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PubMed1990

Ultrastructural comparative distribution of carbohydrates in human tracheal and frog palate mucosa using neuraminidase and lectin-colloidal gold complexes.

We have compared, at the ultrastructural level, the carbohydrate structure of glycoconjugates of the different types of secretory cells of the human tracheal mucosa (HTM) and the frog palate mucosa (FPM), proposed as a model for studying bacterial adherence to mucus-coated respiratory epithelium. In addition to reactivity with Concanavalin A and Lens cullinaris agglutinin, reactivity of Epon-embedded HTM and FPM secretory granules was studied by transmission electron microscopy using neuraminidase-gold complex and colloidal gold-adsorbed lectins with affinity for sugar residues of human mucins, namely the following: Helix pomatia, Lotus tetragonolobus, Ricinus communis II, Wheat germ and Limax flavus agglutinins. The affinity of HTM and FPM mucous and serous cells for the different colloidal-gold complexes was very similar, however Limax flavus agglutinin labelled only HTM and not FPM secretory granules. The FPM mucous and serous secretory granules were nevertheless intensely labelled by the neuraminidase-gold complex, demonstrating the presence of sialic acid residues. The close ultrastructural and histochemical similarities between HTM and FPM suggest that the FPM may be a valuable model for studying the specific interaction between microbial lectins and mucus glycoproteins in the bacterial adherence phenomenon.

Animals↗
PubMedDate not supplied

Effect of colloidal gold size on the conformational changes of adsorbed cytochrome c: probing by circular dichroism, UV-visible, and infrared spectroscopy.

The conformational changes of bovine heart cytochrome c (cyt c) induced by the adsorption on gold nanoparticles with different sizes have been investigated by electronic absorption, circular dichroism (CD), and Fourier transform infrared spectra. The combination of these techniques can give complementary information about adsorption-induced conformational changes. The results show that there are different conformational changes for cyt c adsorbed on gold nanoparticles with different sizes due to the different interaction forces between cyt c and gold nanoparticles. The colloidal gold concentration-dependent conformation distribution curves of cyt c obtained by analysis of CD spectra using the singular value decomposition least-squares method show that the coverage of cyt c on the gold nanoparticles surface also affects the conformational changes of the adsorbed cyt c.

Adsorption↗
PubMedDate not supplied

Colloidal gold labeling of sections and cell surfaces.

Colloidal gold labeling techniques can be simplified enough to become practical for diagnostic use in a hospital electron microscopy laboratory. Methods found to be practical and reproducible are presented, including production of colloidal gold reagents, polar embedding for gold labeling of thin sections, and use of silver enhancement on semithin sections. Methods for cell surface labeling are emphasized; they are technically straightforward and can be applied to investigation of multiple diagnostic problems.

Cell Membrane↗
PubMed2003

Reagentless glucose biosensor based on direct electron transfer of glucose oxidase immobilized on colloidal gold modified carbon paste electrode.

The direct electrochemistry of glucose oxidase (GOD) adsorbed on a colloidal gold modified carbon paste electrode was investigated. The adsorbed GOD displayed a pair of redox peaks with a formal potential of -(449+/-1) mV in 0.1 M pH 5.0 phosphate buffer solution. The response showed a surface-controlled electrode process with an electron transfer rate constant of (38.9+/-5.3)/s determined in the scan rate range from 10 to 100 mV/s. GOD adsorbed on gold colloid nanoparticles maintained its bioactivity and stability. The immobilized GOD could electrocatalyze the reduction of dissolved oxygen and resulted in a great increase of the reduction peak current. Upon the addition of glucose, the reduction peak current decreased, which could be used for glucose detection with a high sensitivity (8.4 microA/mM), a linear range from 0.04 to 0.28 mM and a detection limit of 0.01 mM at a signal-to-noise ratio of 3sigma. The sensor could exclude the interference of commonly coexisted uric and ascorbic acid.

Biosensing Techniques↗
PubMed1980

X-ray microanalysis of colloidal-gold-labelled lysosomes in rat liver sinusoidal cells after incubation for acid phosphatase activity.

The lysosomal apparatus of the Kupffer and endothelial cells of the sinusoidal lining of the rat liver was found to take up colloidal-gold particles with a mean diameter of 5 nm, prepared according to a modified method. After incubation of the glutaraldehyde-perfusion-fixed tissue in a lead-containing medium for the demonstration of acid phosphatase activity, a reaction product was observed in the gold-loaded lysosomes. By X-ray microanalysis of such lysosomes, the presence of osmium, gold and lead was detected qualitatively in the unstained sections from the tissue, which after the incubation had been post-fixed with an OsO4-solution to which K4Fe(CN)6 had been added to enhance the contrast. The quantitative computer-assisted processing of the X-ray microanalytical data from such lysosomes enabled to determine the gold-to-lead ratio and the individual gold and lead peak intensities derived from both the M chi and L chi values in the spectra. On the basis of these results and those obtained similarly in control lysosomes containing either only gold or only lead phosphate precipitate, it was found that only the L chi values were reliable, whereas the M chi values from the same lysosomal spectra were unrealistic, due to deconvolution problems in the computer programs applied. Based upon the L chi values it was found that among the population of lysosomes in single Kupffer cells, studied after a 60-min interval between the injection of the gold colloid and fixation, three types of lysosomal contents could be quantitated by X-ray microanalysis, viz. one type with only gold, one with only lead, one with gold and lead, in various ratios. This quantitative approach might make it possible to detect variations in lysosomal composition associated with ageing.

Acid Phosphatase↗
PubMed1977

Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.

A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.

Animals↗
PubMed2006

A chemiluminescent metalloimmunoassay based on silver deposition on colloidal gold labels.

A sensitive chemiluminescent (CL) immunoassay of human immunoglobulin (IgG) which combined the inherent high sensitivity of CL analysis with the dramatic signal amplification of silver precipitation on colloidal gold tags was developed. First, the sandwich-type complex was formed in this protocol by the primary antibody immobilized on the polystyrene wells, the analyte in the sample, and the secondary antibody labeled with colloidal gold. Second, the colloidal gold was treated by an Ag(+) reduction solution, which resulted in the catalytic precipitation of silver on the surface of colloidal gold. Third, a large number of Ag(+) were oxidatively released in HNO(3) solution from the silver metal anchored on the sandwich-type complexes and then the human IgG was indirectly determined by a sensitive combined CL reaction of Ag(+)-K(2)S(2)O(8)-Mn(2+)- H(3)PO(4)-luminol. The chemiluminescence intensity depends linearly on the logarithm of the concentration of human IgG over the range of 0.02-50ngml(-1) and detection limit (3sigma) is 0.005ngml(-1) (i.e., approximately 3x10(-14)M, 3amol in 100-mul sample). This assay has been successfully applied to the determination of human IgG in human serum samples and showed great potential for numerous applications in immunoassay.

Animals↗
PubMed1992

Detection of fecal blood by colloidal gold agglutination using an anti-human hemoglobin monoclonal antibody.

Monoclonal antibodies to human hemoglobin were produced and a colloidal gold agglutination method has been developed for detection of fecal occult blood. Since hemoglobin is composed of the tetramer, alpha 2 beta 2, a single monoclonal antibody-labeled colloidal gold can agglutinate with hemoglobin. The lowest detectable hemoglobin concentration was 0.5 micrograms/ml. A total of 785 fecal samples were determined using colloidal gold agglutination and compared with latex agglutination. The colloidal gold agglutination detected blood in 75 samples, whereas latex agglutination detected blood in 76 samples, and among them 70 were positive in both methods. Overall agreement between the two methods was 98%.

Agglutination↗
PubMed1984

Colloidal gold and biotin-avidin conjugates as ultrastructural markers for neural antigens.

The use of two new reagents, biotin-avidin conjugates and colloidal gold particles, for ultrastructural immunocytochemistry has been reviewed, and their potential for immunocytochemistry in the central nervous system has been discussed. Although these compounds have been used very little in the C.N.S. in the past, with their high sensitivity, versatility, and potential for simultaneous localization of two antigens with two electron dense markers, these reagents may prove very useful for answering a number of neural questions. Colloidal gold adsorbed to immunoglobulins can be used for light microscopic observation, and owing to its high electron density and ability to readily adsorb to immunoglobulins, protein A, and antigen-carrier complexes, it also serves as an excellent immunomarker at the ultrastructural level. Several procedures have been described for the use of pre- and post-embedding immunostaining with colloidal gold particles. Different size gold particles, adsorbed to different antibodies can be used for pre- and post-embedding staining of different antigens in the same tissue. While at the light microscopic level only the large particles are visible as a pink haze, a fluorescent step can be added to allow easy identification and characterization of neurones labelled with any size colloidal gold, even if the pink colour of the gold is not visible. These same sections can subsequently be studied ultrastructurally. Unlike enzymatic methods, gold particles have less tendency to obscure ultrastructure. Biotin can be easily conjugated to immunoglobulins and, due to its high affinity for avidin (10(15) M-1) which has four biotin binding sites, can be localized with avidin markers, or biotin-avidin-marker complexes. Some procedures such as the avidin-biotin-peroxidase complex are extremely sensitive. Biotin and avidin can be bound to a number of different markers including peroxidase, colloidal gold, and ferritin. Photomicrographs from our hypothalamic work have been used to illustrate the histological results of some of the procedures described.

Animals↗
PubMed1990

Small colloidal gold conjugated to Fab fragments or to immunoglobulin G as high-resolution labels for electron microscopy: a technical overview.

Fab-colloidal gold labelling in conjunction with negative staining and high-resolution electron microscopy was used for targeting single protein units in regular arrays. These were bacteriophage T4 polyheads with Fab-Au2.5, and a specific antibody binding site on the haemagglutinin polypeptide of influenza virus with Fab-Au3, Fab-Au2.5, and Fab-Au1-2. For the latter, IgG-Au3 was also used. Experimental details are summarized to provide generally applicable methods for the preparation of small gold colloids Fab-Au and of labelling. The putative mechanism of protein-gold complex formation and adsorption to preferred sites on Fab and IgG, most probably to sulphur-rich regions, is discussed. The influence of pH during complex formation was found to be of minor importance in the samples investigated. Reported experimental details and our own experiences suggest that the importance of a protein's pI relative to its optimum gold complexing pH critically depends on the nature of the protein in question rather than being of general importance for protein-gold complex stability.

Bacterial Proteins↗
PubMed2001

Characterization of silane-modified immobilized gold colloids as a substrate for surface-enhanced Raman spectroscopy.

Immobilized gold colloid particles coated with a C-18 alkylsilane layer have been characterized as a substrate for surface-enhanced Raman scattering (SERS) studies of adsorption onto hydrophobic surfaces. Atomic force microscopy images, optical extinction spectra, and SERS measurements are reported as a function of accumulation of gold colloid on glass. As the metal particles become increasingly aggregated on the surface, the SERS enhancement increases until the plasmon resonance shifts to wavelengths longer than the excitation laser. The gold colloid substrates are stable and exhibit reproducible SERS enhancement. When octadecyltrimethoxysilane is self-assembled over the gold, the metal surface is protected from exposure to solution-phase species, as evidenced by the inhibition of chemisorption of a disulfide reagent to the overcoated gold surface. The results show that interactions with gold can be blocked by a silane layer so as not to significantly influence physisorption of molecules at the C-18/solution interface. The SERS enhancement from these C-18-overcoated gold substrates is reproducible for different films prepared from the same colloidal suspension; the substrates are also stable with time and upon exposure to laser irradiation.

Journal Article↗
PubMed1996

[Application of monoclonal antibody immuno-colloide gold probe in the study of porphyromonas gingivalis].

Immuno-electron localization of McAb BGG9 was specific for Porphyromonas gingivalis labeled with colloide gold. The monoclonal antibody immuno-colloide gold probe reacted with Porphyromonas gingivalis and 5 species of black pigmented bacteroides groups. The bacteria were fixed and embbed in Epon. sections were examined under transmission electron microscope, and the gold granular was observed near Porphyromonas gingivalis cell membrane. Our conclusion suggests that monoclonal antibody immuno-colloide gold probe is useful tool for studying Porphyromonas gingivalis.

Antibodies, Bacterial↗
PubMed2000

Silver electrodeposition catalyzed by colloidal gold on carbon paste electrode: application to biotin-streptavidin interaction monitoring.

A new electrochemical method to monitor biotin-streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin-streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at + 0.08 V was recorded in NH3 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H2SO4 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at -0.18 V for 2 min and anodic stripping voltammetry were carried out in NH3 1.0 M containing 2.0 x 10(-5) M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25 x 10(-15) to 2.24 x 10(-12) M and a limit of detection of 2.0 x 10(15) M were obtained.

Biotin↗
PubMed1996

[Detection of anti-Treponema pallidum antibodies with colloidal gold immunoprobes].

BACKGROUND: The aim of this study was to evaluate the usefulness of colloidal gold probes widely used in histochemistry and electronic microscopy for the serologic diagnosis of syphilis. METHODS: One hundred eighty-nine sera which had been submitted to the traditional fluorescent trigenemic absorption (FTA-ABS) test and had later been tested with the new colloidal gold probe method (IAT-ABS). RESULTS: Out of a total of 103 positive samples by FTA-ABS all were positive when tested by gold immunoprobes (sensitivity 100%). Eighty-six samples which were negative by immunofluorescence were all negative by IAT-ABS (specificity 100%). CONCLUSIONS: The new method of colloidal gold probes with the current microscopy is useful for confirming serologic diagnosis of syphilis.

Adolescent↗
PubMed1989

Factors affecting efficiency of colloidal gold staining: pH-dependent stability of protein-gold conjugates.

The preparation of stable protein-gold conjugates is important in the quantitative colloidal gold staining. The protein adsorption onto colloidal gold particles and pH-dependent stability of protein-gold conjugates were examined using albumin and fibrinogen as model proteins. Albumin stabilized gold sols at pH values higher than 5.0. The amount of albumin necessary to stabilize gold sols was lowest at pH 5.6 and slightly increased as the pH of the adsorption medium was increased. Albumin was also able to stabilize gold particles even at acidic pH values ranging from 3.5 to 4.5, if a sufficient amount of albumin was added. Stable fibrinogen-gold conjugates were obtained at pH values above 6.5. Unlike albumin, the same amount of fibrinogen was able to stabilize gold sols as pH was increased from 7 to 10. The stability of the prepared protein-gold conjugates was very sensitive to the pH of the storing medium. The protein-gold conjugates were least stable at the isoelectric pH of the protein. The results suggest that protein-gold conjugates should be prepared at pH where conjugates are most stable, instead of pH close to the isoelectric point.

Colloids↗
PubMed1986

Silver-enhanced colloidal gold probes as markers for scanning electron microscopy.

Silver enlargement of small colloidal gold particles has been extensively used for the light microscopical visualization of gold probes. Very recently, a few investigators have employed physical developers in electron microscopy (both pre-embedding and on-grid staining methods). We now demonstrate that physical development of small colloidal gold particles advantageously can be exploited for labelling biological surfaces in scanning electron microscopy. This novel application of silver enhancement of colloidal gold particles is characterized by a high detection efficiency. Thus, specimens are labelled with small gold probes affording high immunocytochemical efficiency but being impossible to detect with the present scanning microscopes. These particles are subsequently scanning electronmicroscopically visualized by silver enhancement.

Animals↗