Immunogold and immunogold/silver staining in the ultrastructural localization of target molecules identified by monoclonal antibodies.

A new method for demonstrating the binding and internalization of target molecules identified by two different monoclonal antibodies (MAbs) is described. This double staining technique utilizes, in a pre-embedding procedure, an immunogold/silver staining and a MAb that recognized cell surface antigens and a post-embedding technique where only immunogold is used to identify intracytoplasmic antigens. We could demonstrate that immunogold and gold followed by silver enhancement are two highly sensitive and accurate techniques. Colloidal gold particles are versatile tracers at the electron microscopic level when used at two different particle sizes (5 and 20 nm in diameter) in a double labelling method for the simultaneous identification of antigenic sites on the same section; the combined use of immunogold and gold/silver staining for the simultaneous localization of breast cancer-associated antigens on the same thin section is more accurate. These techniques have also been used to visualize the internationalization of antigen-antibody complexes by incubating MAb-gold labelled tissue sections for 30 min at 37 degrees C. We conclude that the use of both colloidal gold and gold/silver constitutes a further improvement in immunoelectron microscopy techniques and can help visualize the relative pattern of reactivity of two MAbs on the same cells. Further applications of these techniques will have an important impact on the development and use of MAbs in oncology.

Establishment and characterization of a thymic medullary epithelial cell clone.

Thymic stromal cells were cultured in conditions which select for epithelial cells. These were then transformed in vitro by contact with N-methyl-N'-nitro-N-nitrosoguanidine and cloned at limit dilution. One of the clones was characterized as being of medullary origin on the basis of its reactivity with a battery of antibodies previously shown to distinguish cortical from medullary thymic epithelial cells. The importance of this clone lies in the potential it offers to delineate how various T cell subpopulations acquire their distinct markers and function within the thymus.

Specific adherence of thymocytes to a thymic medullary epithelial cell line.

TMF, a glycoprotein found preferentially at the contact points between thymocytes and thymic medullary epithelial cells, was investigated for its participation in adherence between these two cell types. It was found that thymocytes adhere specifically to a TMF+ cell line, that anti-TMF antibody can inhibit the adherence and that extraneous TMF can effectively compete with the adherence reaction. It is concluded that TMF is indeed involved in the temporary contact which occurs between thymocytes and medullary epithelial cells.

Immunocytochemical localization of a medullary thymic epithelial glycoprotein.

The thymic microenvironment is known to play a key role in T-cell differentiation, but the exact nature of the interactions between epithelial and lymphoid cells has not been fully elucidated. With a monoclonal antibody to a thymic epithelial glycoprotein, we report the localization of an antigen specific for medullary epithelial cells of the mouse thymus. This antigen is found in the Golgi apparatus of epithelial cells, and on their borders with adjoining lymphocytes. This location is compatible with the previously reported observation that differentiation signals transmitted to thymic lymphocytes by thymic epithelial cells require actual contact between these two cell types.

Ultrastructural localization of factor VIII procoagulant antigen in human liver hepatocytes.

Factor VIII is generally believed to circulate in blood as a multimeric complex of two glycoproteins which are physiologically and immunologically distinct. One component of the factor VIII complex is factor VIII procoagulant activity (FVIII:C) which is associated with factor VIII/procoagulant antigen (FVIII:Ag, formerly FVIII/CAg). The second, larger unit of the complex is factor VIII/von Willebrand factor (vWF:Ag, formerly factor VIII-related antigen or FVIIIRAg). FVIII:C has anti-haemophilic activity and is defective or deficient in patients with classical haemophilia, and vWF:Ag is absent in patients with von Willebrand disease. FVIII:Ag was demonstrated recently in endothelial cells lining hepatic sinusoids, by using immunoperoxidase staining and light microscopy, whereas biochemical data had indicated its presence predominantly in the hepatocyte fractions and in lesser amounts in endothelial cells. Moreover, recent hybridization experiments detected FVIII:C messenger RNA in liver and kidney tissues. Despite several efforts, the cells responsible for FVIII:C synthesis have not been unequivocally identified. Here we use protein A-gold complex labelling to demonstrate the ultrastructural localization of FVIII:C in human liver cells; the results indicate that hepatocytes may synthesize FVIII:Ag.

Isolation and expression of Rhizobium japonicum cloned DNA encoding an early soybean nodulation function.

A first visible step in the nodulation of legumes by Rhizobium spp. is the deformation and curling of root hairs. We have identified and cloned DNA sequences encoding this function from two strains of Rhizobium japonicum (USDA 122 and USDA 110) with a weakly homologous probe from Rhizobium meliloti. Root hair curling encoded by the cloned DNA fragments was examined on soybeans (Glycine soja ) after conjugative transfer of these sequences in broad-host-range vectors to various bacterial genera. Pseudomonas putida gave unambiguous expression of the root hair curling genes. This enabled us to identify the 8.7-kilobase EcoRI fragments encoding root hair curling from each strain. The phenotypes encoded by the plasmids pBS1 (derived from strain USDA 122) and pBS2 (derived from strain USDA 110) are distinct and represent a phenotype characteristic of their parent R. japonicum strains. Subclones of pBS1 and pBS2 were generated in single and multicopy vectors, and their expression was analyzed in P. putida. We established that a 4.2-kilobase internal Sa/I fragment of pBS1 and a 3.5-kilobase SstI -EcoRI fragment of pBS2 are sufficient to confer root hair curling on soybeans.