A Covalently Linked 10 Nm Gold Immunoprobe

1999 ◽  
Vol 5 (S2) ◽  
pp. 1324-1325
Author(s):  
Edmund Gutierrez ◽  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Peter M. Takvorian
Keyword(s):  
Colloidal Gold ◽  
Metal Cluster ◽  
Molecular Wires ◽  
Cluster Compounds ◽  
Nonspecific Binding ◽  
Double Labeling ◽  
Tissue Sections ◽  
Wide Range ◽  
Covalently Linked ◽  
Source Of Error

Chemically functionalized metal cluster compounds have demonstrated important advantages over colloidal gold as biological microscopy labels. They are covalently cross-linked to the targeting biomolecule, and therefore may be conjugated to a wide range of molecules which cannot be labeled with colloidal gold. The 1.4 nm Nanogold® cluster has been conjugated to peptides, lipids and oligonucleotides, some of which have been proposed as elements of novel molecular wires and novel materials. Dissociation of colloidal gold particles from the conjugate probe, a source of error in quantitative immunogold studies, is greatly reduced by covalent cross-linking. Nanogold® is an uncharged molecule, and because its surface is completely coordinated by organic ligands, nonspecific binding is greatly reduced. Nanogold® conjugates also show greatly enhanced penetration into cells and tissue sections. However, gold probes larger than Nanogold® are desirable for improved visualization in specimens with electron-dense regions or staining, or for applications such as double labeling studies with different sized gold labels, or visualizing wider antigen distributions.

scholarly journals Double immunocytochemical labeling of cell and tissue samples with monoclonal anti-bromodeoxyuridine.

1989 ◽  
Vol 37 (10) ◽  
pp. 1517-1527 ◽  
Author(s):  
J P Magaud ◽  
I Sargent ◽  
P J Clarke ◽  
M Ffrench ◽  
R Rimokh ◽  
...  
Keyword(s):  
Antigen Expression ◽  
Lymphoid Cells ◽  
Brdu Incorporation ◽  
Animal Cells ◽  
Human Tonsil ◽  
Double Labeling ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Mouse Tissues ◽  
Wide Range

We describe a new monoclonal antibody (designated Bu20a) against bromodeoxyuridine (BrdU). This antibody was selected by screening against human tissues using the APAAP technique, and shows no crossreactivity with normal nuclei. It stains BrdU incorporated into the nuclei of a wide range of cell types, including human tonsil lymphoid cells, normal mouse tissues, and human tumors growing in nude mice. A double-labeling technique is described using this antibody in which cell smears or tissue sections are first labeled by an immunoperoxidase procedure for a cellular antigen (e.g., mouse or human histocompatibility class II antigen, T-lymphocyte antigen, keratin) and BrdU is then detected by indirect immunofluorescence. This procedure, which was applied to a variety of human and animal cells and tissues, is of wide potential value in analyzing the phenotype of S-phase cells and in co-localizing antigen expression and BrdU incorporation in tissue sections.

The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

1985 ◽  
Vol 43 ◽  
pp. 426-429
Author(s):  
George H. Herbener ◽  
Antonio Nanci ◽  
Moise Bendayan
Keyword(s):  
Tissue Section ◽  
Colloidal Gold ◽  
Unit Area ◽  
Protein A ◽  
Extracellular Proteins ◽  
Gold Complex ◽  
Second Step ◽  
Igg Antibodies ◽  
Tissue Sections ◽  
Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

The chemistry of stabilized clusters

1982 ◽  
Vol 308 (1501) ◽  
pp. 27-34 ◽  
Keyword(s):  
Electron Donor ◽  
Organic Molecules ◽  
Metal Cluster ◽  
Organic Substrates ◽  
Insertion Reactions ◽  
Donor Ligand ◽  
Small Organic Molecules ◽  
Wide Range ◽  
Metal Metal ◽  
High Yields

Studies of the chemistry of metal cluster complexes and, in particular, their reactions with small organic molecules, have been confined to relatively few systems. Among the reasons for this are: (i) not many clusters are easily synthesized in high yields; (ii) their reactions often give a multitude of products that are difficult to separate and characterize; (iii) the conditions required to bring about reactions often lead to fragmentation of the cluster into lower nuclearity (often mononuclear) species. One cluster whose chemistry has been extensively studied is [Os 3 H 2 (CO) 10 ]. This can be synthesized in high yields from [Os 3 (CO) 12 ] + H 2 (Knox et al. 1975) and reacts readily under mild conditions with a wide range of electron-donor molecules by virtue of its coordinative unsaturation (Shapley et al. 1975; Deeming & Hasso 1976; Adams & Golembeski 1979). Formally, one may consider that a metal—metal double bond is present, which is reduced to a single bond on coordination of an additional two-electron donor ligand such as an organophosphine. The presence of metal—hydrogen bonds in this cluster and the cluster’s ability to coordinate organic substrates enable it to undergo a wide variety of insertion reactions, leading to products that may be regarded as intermediates in the reduction of organic molecules by clusters (Deeming & Hasso 1975; Keister & Shapley 1975).

Computer Graphics in the Study of Metal Cluster Compounds

1986 ◽  
Vol 25 (10) ◽  
pp. 853-860 ◽  
Author(s):  
Kim Henrick ◽  
Mary McPartlin ◽  
Jill Morris
Keyword(s):  
Computer Graphics ◽  
Metal Cluster ◽  
Cluster Compounds

scholarly journals Monoclonal antibodies to type V collagen for immunohistological examination of new tissue deposition associated with biomaterial implants.

1991 ◽  
Vol 39 (9) ◽  
pp. 1215-1220 ◽  
Author(s):  
J A Werkmeister ◽  
J A Ramshaw
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Experimental Models ◽  
Type V Collagen ◽  
Tissue Sections ◽  
Novel Approach ◽  
Wide Range ◽  
Conventional Elisa ◽  
Pre Treatment ◽  
Type V

We developed a panel of highly specific monoclonal antibodies (MAb) against dog Type V collagen. Each antibody showed differential reactivities towards Type V collagen from other species. All the antibodies were highly reactive in conventional ELISA, as well as with electroblots of collagen after polyacrylamide gel electrophoresis using non-denaturing conditions. The MAb were shown to be suitable for the immunohistological detection of Type V collagen in tissue sections, although this normally required pre-treatment of sections with 50 mM acetic acid. In particular, the antibodies were shown to be useful for examining samples of a collagen-based biomaterial, a vascular prosthesis, after explant from evaluation in an animal model. This showed that Type V collagen was most prominent in regions of new tissue formation within the neointima, close to the inner surface of the prosthesis. The broad spectrum of differential reactivities allows the antibodies to be used for a wide range of experimental models. These MAb therefore provide a novel approach for the evaluation of biomaterial performance, particularly for collagen-based implants.

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