Virus morphology as an aid for rapid diagnosis.

Standard methods of virus diagnosis may take many days to complete. As antiviral drugs are being used with more effectiveness, it becomes more important to develop rapid diagnostic methods. It takes only a few minutes to prepare and examine a specimen for electron microscopy (EM), using the negative staining technique. Viruses in the specimen can readily be identified by their morphology. In order to be detected by EM there must be at least 10(7) virus particles per milliliter of sample. This concentration is frequently found in certain types of specimens. The sensitivity of EM is increased 100-fold if homologous antibody is used to aggregate the virus. Visualization of virus-antibody aggregates forms the basis for serotyping by immunoelectron microscopy (IEM).     

Sphere-linked immunodiagnostic assay (SLIDA): an electron microscopic method for detecting specific antibodies

We have developed a sensitive method, sphere-linked immunodiagnostic assay, using specific antigens covalently bonded to microspheres for the detection of antibodies in serum. In this method, specific antigens, such as the capsid proteins of tobacco mosaic virus and tobacco etch virus, were independently, covalently bonded to plastic micropheres of 0.5 microns or 0.9 microns in diameter. The antigen-linked spheres were then exposed to normal serum or serum containing specific antibody, followed by treatment with gold-labeled secondary antibodies. The binding of the gold-labeled secondary antibodies to the specific primary antibodies on the spheres acted as an indication of the presence of the specific primary antibodies. The spheres were then examined and photographed by transmission electron microscopy. The number of gold particles bound to the spheres was counted manually using the photographs. The gold labeling was found to be specific and sensitive, enabling detection of antibodies present in highly diluted antisera. The efficiency and sensitivity of the technique for detection of antibodies were compared with those of the enzyme-linked immunosorbent assay and found to be highly sensitive. The technique was also used for testing for the presence of antibodies to herpes simplex virus as well as antibodies to Staphylococcus enterotoxin using microspheres coated with the respective antigens. We believe that this technique could be applied clinically when needed for detection of antibodies to other viruses, such as the Human Immunodeficiency Virus.     

Immunohistochemical localization of barley stripe mosaic virions in infected wheat cells

Barley stripe mosaic virus particles were localized in ultrathin sections with colloidal gold-labeled specific IgG or antiserum followed by gold-labeled goat anti-rabbit IgG. On the average, 1.5 gold particles were attached per virus rod. A statistical analysis of counts of gold and virus particles showed that the staining procedure was highly reproducible from experiment to experiment and after several independently prepared colloidal gold solutions. The procedure should be useful for the intracellular localization of any protein to which an antibody can be prepared.     

Evaluation of immunoelectron microscopic techniques in the study of basement membrane antigens

 Recent technical advances in immunoelectron microscopy (IEM), including methods of pre- and postembedding IEM and cryoultramicrotomy, have helped to elucidate the precise ultrastructural localization of various basement membrane-related molecules. Our objective was to evaluate the advantages and disadvantages of several different techniques for studying the ultrastructural organization of basement membrane components. We found that, while ”on-surface” immunolabeling of postembedding IEM and cryoultramicrotomy with anti-type IV collagen or anti-laminin-5 antibody clearly demonstrated dense labeling on the lamina densa, preembedding IEM with a 1-nm ultra-small gold probe showed labeling only on the epidermal and/or dermal surfaces of the lamina densa, with no specific gold particles being seen within the lamina densa itself. These results indicate that even ultra-small colloidal gold-labeled antibody fails to penetrate the lamina densa in preembedding IEM. However, labeling with a GB3 monoclonal antibody against laminin-5 was demonstrable with preembedding IEM and cryoultramicrotomy, but not with post-embedding IEM, probably due to a loss of antigenicity. These results confirm the advantages and limitations of these techniques of IEM and emphasize the importance of using different techniques of IEM in determining the precise ultrastructural distribution of basement membrane antigens. Accepted: 30 January 1998     

Immunocytochemical localization of nuclear antigens in the unicellular green alga,Chlamydomonas reinhardtii,processed by cryofixation and freeze-substitution

Summary We describe the preparation of monoclonal antibodies to nuclear antigens in the green alga,Chlamydomonas reinhardtii, and their localization at the light and electron microscope level. Supernatants from hybridomas were screened by the ELISA method and the four antibodies giving the strongest signal were subjected to further analysis. At the LM level immunogold silver staining was used on semi-thick resinless sections. We have examined at the EM level the distribution of these antigens by post-embedding immunocytochemical techniques on sections of conventionally fixed specimens compared to cryofixed and freeze-substituted ones. Enhanced ultrastructural preservation was observed in cells which were cryofixed, freeze-substituted and embedded at –35°C in Lowicryl K4M. Different preparative procedures involving cryofixation and substitution are described. Of the four antibodies three were localized under light and electron microscopy. All three were distributed in the interchromatin space. One of these antigens (QUL4D2, 54 kDa) is also found in the dense fibrillar component and fibrillar centers of the nucleolus.Abbreviations DFC dense fibrillar component - EM electron microscope - FC fibrillar center - GAM5 goat anti-mouse IgM coupled to 5 nm colloidal gold - Ig immunoglobulin - LM light microscope - MAb monoclonal antibody - PAG protein A-gold - PBS phosphate buffered saline - PEG polyethylene glycol     

An Occurrence of Diarrheal Cases Associated with Group C Rotavirus in Adults

Six of the 23 college students who joined a group trip in February of 1991 developed acute nonbacterial gastroenteritis with severe diarrhea. The causal agent was identified as group C rotaviruses by electron microscopy (EM), immune-EM (IEM) and the molecular examinations including polyacrylamide gel electrophoresis (PAGE) and polymerase chain reaction (PCR) on virus particles detected in the extract of watery fecal specimens of the patients. The patients positive for virus isolation showed significant increase in IEM antibody to the isolated virus in their paired sera. These findings suggest that the group C rotavirus is an important etiological agent of diarrhea and may also cause serious food-borne diarrheal disease in adults.     

Immunogold staining method for the light microscopic detection of leukocyte cell surface antigens with monoclonal antibodies: its application to the enumeration of lymphocyte subpopulations

Colloidal gold was used as a marker for the light microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies. Suspensions of peripheral blood leukocytes were first incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. The cells were fixed and cytocentrifuge preparations or smears were made. Granulocytes and monocytes were then labeled by the cytochemical staining of their endogenous peroxidase activity. Lymphocytes reacting with the monoclonal antibody had numerous dark granules around the surface membrane. With electron microscopy, these granules appeared as patches of gold particles. This immunogold staining method proved to be a reliable tool for the enumeration of T-lymphocyte subpopulations in peripheral blood. The results were almost identical to those obtained with immunofluorescence microscopy. The procedure can also be applied on small volumes of capillary blood. This constitutes a good microtechnique for the determination of lymphocyte subsets in children.     

Enhancement of Antigenic Site Detection with Gold Labeled Secondary and Tertiary Antibodies Using the Immunogold-Silver Staining Method

We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 μm frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy.     

Use of protein A in the serum-in-agar diffusion method in immune electron microscopy for detection of virus particles in cell culture

A modified technique using protein A in the serum-in-agar (SIA) method for immune electron microscopy (IEM) was presented. Grids coated with staphylococcal protein A were floated on samples mounted on agar containing 2% antiserum and incubated at 37 C, for 60 min. After washing and staining, the grids were observed in an electron microscope. The effects of protein A on virus detection were evaluated using poliovirus and bovine rotavirus infected cell culture fluids. The results showed that the technique using protein A (PA-SIA) had at least 10-fold higher sensitivity for virus detection than the original SIA. The optimal concentration of protein A was 1 to 10 micrograms/ml for coating the grids to trap virus particles. The PA-SIA method was also compared with immunosorbent electron microscopy (ISEM). The former showed higher or at least the same sensitivity and some advantages in detecting antigen-antibody reaction than the latter method. These results indicate that our PA-SIA method may be superior to other IEM techniques presented previously for the detection and identification of viruses.     

Immunocytochemistry: its evolution and criteria for its application in the study of epon-embedded cells and tissue

The word immunocytochemistry is currently used to describe a number of methods that can be employed to localize antigens within cells by means of antigen-specific antibodies. In this article we will review a number of these methods, including immunofluorescence, immunoperoxidase, avidin-biotin, and colloidal-gold techniques. The advantages and disadvantages of the various methods are discussed, special attention being focused upon immunocytochemical staining of plastic-embedded tissue. Studies on the light microscope level show that embedding tissue in plastic prior to immunoperoxidase staining not only improves visualization of antigen-specific staining but also provides an accurate and efficient means of prescreening tissue for antigen prior to immunocytochemical staining on the electron microscope level. Varying section thickness between 1 and 3 microns does not significantly influence staining, whereas the fixative used to preserve the tissue under study does. On the electron microscope level, the colloidal gold technique appears superior to immunoperoxidase staining. It is both esthetically more pleasing and highly sensitive. Of five different colloidal gold methods tested, the most sensitive is the two-step technique that employs an antigen-specific primary antibody followed by a gold-labeled secondary antibody. Throughout this article, special emphasis is placed on the use of proper controls, both on the light and electron microscope levels. Where possible, such controls should include substitution of specific antiserum with normal serum; the use of antigen-adsorbed antiserum; the use of antisera with specificities for antigens not present in the tissue being studied; the use of tissue previously shown to be stainable for the antigen; and if cultured cells are being studied, the use of a number of cell types that do not contain the antigen.     

Localization of a phytohormone using immunocytochemistry

The localization of cytokinins in corn root tips was investigated using antibodies or antibody fragments directed against dihydrozeatin riboside and labeled with rhodamine or colloidal gold. Roots were sectioned at -30 degrees to -40 degrees for immunofluorescence or freeze-substituted in ethanol or acetone and embedded in plastic for electron microscopy. Meristematic cells surrounding the quiescent center as well as root cap cells were specifically labeled using direct immunofluorescence techniques, whereas cells of the quiescent center did not bind label. Tissue sections treated with colloidal gold-labeled antibody fragments had gold particles widely distributed in the cytoplasm. The results show that the quiescent center is not the major site of cytokinin localization in root tips.     

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35–1.0 m) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 m. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.     

Lectins for electron microscopic distinction of eosinophils from other blood cells

Colloidal gold-labeled soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), Dolichos biflorus agglutinin (DBA), and Griffonia simplicifolia lectin (GS-1) were used for electron microscopic observation of blood cells. Colloidal gold-labeled SBA, HPA, and DBA showed marked deposition on eosinophil granules at all stages of maturation. Gold particles were not deposited on basophils, neutrophils, monocytes, lymphocytes, or other blood cells. Only a few colloidal gold-labeled GS-1 were deposited on eosinophil granules. Eosinophil granules are rich in N-acetyl-D-galactosamine compounds, and the colloidal gold-labeled SBA, HPA, and DBA are useful for electron microscopic detection of eosinophil granules.     

Use of immunogold labelling technique for immunoelectron microscope localization of proteins in Drosophila polytene chromosomes

Using gold labeled antibodies, we developed and tested an immunoelectron microscope (IEM) method for detection of protein localization in Drosophila melanogaster polytene chromosomes. This method is based on procedures widely used for indirect immunofluorescent (IF) staining of salivary gland polytene chromosome squashes. The application of IEM was evaluated by using specific antibodies against proteins earlier localized in both decondensed (interbands and puffs) and compact (bands) regions of polytene chromosomes. In all the experiments, IEM and IF images for homologous chromosome regions were compared. When applied to regions of loose structures, IEM enabled us to localize, with high precision, signals in fine bands, interbands and puffs. There was a good correspondence between immunogold EM and IF data. However, there was no correspondence for dense bands: gold particles were distributed at their boundaries, while the entire bands showed bright fluorescence. This discrepancy probably resulted from a poor penetration of antibodies conjugated to gold particles in the tightly packaged structures. From the results obtained it may by concluded that the IEM method is advantageous for studying the fine protein topography of loose decompacted regions of polytene chromosomes. And this must be taken into consideration when protein localization in polytene chromosomes is performed.     

基于N蛋白单克隆抗体的小反刍兽疫病毒抗体检测胶体金试纸条的研制

本研究旨在建立一种简便、快捷、可直观检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)抗体的检测方法。将pET-32a-N重组质粒转化至大肠杆菌(Escherichia coli) Rosetta(DE3)感受态细胞中进行诱导表达,以纯化的PPRVN蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,间接酶联免疫吸附试验(enzyme-linked immunosorbent assays, ELISA)筛选及亚克隆,获得了抗PPRV N蛋白的单克隆抗体。将PPRV N蛋白分别作为金标抗原及检测线(T线)包被抗原、单克隆抗体作为质控线(C线)包被抗体,组装成检测PPRVN蛋白抗体的胶体金免疫层析试纸条。结果显示：成功获得1株能稳定分泌抗N蛋白抗体的杂交瘤细胞株,命名为1F1;间接ELISA检测1F1腹水效价为1:128000;亚类鉴定结果为IgG1,轻链为kappa链。Westernblotting结果显示,1F1能与PPRV N蛋白特异性结合;间接免疫荧光(indirect immunofluorescent ass...     

Optimizing parameters for correlative immunogold localization by video-enhanced light microscopy, high-voltage transmission electron microscopy, and field emission scanning electron microscopy

Correlative video-enhanced light microscopy, high-voltage transmission electron microscopy, and low-voltage high resolution scanning electron microscopy were used to examine the binding of colloidal gold-labeled fibrinogen to platelet surfaces. Optimal conditions for the detection of large (18 nm) and small (3 nm) gold particles are described.     

Lectin--digoxigenin conjugates: a new hapten system for glycoconjugate cytochemistry.

We report the use of a novel hapten system for lectin cytochemistry. Various lectins conjugated to the steroid hapten digoxigenin (DIG) and monospecific anti-digoxigenin antibodies were applied for the light and electron microscopic detection of glycoconjugates in tissue sections. Both IgG and Fab' anti-DIG antibodies were complexed to particles of colloidal gold and compared to commercially available alkaline phosphatase and horseradish peroxidase conjugated Fab' as general second step reagents. The three different markers performed equally well on paraffin sections whereas the gold-labeled antibodies were superior reagents for semithin and ultrathin sections of Lowicryl K4M embedded tissues. In conjunction with the latter marker, no pretreatment to abolish endogenous enzyme activity was necessary. At the light microscope level, gold signal amplification by the photochemical silver reaction was required. DIG, in contrast to biotin, does not occur in animal tissues thus eliminating the need for blocking reactions prior to lectin incubation. Compared to affinity techniques using glycoprotein-gold complexes as second step reagent the DIG hapten system required smaller amounts of lectins. The staining patterns were indistinguishable from those obtained in other lectin-gold techniques and the specificity of the labeling could be demonstrated in sugar inhibition tests.     

Lectin — digoxigenin conjugates: a new hapten system for glycoconjugate cytochemistry

Summary We report the use of a novel hapten system for lectin cytochemistry. Various lectins conjugated to the steroid hapten digoxigenin (DIG) and monospecific anti-digoxigenin antibodies were applied for the light and electron microscopic detection of glycoconjugates in tissue sections. Both IgG and Fab' anti-DIG antibodies were complexed to particles of colloidal gold and compared to commercially available alkaline phosphatase and horseradish peroxidase conjugated Fab' as general second step reagents. The three different markers performed equally well on paraffin sections whereas the gold-labeled antibodies were superior reagents for semithin and ultrathin sections of Lowicryl K4M embedded tissues. In conjunction with the latter marker, no pretreatment to abolish endogenous enzyme activity was necessary. At the light microscope level, gold signal amplification by the photochemical silver reaction was required. DIG, in contrast to biotin, does not occur in animal tissues thus eliminating the need for blocking reactions prior to lectin incubation. Compared to affinity techniques using glycoprotein-gold complexes as second step reagent the DIG hapten system required smaller amounts of lectins. The staining patterns were indistinguishable from those obtained in other lectin-gold techniques and the specificity of the labeling could be demonstrated in sugar inhibition tests.     

Multiple correlative immunolabeling for light and electron microscopy using fluorophores and colloidal metal particles.

Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric microscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and alpha-actinin bands of skeletal muscle tissue and also actin and alpha-actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM.     

A Classification of Virus Particles Based on Morphology

Recent improvements in electron microscope techniques which allow the study of virus fine structure have permitted the grouping of many viruses on a purely morphological basis. Briefly the techniques used in electron microscopy for the study of viruses are reviewed and the symmetry properties of virus particles as revealed by negative staining are discussed somewhat more fully.Finally, virus particles are grouped on two bases, firstly the site of formation of the virus within the cell as seen by thin sectioning techniques, and secondly the symmetry property of the virus as seen by negative staining. Consideration of the groupings obtained in this way reveals that the biochemical and physical properties of a virus can be deduced from the readily established morphological characteristics.