Simplified purification and testing of colloidal gold probes

A novel efficient method for purifying and testing colloidal gold probes has been developed. The method consists of concentrating colloidal gold particles conjugated to IgG or protein A in dialysis bags over silica gel and purifying them by gel chromatography on small columns of Sephacryl S-400. Fractions collected are tested by paper immunocytochemical models. Comparisons to gold probes purified by conventional ultracentrifugation documents that ultrastructural staining intensities and total yield of gold probes is the same, but that the chromatographically purified gold probes are less prone to aggregation or clumping. The method has been extensively used for preparing conjugates of 5, 10 or 15 nm gold particles with antirabbit immunoglobulins but has also been exploited for preparing streptavidin-gold conjugates, protein A-gold conjugates and antirabbit immunoglobulin-silver conjugates.     

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.     

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

Summary 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 electronmicro-scopically visualized by silver enhancement.Presented in part at the International Symposium on Biological Regulation of Cell Proliferation, 9th International Chalone Conference, Milano, Italy, March 3–6, 1986     

Analysis of colloidal gold probes by isoelectric focusing in agarose gels

Colloidal gold particles of different size (3-20 nm in diameter) were prepared by tannic acid-citrate and citrate reduction methods. From these colloids, different probes were prepared using sheep anti-rabbit antiserum, sheep anti-rabbit IgG, bovine serum albumin, polyethylene glycol, and protein A as the primary stabilizers and polyethylene glycol and/or bovine serum albumin as secondary and tertiary stabilizers, in different combinations. The probes were analyzed by isoelectric focusing in agarose gels, which allow the migration of particles in the size range 3-20 nm. (P. Sewer and S. J. Hayes, 1986, Anal. Biochem. 158, 72-78). Isoelectric focusing revealed that the surface charge of colloidal gold probes is dependent upon the size of the gold particle, the reduction method used, the primary ligand, and the pH at which this is adsorbed, as well as upon the secondary and tertiary stabilizers used. It is proposed that such differences in surface charge may underlie the different results which may sometimes be observed in colloidal gold labeling, especially when novel ligands are used.     

DNA probes on chip surfaces studied by scanning force microscopy using specific binding of colloidal gold

Single-stranded DNA was covalently bound on chip surfaces using two different silanization procedures. The resulting surfaces were characterized by fluorescence and scanning force microscopy using sequence-complementary DNA molecules with labels. Colloidal gold (30 nm) was used as the topographic label. Scanning force microscopy revealed the individual labels on the surface and their distribution. Steps of silane layers or DNA-modified surfaces prepared using an elastomeric mask provided internal controls for comparison of modified with unmodified surfaces.     

Protein blotting: detection of proteins with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes

Methods to detect "native" proteins immobilized on nitrocellulose membranes in spot tests or on blots prepared from polyacrylamide slab gels after electrophoretic separation are described. Gold sols were found to be useful as general stains for proteins: They are polychromatic, yield an indelible record, and are complementary to india ink as protein stains because these two stains have different sensitivities for a number of proteins tested. For detection of wheat germ lectin (WGL)-binding glycoproteins, avidin-peroxidase was an effective enzyme probe, because the glycoportion of the avidin moiety possesses binding affinity to WGL. Glycocomponents in human parotid saliva were detected with this probe and with the following biotin-conjugated lectins as intermediary probes: soybean lectin, Bandeiraea simplicifolia lectin, Lotus tetragonolobus lectin, and kidney bean lectin. Autoclaving blots prior to probing eliminated endogenous peroxidase activity. Concanavalin A and WGL were separated by isoelectric focusing and detected on blots with horseradish peroxidase and avidin-peroxidase, respectively. The versatility of the biotin/avidin system was used to detect other lectins on similar blots using biotin-conjugated glycoproteins as intermediary probes: Helix pomatia lectin and B. simplicifolia lectin were detected with biotinyl neoglycoproteins, and kidney bean lectin with biotin-conjugated components of parotid saliva.     

Spectroimmunochemistry using colloidal gold bioconjugates

Using surface-enhanced infrared absorption (SEIRA) spectroscopy of dry films of colloidal gold (CG) bioconjugates with protein A, it is shown that certain characteristic bands of the protein (e.g., amide I, amide II and some other vibration modes) are essentially affected by the metal surface. Thus, the method may be used for controlling the quality of such bioconjugates. Moreover, it is demonstrated that the biospecific reaction of protein A attached to CG particles with human immunoglobulin G (IgG) results in further essential changes in SEIRA spectra, providing a means for an easy and rapid IR spectroscopic detection of biospecific immunochemical interactions (i.e., spectroimmunochemistry). The results obtained can form a basis for developing test systems for detecting various biospecific interactions.     

Diffusing colloidal probes of protein and synthetic macromolecule interactions

A new approach is described for measuring kT and nanometer scale protein-protein and protein-synthetic macromolecule interactions. The utility of this method is demonstrated by measuring interactions of bovine serum albumin (BSA) and copolymers with exposed polyethyleneoxide (PEO) moieties adsorbed to hydrophobically modified colloids and surfaces. Total internal reflection and video microscopy are used to track three-dimensional trajectories of many single diffusing colloids that are analyzed to yield interaction potentials, mean-square displacements, and colloid-surface association lifetimes. A criterion is developed to identify colloids as being levitated, associated, or deposited based on energetic, spatial, statistical, and temporal information. Whereas levitation and deposition occur for strongly repulsive or attractive potentials, association is exponentially sensitive to weak interactions influenced by adsorbed layer architectures and surface heterogeneity. Systematic experiments reveal how BSA orientation and PEO molecular weight produce adsorbed layers that either conceal or expose substrate heterogeneities to generate a continuum of colloid-surface association lifetimes. These measurements provide simultaneous access to a broad range of information that consistently indicates purely repulsive BSA and PEO interactions and a role for surface heterogeneity in colloid-surface association. The demonstrated capability to measure nonspecific protein interactions provides a basis for future measurements of specific protein interactions.     

Labelling of colloidal gold with IgE

Summary Immunocytochemical markers prepared by labelling colloidal gold with antibodies are gaining wide acceptance both in transmission and scanning electron microscopy. However, detailed information on the process and extent of adsorption of IgG and IgE in particular are still lacking. The adsorption isotherm of mouse monoclonal ¹²⁵I-IgE antibovine milk -lactoglobulin was studied quantitatively with colloidal gold buffered at pH 6.1–8.8 (28 nm in particle diameter). At low coverage of the particles (5 molecules per particle), the isotherm was independant of pH. In the presence of a large excess of IgE, the highest coverage was obtained at pH 6.1 near the pI of IgE (5.2–5.8). The binding constants were higher at low coverage (side-on adsorption) than at high coverage where desorption was observed. IgE-Au markers were unreactive towards the immobilized antigen and did not bind to receptors for IgE of rat basophilic leukemia cells (RBL-1). The reactivity of immobilized anti-IgE antibodies with IgE-Au markers increased as a function of particle coverage. Mappings of RBL-1 cell membrane IgE receptors was achieved by incubating successively IgE-sensitized RBL-1 cells with anti-IgE antibodies and a protein A-gold marker at 4°C. Surface clusters developed when the cells were incubated at 37°C.     

Labelling of colloidal gold with protein

Summary Markers prepared by labelling colloidal gold with macromolecules such as lectins, antibodies and protein A are gaining wise acceptance both in transmission and scanning electron microscopy. However detailed information on the process and extent of adsorption of macromolecules onto gold particles are still lacking. The adsorption isotherm of protein onto gold particles was studied quantitatively using goat -lactoglobulin (L) tritiated in vivo. When this protein was modified chemically by iodination with ¹²⁵I, the adsorption isotherm was not significantly different (Langmuir type for monolayer). In the presence of saturating amount of L, a maximum of 13–14 molecules was adsorbed per particle of 12 nm in diameter for a theoretical maximum of 20 (compact monolayer). Ellipsometric measurements on nickel-coated slides indicated that L was adsorbed onto metallic surfaces as a compact monolayer. The molecules were irreversibly adsorbed on gold particles, kept to a large extent their capacity to bind anti-L antibodies and could not be displaced by polyethylene glycol, a stabilizer commonly used in the preparation of gold markers. Only markers labelled with more than 5 L molecules per particle could be completely bound by immobilized anti-L antibodies. Preliminary data indicated that the energetics of adsorption of L onto colloidal gold was in agreement with that expected from the mutual interaction of surface and adsorbate.     

分析紫杉醇与三尖杉宁碱的简易方法

以粒径单分散聚苯乙烯反相色谱填料为色谱柱固定相,即采用MKF-DK-RP型分析柱(300mm×7.8mm,8μm),流动相为乙腈-水溶液,流速为1.0mL/min,检测波长为229nm,建立了一种分析紫杉醇与三尖杉宁碱的简易方法。结果表明,本方法可使紫杉醇与三尖杉宁碱达到有效分离,且紫杉醇的检测浓度在0.0625~2.0mg/mL范围内线性关系良好(r=0.9999)。本方法简单,重复性好。     

氨苄青霉素标记的胶体金颗粒的制备及活性研究

采用微波加热法制得15 nm的胶体金颗粒,并将小分子抗原氨苄青霉素分别以戊二醛和碳化二亚胺为偶联剂与牛血清蛋白偶联制成全抗原,再通过最佳标记量的确定分别将氨苄青霉素、戊二醛法全抗原、碳化二亚胺法全抗原同胶体金进行结合,红外检测和杯碟法的抑菌试验表明采用仅有戊二醛作为偶联剂的全抗原能够很好地与胶体金结合,并保持良好抗原活性。     

Application of colloidal gold to ultrastructural studies

Progress in recognition of structure and function in a variety of cells has always been linked to development of new research techniques. Beyond doubt, immunocytochemical techniques belong to such new methods. However, the techniques had not been widely applied to electron microscopy until colloidal gold was introduced as a label. This paper presents the pre-embedding and the post-embedding techniques and the applied markers, which are used in immunocytochemical techniques at the ultrastructural level. The potential of colloidal gold techniques is discussed. Particular attention is paid to methods of reaction amplification. Application of gold is illustrated with our own results.     

Interactions of lectins with specific cell types in toad urinary bladder. Surface distribution revealed by colloidal gold probes and label fracture

Colloidal gold probes were used in conjunction with pre-embedding labeling and label-fracture to show the plasma membrane distribution of Helix pomatia lectin (HPL) and wheat germ lectin (WGL) binding sites on different epithelial cell types of toad urinary bladder. Mitochondria-rich cells were virtually unlabeled with HPL, but showed a strong affinity for WGL. Granular cells were weakly labeled with WGL but had a variable affinity for HPL. Strongly labeled granular cells were arranged in either chains or clusters that were surrounded by poorly-stained granular cells. By label-fracture, the distribution of gold-labeled lectins was related to other membrane features seen in freeze-fracture. Neither HPL nor WGL binding sites appeared to be specifically related to the large intramembrane particles that characterize granular cells, or to the rod-shaped intramembrane particles that are a feature of membranes of mitochondria-rich cells. The preferential lectin binding affinity of these functionally distinct cell types provides an important starting point for their isolation and the characterization of their plasma membranes. Furthermore, the label-fracture approach can now be used to examine the plasma membrane modifications that occur in these cells under different physiologic conditions affecting epithelial transport processes.