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Complexes of triiodothyronine-bovine serum albumin-colloidal gold were specifically internalized in human peripheral leukocytes after 5 min of incubation. The specificity was partially lost after a longer incubation.
SELEX procedure is a methodology in which single stranded oligonucleotides are selected from a wide variety of sequences based on their interaction with a target molecule. We have designed a novel SELEX methodology using colloidal gold to select high affinity single stranded DNA aptamers against Leishmania infantum KMP-11. Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell membrane of kinetoplastid parasites. Although its function is not known, the fact that KMP-11 is a cytoskeleton-associated protein suggests that it may be involved in mobility or in some other aspects of the flagellar structure. We have isolated a single stranded DNA aptamer population that binds specifically to L. infantum KMP-11. This population has been characterized in a series of in vitro experiments suggesting that it may be used as a powerful tool to further investigate the role of KMP-11 during Leishmania development and/or as a diagnostic tool in Leishmania infection.
Results of studies demonstrating the identification of the interleukin-2 receptor (IL-2R; i.e., anti-Tac) on the membrane ultra-structure of human leukemic T cells with an antibody carrying an electron dense colloidal gold microsphere (e.g., immunogold) that was visualized using a scanning electron microscope (SEM) are reported. Our IL-2R model system employed HTLV-1 retrovirus-infected lymphoblastoid cells of the long-term human leukemic T cell line HUT-102B2. The presence of the IL-2R on these cells was defined using a double antibody procedure that employed as the primary antibody a purified mouse monoclonal anti-Leu-IL-2R antibody (mIgGlk, anti-Tac, CD25), and used as the secondary antibody a goat anti-mouse IgG (gamma-chain specific) antibody that had been covalently bonded to a 40 nm colloidal gold particle. More than 95% of the HUT-102B2 were IL-2R+, and there was a uniform distribution of the IL-2R over the surface of the cells. Corresponding controls were employed in all examinations and included IL-2R- Jurkat human leukemic T cells and isotype identical immunoglobulins. The primary and secondary antibody reagents contained whole human serum and bovine serum albumin, and there was no evidence of the non-specific binding of these antibodies. These studies are the first to demonstrate the presence of a lymphokine receptor on the surface architecture of a cell. We anticipate no difficulty in applying the immunogold/SEM technology to define both normal and malignant cell membrane receptors for other cytokines.
Rat hepatic sinusoidal cells absorption of native high-density lipoproteins was studied by electron microscope. Visualization of the endocytosis process was achieved by binding of lipoprotein particles and colloidal gold. The study was carried out on isolated liver perfusing by oxygenated nutritional media which consist of colloidal gold-lipoproteins conjugates. Electron microscope analysis testifies that high-density lipoproteins are absorbed by hepatic sinusoidal cells by means of receptor endocytosis. Both Kupffer cells and endothelial cells have such receptors. Lipoproteins of this class reach hepatic cells in Disse's space through the endothelium, i.e. transcellularly unlike low-density lipoproteins which penetrate paracellularly--through the fenestrae and intracellular contact's ares.
OBJECTIVE: To develope an easy to use, rapid and accurate test for detecting buprenorphine based on the principle of competitive immunoassay. METHODS: Monoclonal antibody against buprenorphine was conjugated with colloidal gold and dispensed on the glass fiber. The complete antigen Buprenorphine-BSA and the goat anti-mouse IgG polyclonal antibody were separately sprayed on the nitrocellulose membrane as the test line (T line) and the control line (C line). The rapid test kit was the final assembled product of test strip with the plastic cover. RESULTS: A total of 100 urine samples were tested for buprenorphine by immunochromatographic and GC/ MS methods. The accuracy was 99.0%. It is found the test kit can only detect by cross reaction with other 45 kind drugs. CONCLUSION: Rapid test kit can detect buprenorphine in the samples in 5 minutes. The cut-off value of the test is 100 ng/mL.
A critical presentation of basic methods for the preparation of colloid gold and of gold particle complexes with macromolecules to be used on the ultrastructural level.
A method of the competitive immunochromatographic assay of the pesticides 2,4-D (2,4-dichlorophenoxyacetic acid) and simazine (2-chloro-4,6-bis(N-ethylamino)-1,2,5-triazine) in aqueous samples was developed. Monoclonal antibodies to these pesticides labeled with colloidal gold were used to visualize the results. The sensitivity of the 2,4-D and simazine assay is 12 ng/ml, and the time of analysis is 3-7 min. The method does not differ in sensitivity from the competitive EIA using conjugates of monoclonal antibodies to the pesticides with horseradish peroxidase; however, the time of the EIA is 1.5 h. The immunochromatographic method of the pesticide detection is available and simple and may be recommended for the development of assays of any other low-molecular compounds. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.
A new technique for localising the centre of sites of microinjection of drugs in the brain using the silver intensification of colloidal gold is described. The technique is compatible with immunocytochemistry and in this study was used in Wistar-Kyoto rats to investigate the relationship between the location of phenylethanolamine-N-methyltransferase-like immunoreactive (PNMT-LI) cell bodies in the medulla and the precise sites where microinjection of L-glutamate elicited pressor responses. Colloidal gold provided a simple technique for localising the centre of sites of microinjection with a high degree of spatial resolution and allowed pressor sites to be precisely localised in the region of ventrolateral medulla with the highest density of PNMT-LI cells.
The hypothesis that the retrieval of membranes of neurohypophysial neurosecretory granules (NSG) and small electron-lucent microvesicles occurs by different routes was tested by incubating neurohypophysial neurosecretosomes with colloidal gold particles of various sizes. Neurosecretosomes derived from normal Long Evans rats and incubated in media of normal ionic composition endocytosed a few small (less than 25 nm) gold particles into 40-50 nm electron-lucent microvesicles. After depolarisation, more small gold particles were found in microvesicles, and small and large (greater than 25 nm) gold particles in vacuoles. Oxytocin-containing neurosecretosomes derived from Brattleboro rats, which contain 160 nm-diameter NSG, endocytosed gold particles in a pattern indistinguishable from that of neurosecretosomes from Long Evans rats. However, neurosecretosomes derived from defective vasopressin neurones of Brattleboro rats, which contain microvesicles, small vacuoles, and a few 100 nm dense-cored vesicles, but no 160 nm NSG, endocytosed only small colloidal gold particles. Early after depolarisation the gold particles were present only in microvesicles, but later some could be found in vacuoles and lysosome-like structures. Immunogold cytochemistry using a polyclonal antiserum raised against microvesicle-rich neurosecretosomes derived from Brattleboro rats labelled microvesicles in the posterior pituitary strongly, NSG weakly, and vacuoles to a variable extent. These data together indicate that, after exocytosis, the membranes of NSG are recaptured as large vacuoles. Microvesicles are exocytosed and endocytosed separately.
Rabbit antibodies against a neutral Mn(2+)-dependent rat liver DNAse were obtained, whose specificity towards DNAse was ascertained by suppression of the enzyme activity both in vitro system and immunoblotting assays. Procedures of synthesis of ferritin and colloidal gold conjugates with antibodies are described. The biological activity of the conjugates proved to be similar to that of the original antibodies.
Colloidal gold was used as a marker for the light microscopic detection of leukocyte cell surface antigens with monoclonal antibodies. Leukocytes were first incubated with monoclonal mouse antibodies, and then with colloidal gold-labeled goat-anti-mouse antibodies. The cells were fixed and stable preparations were made. Positive cells displayed numerous dark granules all over their surface membrane. In 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-lymphocytes and their subclasses. Accurate cell recognition, based on morphology and cytochemistry, permitted rapid cell counting. The procedure was also applied on small volumes of whole blood. This approach was used as a microtechnique for the enumeration of lymphocyte subpopulations in pediatric patients. In normal peripheral blood, the intracellular enzymatic activities of the immunogold-labeled lymphocytes were detected by the classical cytochemical reactions. The staining patterns for acid alpha-naphthyl acetate esterase, acid phosphatase and beta-glucuronidase were identical to those found in smears of unlabeled cells. The cytochemical profile of the OKT-defined normal lymphocyte subpopulations was established.
We characterized the mucus glycoconjugates produced by goblet and non-goblet epithelial cells in asymptomatic contact lens (CL) wearers. We employed four lectins (proteins of vegetal origin which specifically recognize glycosidic residues: WGA, PNA, SBA and ConA) conjugated with colloidal gold as ultrastructural marker, at Transmission Electron Microscopy. A computerized quantitative analysis was carried out in order to compare the results from the CL wearers to those from the control patients. Goblet cells produce different amount of glycosidic residues, in particular, a significant decrease in the distribution of sialic acid, N-acetylglucosamine, N-acetylgalactosamine, galactose-N-acetylgalactosamine and mannose was observed. The content of glycosidic residues in the mucus vesicles of the non-goblet epithelial cells appeared unchanged as to the normal situation. We speculate that the CL could possibly contribute to the failure of the tear film stability by altering the production of mucus.
We used 46 rabbits for the study of blood-brain barrier (BBB) permeability quantitatively in early-stage brain injury by a dropping weight method. The animals were divided into two groups: control group and brain injury group. Both Evans' blue and colloidal gold (CG) particles were utilized for evaluation of BBB permeability alteration. Three different sizes of CG 5, 10 and 15 nm in diameter were employed as tracers for a quantitative investigation of BBB changes by light and electron microscopes respectively. In addition, the brain water contents were determined. The preliminary data indicated that an increase of BBB permeability began 30 minutes after the injury with a few of 5 or 10 nm CG particles present in the endocytic pits and endothelial microvilli, and the augmentation of BBB permeability reached its peak 6 hours after the injury. More CG tracers penetrated into the precapillary arterioles, venules or capillaries through the opening of tight junctions of BBB and by vesicular transportation. The variation of the brain water contents was closely correlated to the above-mentioned BBB changes. It is suggested that the vasogenic cerebral edema was mainly caused by the augmentation of BBB permeability.
Ultrastructural distribution of laminin within renal glomerular (GBM) and tubular basement membranes (TBM) was investigated using post-embedding immunolocalization with colloidal gold. Rat kidneys were fixed with 4% formaldehyde and embedded at 4 degrees C in Lowicryl K4M medium. Thin sections were then sequentially treated with affinity-purified rabbit anti-laminin IgG and anti-rabbit IgG conjugated to 10 nm diameter colloidal gold. Gold bound specifically to the GBM and TBM with particle densities of 690/micron2 and 731/micron2, respectively. In the GBM, the number of gold particles bound/micron2 of lamina densa greater than lamina rara externa greater than lamina rara interna. Closely similar binding patterns were found when kidneys were fixed with 0.5% glutaraldehyde plus 3% formaldehyde and embedded at 60 degrees C in L.R. White resin, but slightly less gold bound to sections overall than that seen with formaldehyde alone and Lowicryl. Taken together, these results illustrate that anti-laminin IgG, whether applied to fixed sections in vitro or introduced in vivo, bound to the lamina rara interna, lamina densa, and lamina rara externa of the GBM and throughout the TBM.
We present a new autometallographic technique for demonstrating vessels and other small cavities at light microscopy (LM) and electron microscopy (EM) levels. It is possible to obtain detailed knowledge of the 3-D appearance of the vascular system by exchanging blood with a 40 degrees C, 8% gelatine solution containing colloidal gold particles (gold gelatine solution, GGS) and ensuing silver enhancement of the gold particles by autometallography (AMG). The GGS-AMG technique demonstrates the vascular system as a dark web that can be studied in cryostat, vibratome, methacrylate, paraffin and Epon sections at all magnifications. The infused GGS becomes increasingly viscous and finally becomes rigid when the temperature falls below 20 degrees C. An additional advantage of this technique is the fact that none of the tested counterstains or immunotechniques interfere with this AMG approach. The GGS-AMG technique is demonstrated on rat brains but can be applied to any organ. We believe that the present technique is valuable for both experimental studies and routine pathology.
OBJECTIVE: To seek for a simple, sensitive and rapid assay to detect specific antibody of patients suffering from hemorrhagic fever renal syndrome (HFRS). METHODS: Serum anti-HFRS IgM (sIgM) and IgG (sIgG) antibody were detected in 186 patients with HFRS using colloidal gold immuno-dot assay (CGIDA), and compared with those detected with enzyme linked immunosorbant assay (ELISA) and indirect fluorescent antibody test (IFAT). Kuhuang Shenmai injection (KHSM) combined Astragalus decoction (AD) treatment was applied to 101 HFRS patients in a treated group (n = 50), and the effect of therapy was compared with that in a control group (n = 51) treated with ribarvirin and ganlixin injection. RESULTS: CGIDA showed positive sIgM antibody in 132 cases (70.9%) and positive sIgG antibody in 163 (87.1%) of the 186 HFRS patients. As compared the two groups in crossing stage, the rate of crossing shock stage in the treated group was higher than that in the control (P < 0.05). As for rate of crossing oliguria stage and from febrile stage directly developed into diuretic stage, the difference between the two groups was insignificant (P < 0.05). CONCLUSION: CGIDA shows the merits of quick, simple and convenient in detecting sIgM and sIgG antibody in HFRS patients. The effect of KHSM combined AD treatment was not significantly different from that of ribarvirin and ganlixin injection.
Gold particles in colloidal solutions often vary considerably in size. The finest sols (diameter less than 15 nm), especially, are very heterogeneous, as is indicated by coefficients of variance (CV) of 25-35%. We have complexed staphylococcal protein A with gold particles (PA/Au) and then fractionated the preparations by glycerol or sucrose gradient centrifugation into very homogeneous subfractions. In this way, PA/Au probes of almost any size between 4.5 and 15 nm could be prepared. The variation of the gold particles in these fractions resulted in CV's between 9 and 16%. The reactivity of the PA/Au complex was not affected by the gradient procedure, as was shown by single- and double-labeling immunocytochemistry of ultrathin cryosections of rat pancreatic tissue.
The relationship between unbound and bound proteins prepared during labelling with colloidal gold (Au) was investigated. For this aim, labelled 125I-bovine serum albumin and 125I-rabbit immunoglobulin were employed. The procedures associated with the washing of the Au labelled proteins (i.e. albumin) had a marked influence on the dissociation of the bound ligand. This was most evident when the concentration of albumin that was used for labelling was too high (0.1 or 1.0 mg/ml Au sol). We suggest that purification of labelled proteins be conducted shortly before use so as to avoid a significant amount of dissociation during the time when the solution is coming to equilibrium.