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At least 307 records · Page 17Linked to original sources
PubMed1989

Immunoelectron microscopy of Proteus vulgaris by the plasma polymerization metal-extraction replica method: differential staining of flagellar (H) and somatic (O) antigens by colloidal golds.

Flagellar (H) and somatic (O) antigens of Proteus vulgaris were differentially stained with antibodies coupled to different sizes of colloidal gold particles, and the distribution of these antigens was visualized by the plasma polymerization metal-extraction replica (PMR) method. The H antigen, labeled with 5 nm colloidal gold, was almost exclusively located on the flagella, whereas the O antigen, labeled with 10 nm colloidal gold, was almost exclusively located on the bacterial body. The marker gold particles were clearly observed as electron-dense particles on the relatively low contrast background of three-dimensional replica image of the flagellated bacteria. Thus, the PMR method may prove to be a useful tool for studying the localization of multiple substances on the cell surface, at a high resolution and in three dimensions. The diameter of the flagella measured by the replica method was about 15 nm, close to the value obtained by negative staining (16 nm). When treated with anti-flagellar (H) factor serum and protein A-gold, the diameter of flagella was significantly increased to about 35 nm. This increase in diameter was presumably caused by binding of immunoglobulins to H antigens of flagella.

Antigens, Bacterial↗
PubMed2005

[A colloidal gold immunochromatographic assay for detecting p38 antigen of Schistosome japonicum].

OBJECTIVE: To establish a colloidal gold immunochromatographic assay (GICA) for detecting Schistosome japonicum (Sj). METHODS: Eight monoclonal antibodies (mAbs) against Sj p38 antigen (1A6, 3C4, 3D12, 6F10, 6G12, 9H6, 9G7 and A5H) prepared previously were purified by protein-G affinity chromatography. The affinity constant (K(aff)) was determined by indirect enzyme-linked immunosorbent assay. All the mAbs were labeled with horseradish peroxidase by sodium oxidation method and the mAb pairs with high affinity and stability were identified according to their optical density at 450 nm (OD450). Four mAbs (1A6, 6G12, 9H6 and 9G7) were chosen for colloidal gold labeling. GICA was then performed by further optimization of the labeling and the conditions were determined. The sera of mice at different infection stages were examined with GICA dipstick, with the sera collected before infection and those of Toxoplasma gondii-infected mice as negative controls. RESULTS: The purity of the 8 mAbs was higher than 95% with K(aff) ranging from 2.8x10(-10) to 1x10(-8) mol/L. 9G7 coating (2.5 mg/ml) as the capture antibody and detection with 1A6 (diluted at 1:4) as the labeling antibody was determined as the best reaction model. With this combination, the positivity rates of the detection were 40%, 50%, 60% and 80% for mouse sera collected at 3, 4, 5 and 6 weeks after Schistosome japonicum infection, respectively, without positive results for the negative control samples. CONCLUSION: GICA established in this study is characterized by simplicity, rapidity and good sensitivity, and the prepared rSjP38 dipstick can test the circulating antigen SjP38 in early stage of infection.

Animals↗
PubMed2005

Silver-enhanced colloidal gold metalloimmunoassay for Schistosoma japonicum antibody detection.

A silver-enhanced colloidal gold metalloimmunoassay has been proposed for the determination of Schistosoma japonicum antibody (SjAb) in rabbit serum. The adult worm antigen of S. japonicum (SjAg) was adsorbed passively on the walls of a polystyrene microwell and then reacted with the desired SjAb. The colloidal gold-labeled goat anti-rabbit IgG secondary antibody was adsorbed on the walls of the polystyrene microwells through the reaction with SjAb, followed by the silver enhancement process, dissolution of silver metal atoms in an acidic solution, and determination of dissolved silver ions by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. Assay conditions were optimized, including the reaction time of SjAg with SjAb, the interaction of SjAb with the colloidal gold-labeled secondary antibody, the dilution ratio of the colloidal gold-labeled secondary antibody and the silver enhancement time. The integration of the anodic stripping peak current depended linearly on the SjAb logarithmic concentration over the range of 6.4 ng/ml to 100 microg/ml. A detection limit as low as 3.0 ng/ml SjAb was achieved, which was better than the piezoelectric body acoustic wave sensor (detection limit of 7.2 microg/ml) and the renewable amperometric immunosensor (detection limit of 0.36 microg/ml). Rabbit serum samples with various degrees of infection were analyzed, and the results demonstrate that the proposed method meets the requirements of clinical analysis.

Animals↗
PubMed1981

Cell surface labelling with gold colloid particulates: the use of avidin and staphylococcal protein A-coated gold in conjunction with biotin and fc-bearing ligands.

Procedures for preparing gold colloid particles stabilized with either avidin or protein A are described. Methods of using these general utility tracers for localizing biotinylated and fc bearing immunoglobulins are outlined and, as examples of the way in which these methods can be applied, procedures for identifying epidermal growth factor receptors and surface fibronectin on ovarian granulosa cells are described.

Animals↗
PubMed1993

Colloidal gold particles as an incompressible atomic force microscope imaging standard for assessing the compressibility of biomolecules.

Colloidal gold particles have multiple uses as three-dimensional atomic force microscopy imaging standards because they are incompressible, monodisperse, and spherical. The spherical nature of the particles can be exploited to characterize scanning tip geometry. As uniform spheres, colloidal gold particles may be used to calibrate the vertical dimensions of atomic force microscopy at the nanometer level. The monodisperse and incompressible nature of the gold can be used to characterize the vertical dimensions of coadsorbed biomolecules. Simultaneous measurements of gold with tobacco mosaic virus show that, at the same applied vertical force, the tobacco mosaic virus is undamaged by blunt tips but is compressed or disintegrated under sharper scanning styli, suggesting that specimen degradation is partly a pressure-dependent effect.

Biophysical Phenomena↗
PubMed1991

Binding and endocytosis of thrombospondin and thrombospondin fragments in endothelial cell cultures analyzed by cuprolinic blue staining, colloidal gold labeling, and silver enhancement techniques.

We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.

Ammonium Chloride↗
PubMed1985

Labelling of colloidal gold with protein A. A quantitative study.

Colloidal gold complexes with protein A are extensively used in immunocytochemistry as secondary reagents for the localization of antigens. However detailed information on the process and extent of adsorption of protein A onto gold particles, the optimal condition of preparation and the stability of such complexes are lacking. The adsorption isotherm of 125I-protein A onto gold particles (11.2 nm in diameter) was studied quantitatively with gold sols buffered at pH 4-7. At low coverage of the particles, the isotherm was independent of pH. However in the presence of a large excess of protein A, the highest coverage was obtained with a gold sol buffered at pH 5.1, the isoelectric point of the protein. The association constant was decreased at high coverage of the particles. Maximum binding of the complex to immobilized IgG occurred with particles labelled with at least 9 molecules of protein A. The complex was stable under storage with up to 12 molecules adsorbed per particle. At high coverage (26 molecules per particle), a progressive loss of protein A was observed. The optimum condition for preparing the complex are reported.

Adsorption↗
PubMed1985

Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis.

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.

Asialoglycoproteins↗
PubMed1991

An electrophoretic method for selection of conditions for production of electrophoretically uniform protein colloidal gold complexes.

An electrophoretic method was developed to determine the conditions required for production of electrophoretically uniform protein-colloidal gold complexes from monodisperse colloidal gold (Au) and electrophoretically uniform protein. The method is based on the electrophoretic migration of protein-Au complexes in agarose. The results demonstrate that two variables, the pH of adsorption and the quantity of protein added, can be manipulated to vary the electrophoretic mobility of the resulting protein-Au complexes. Thus, agarose gel electrophoresis can be used to select the pH of adsorption and the quantity of protein required to produce electrophoretically uniform protein-Au complexes. This new electrophoretic mobility test can be used in place of or in addition to the classical procedure of Zsigmondy and its many variations, both visual and spectrophotometric. The procedure described is also useful for electrophoretic comparison of small quantities of various protein-Au samples.

Adsorption↗