胶体金免疫电镜技术检测番茄环纹斑点病毒

本实验利用胶体金免疫电镜技术检测番茄环纹斑点病毒（ tomato zonate spot virus, TZSV）,分别以TZSV的N 蛋白多克隆抗体及TZSV的Gn蛋白多克隆抗体为一抗,然后以胶体金标记羊抗兔IgG?gold（10 nm）为二抗对病毒进行胶体金标记,电镜观察用TZSV Gn蛋白抗体标记的胶体金颗粒能很好地结合到病毒粒体上。结果表明该方法可以实现对TZSV更为准确可靠地检测。     

食品中单增李斯特氏菌检测PCR-免疫胶体金 试纸条方法的建立

目的 建立食品中单核增生李斯特氏菌快速检测PCR-免疫胶体金试纸条法。方法 通过设计特异性引物建立单增李斯特氏菌检测PCR方法并使用免疫胶体金技术建立PCR产物快速检测试纸条; 用试验菌株检测PCR-免疫胶体金试纸条方法的检测特异度与敏感度。使用新建方法对市售肉制品和乳制品中单核增生李斯特氏菌进行检测, 验证该方法在食品检测中的可行性。结果 PCR-免疫胶体金法具有良好的特异度, 敏感度比标准琼脂糖凝胶电泳法高100倍。采集乳品样品131份, 阳性样品1份, 检出率1.53%; 肉制品224份, 阳性样品4份, 检测率1.79%。结论 建立的单增李斯特氏菌检测PCR-免疫胶体金试纸条法特异度好, 敏感度高, 适用于食品中单增李斯特氏菌的检测。     

免疫胶体金标记电镜技术在研究埃希氏大肠杆菌987P粘着素抗原结构上的应用

作者以单克隆抗体为第一抗体,应用免疫胶体金标记电镜技术研究了埃希氏大肠杆菌987P粘着素抗原结构。结果表明,该粘着素由相同的亚单位组成,并呈螺旋状结构。每个亚单位包含着几个相同的抗原决定簇分子,与酶联免疫吸附电镜技术相比较,该法除具有单克隆抗体极高特异性的特点外,还其有大小一致的胶体金颗粒致密附着在抗原相关位点上,清晰易于识别的优点。     

Silver-enhanced colloidal-gold labelling of rabbit kidney collecting-duct cell surfaces imaged by scanning electron microscopy

The luminal cell surfaces of rabbit kidney cortical collecting-duct cells were labelled with peanut lectin (PNA) and investigated by scanning electron microscopy. Labelling was performed either on 20-μm-thick cryostat sections from prefixed and cryoprotected rabbit kidney tissue or on cultured collecting-duct epithelium using biotinylated PNA and a 6-nm colloidal-gold-coupled antibody against biotin. Colloidal-gold labels were detected at low magnification (2000–4000 x) using silver enhancement. Coating with chromium allowed simultaneous imaging of both cell-surface morphology and labelling topography in the backscattered electron imaging mode. Our results show that PNA binding is specific for a subtype of intercalated cells equipped with microvilli on the luminal surface. The presented method promises to be useful for the identification of specific cell types in heterogeneous tissues.     

Amplification of Resonant Rayleigh Light Scattering Response Using Immunogold Colloids for Detection of Lysozyme

A strategy for attomolar‐level detection of small molecule‐size proteins is reported based on Rayleigh light scattering spectroscopy of individual nanoplasmonic aptasensors by exploiting the outstanding characteristics of gold colloids to amplify the nontransparent resonant signal at ultralow analyte concentrations. The fabrication method utilizes thiol‐mediated adsorption of a DNA aptamer on the immobilized Au nanoparticle surface, the interfacial binding characteristics of the aptamer with its target molecules, and the antibody–antigen interaction through plasmonic resonance coupling of the Au nanoparticles. Using lysozyme as a model analyte for disease detection, the detection limit of the aptasensor is ～7 × 10³ aM, corresponding to the LSPR λ_max shift of ～2.25 nm. Up to a 380% increase in the localized resonant λ_max shift is demonstrated upon antibody binding to the analyte compared to the primary response during signal amplification using immunogold colloids. This enhancement leads to a limit of detection of ～7 aM, which is an improvement of three orders of magnitude. The results demonstrate substantial promise for developing coupled plasmonic nanostructures for ultrasensitive detection of various biological and chemical analytes.     

In situ localization of beta-glucans in the cell wall of Schizosaccharomyces pombe

The chemical composition of the cell wall of Sz. pombe is known as beta-1,3-glucan, beta-1,6-glucan, alpha-1,3-glucan and alpha-galactomannan; however, the three-dimensional interactions of those macromolecules have not yet been clarified. Transmission electron microscopy reveals a three-layered structure: the outer layer is electron-dense, the adjacent layer is less dense, and the third layer bordering the cell membrane is dense. In intact cells of Sz. pombe, the high-resolution scanning electron microscope reveals a surface completely filled with alpha-galactomannan particles. To better understand the organization of the cell wall and to complement our previous studies, we set out to locate the three different types of beta-glucan by immuno-electron microscopy. Our results suggest that the less dense layer of the cell wall contains mainly beta-1,6-branched beta-1,3-glucan. Occasionally a line of gold particles can be seen, labelling fine filaments radiating from the cell membrane to the alpha-galactomannan layer, suggesting that some of the radial filaments contain beta-1,6-branched beta-1,3-glucan. beta-1,6-glucan is preferentially located underneath the alpha-galactomannan layer. Linear beta-1,3-glucan is exclusively located in the primary septum of dividing cells. beta-1,6-glucan only labels the secondary septum and does not co-localize with linear beta-1,3-glucan, while beta-1,6-branched beta-1,3-glucan is present in both septa. Linear beta-1,3-glucan is present from early stages of septum formation and persists until the septum is completely formed; then just before cell division the label disappears. From these results we suggest that linear beta-1,3-glucan is involved in septum formation and perhaps the separation of the two daughter cells. In addition, we frequently found beta-1,6-glucan label on the Golgi apparatus, on small vesicles and underneath the cell membrane. These results give fresh evidence for the hypothesis that beta-1,6-glucan is synthesized in the endoplasmic reticulum-Golgi system and exported to the cell membrane.     

Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling

Electron microscopy (EM) is traditionally employed as a follow‐up to fluorescence microscopy (FM) to resolve the cellular ultrastructures wherein fluorescently labelled biomolecules reside. In order to translate the information derived from FM studies to EM analysis, biomolecules of interest must be identified in a manner compatible with EM. Although fluorescent signals can serve this purpose when FM is combined with EM in correlative light and electron microscopy (CLEM), the traditional immunogold labelling remains commonly used in this context. In order to investigate how much these two strategies relate, we have directly compared the subcellular localization of on‐section fluorescence labelling with on‐section immunogold labelling. In addition to antibody labelling of LAMP‐1, bioorthogonal click labelling was used to localize soluble cysteine cathepsins or membrane‐associated sialylated glycans. We reveal and characterize the existence of inherent discrepancies between the fluorescence signal and the distribution of gold particles in particular in the case of membrane‐associated antigens.     

En bloc optical sectioning of resin-embedded specimens using a confocal laser scanning microscope

Reconstruction of 3D structures of specimens embedded for light or electron microscopy is usually achieved by cutting serial sections through the tissues, then assembling the images from each section to reconstruct the original structure or feature. This is both time-consuming and destructive, and may lead to areas of particular interest being missed. This paper describes a method of examining specimens which have been fixed in glutaraldehyde and embedded in epoxy resin, by utilising the autofluorescence preserved or enhanced by aldehyde fixation, and by using a confocal laser scanning microscope to section optically such specimens in the block down to a depth of about 200 μm. In this way, the accurate estimation of the depth of particular features could be used to facilitate subsequent sectioning at the light microscope or electron microscope level for more detailed studies, and 3D images of tissues/structures within the block could be easily prepared if required.     

福氏2a痢疾杆菌膜抗原的免疫电镜定位

本文报告应用抗福氏2a痢疾杆菌膜成份McAb及胶体金探针对细菌膜抗原进行了定位。结果显示4种抗脂多糖McAb(3A6、2E6、4F1、2A3)可不均一的识别位于细菌壁表面的抗原,而另外一种抗LPS的McAb种(1G8)和一种抗膜蛋白McAb(1A1)识别的抗原未暴露于细菌表面。应用细菌膜碎片包埋前染色成功定位了抗膜蛋白McAb(1A1)位于细菌内膜的抗原。与McAb的生物活性比较表明阳性标记细菌表面抗原的McAb的抗原与其阻段志贺氏菌接触性溶血试验和对小鼠的被动保护力密切相关。提示免疫电镜标记技术确定LPS抗原表位在细菌表面的可及性和拷贝数对构建和筛选痢疾工程菌苗具有重要意义。     

A review of methods for the production and use of monoclonal antibodies to study zoosporic plant pathogens

For many species of Oomycetes, the infection of host plants is initiated by motile biflagellate zoospores. In recent years, studies of these zoospores have utilized monoclonal antibodies directed towards a variety of zoospore components. Although only two species of fungi, Phytophthora cinnamomi and Pythium aphanidermatum, have been used for immunization and initial screening, the reactions of these antibodies with over thirty species of fungi in the Peronosporales or Saprolegniales have now been determined. The present paper reviews the methods employed to produce the monoclonal antibodies and to use them to study the biology of the zoospores and the infection process. The lack of a cell wall means that fixation protocols for zoospores for immunization and screening must be chosen carefully so that cell-surface or intracellular sites will be accessible to the antibodies. The inclusion of glutaraldehyde in the fixative helps keep the zoospore plasma membrane as intact as possible, and screening with cells fixed in the presence of glutaraldehyde selects for antibodies that bind to the surface of the zoospores. Five different patterns of labelling to the zoospore surface have been found. Other antibodies bind with three distinct patterns to the surface of zoospores and/or cysts. The use of formaldehyde alone in the fixative solution allows fragmentation of the plasma membrane and the exposure of intracellular components. In P. cinnamomi attempts to obtain antibodies directed against intracellular antigens were hampered by the presence of an immunologically dominant component that is stored in small vesicles in the zoospore cortex and secreted onto the surface of cysts. This problem was resolved by immunotolerizing mice neonatally before proceeding with immunization 2 months later. Antibodies directed towards a number of novel sites were obtained in this way. Monoclonal antibodies generated by these methods have been used to identify taxonomically specific spore components, to locate surface molecules that might be responsible for the induction of zoospore encystment and to characterize molecules involved in spore adhesion to potential hosts.