plays important roles in various extracellular proteolytic processes. In humans, protein C inhibitor (PCI) is known to regulate the activity of the serine proteases involved in blood coagulation, wound healing, and tumor metastasis, whereas PCI is not present in murine plasma or tissues other than the reproductive tissues. The large amount of uPA–PCI complexes found in human urine suggests that these complexes are formed in the kidneys. In the present study, we performed immunofluorescence double labeling and electron microscopic immunocytochemistry using renal tissues from humans and human PCI gene transgenic (PCI-TG) mice. In human renal tissues, PCI and uPA colocalized in the cytoplasm of renal proximal tubular epithelial cells (RPTECs), and juxtaposition of PCI and uPA immunoreactive particles was detected in the microvilli and lysosomes in the RPTECs. The intracellular distributions of PCI and uPA in the RPTECs from PCI-TG mice were similar to those observed in human RPTECs. These findings hint at the physiological roles of uPA and PCI in human kidneys, and also suggest that the PCI-TG mice will be useful for evaluating the roles of PCI in human physiological and pathological conditions.     

Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections

We developed a protein microarray methodology that has the ability of serodiagnosis of IgM antibodies directed against TORCH pathogens. Six chemical surface modifications were validated by a dimension atomic force microscope (AFM) and contact angle measurement, agarose modified surface of which offered an appropriate platform for detecting IgM antibody. Further, signal amplification sensitivities on agarose modified microarrays were detected by Cy3-labeled biotin-streptavidin and immunogold-based assays. The detection limits of IgM antibody on the microarrays were 0.48 and 0.24mug/ml, quantitatively equal to 0.25 and 12.5pg, respectively, on each spot as ascertained by the two assays. Satisfactory linear correlations between the signal intensity and the logarithm of the IgM concentration were obtained. Finally, 60 serum samples characterized by a commercial ELISA were evaluated by the protein microarray. There were good concordances between the results of the protein microarray and ELISA assay for sorting of the TORCH infected sera (95.0% by fluorescence-based assay and 96.7% by immunogold-based assay). Clearly, the potential application of this protein microarray format facilitates clinical detection of not only the antibodies directed against TORCH pathogens but also other autoimmune diseases.     

Previous Enteric Adenovirus Infection Does Not Protect against Subsequent Symptomatic Infection: Longitudinal Follow-up of Eight Infants

Eight infants followed longitudinally were found to have enteric adenovirus (EAdv) infections: in 5 infants with diarrhea and in 3 with no accompanying diarrhea. Sequential stool samples prior to EAdv infections were tested for adenovirus antigen, anti-adenoviral IgA and neutralizing antibodies to serotypes 40, 41 and 2 in order to ascertain whether protection from symptoms was due to prior infection. No difference was found in the number of adenoviral infections among infants prior to their EAdv infections with or without accompanying diarrhea. However, in 3 of the 5 infants in whom EAdv infection was accompanied by diarrhea and 2 of 3 control infants, previous EAdv infections had occurred as detected by serotype-specific antibody rises.     

Development of a dot immunobinding assay to detect Phytophthora spp. in naturally dark-rooted woody plants

An indirect dot immunobinding assay (DIBA) with a PAb raised against an isolate of P. cinnumomi was evaluated to detect Phytophthora spp. in naturally dark-rooted woody plants. Screening of different modifications of the procedure were done with root samples of Chamaecyparis lawsoniana non-infected and infected with P. cinnamomi. With the optimised DIBA procedure, root infection could be diagnosed by a clearly defined halo around the spot and by a colour change in the spot using Fast Red or Fast Blue substrate. Detection of different Phytophthora species in Chamaecyparis plants from commercial nurseries was successful with the optimised procedure.     

Membrane topology of insulin receptors reconstituted into lipid vesicles

Insulin receptors were incorporated into liposomes by two different procedures, one using dialysis and one using detergent removal by Bio-Beads. Receptor incorporation was analyzed by gradient centrifugation and electron microscopy. Reconstituted receptors projected up to 12 nm above the membrane and exhibited a T-shaped structure compatible with that previously described for the solubilized receptor. Insulin binding and autophosphorylation experiments indicated that approx. 50% of the receptors were incorporated right-side out. Such random orientation was confirmed by immunogold labeling of the - and the -subunit of the receptor. Immunogold labeling of the C-terminus of the -subunit indicates that it resides about 6 nm off the membrane, while two -subunit epitopes were labeled at about twice this distance, confirming that the -subunit is harbored in the cross-bar of the T-structure.We thank Ms. Birthe Nystrøm, Lisette Hansen and Ulla Blankensteiner for excellent technical assistance and Ms. Birgit Risto for skillful work with the photographic prints.     

Localization on the cell surface of streptococcal protein antigen A

Abstract The cellular localization of the major surface protein SpaA of Streptococcus sobrinus 6175 was examined by immunoelectron microscopy with rabbit polyclonal antibodies directed against purified SpaA protein. Immunoelectron microscopic analysis of thin sections of S. sobrinus cells revealed that the SpaA protein is associated with the fibrillar fuzzy coat of S. sobrinus cells and appears to be distributed over the entire surface of S. sobrinus cells.     

耐热性碱性磷酸酯酶作为非放射性标记物的研究

通过生物素与亲和素－酶复合物系统或地高辛与抗地高辛－酶复合物系统可把酶间接标记到探针上．Ｒｅｎｚ等通过不同的化学方法直接把酶标记到探针上［１～３］．耐热性碱性磷酸酯酶ＦＤ－ＴＡＰ（ｔｈｅｒｍｏｓｔａｂｌｅａｌｋａｌｉｎｅｐｈｏｓｐｈａｔａｓｅ）具有耐...