Isolation and ultrastructural localization of a soluble protein from Ophiostoma ulmi

1990 ◽  
Vol 68 (11) ◽  
pp. 2517-2524 ◽  
Author(s):  
R. S. Jeng ◽  
A. M. Svircev
Keyword(s):  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Leading Edge ◽  
Ultrastructural Localization ◽  
Immunogold Labelling ◽  
Two Dimensional ◽  
Thin Sections ◽  
Ophiostoma Ulmi ◽  
Gold Label

Two-dimensional polyacrylamide gel electrophoresis was used to identify and isolate a soluble polypeptide, the QP1 protein, which is characteristic of the vegetative hyphae of nonaggressive isolate Q412 of Ophiostoma ulmi. Individual QP1 spots were excised from 16 two-dimensional gels. Polypeptides were eluted from the gel spots by electroelution and lyophilized. The protein was injected into rabbits for the production of polyclonal antibodies. Antiserum specificity was tested by transferring polypeptides from a two-dimensional gel onto nitrocellulose and treating with QP1 serum. The resulting immunoblot contained a single spot that corresponded in shape and location to that of the QP1 polypeptide. Thin sections of fungal mycelia, from nonaggressive isolate Q412 and the aggressive isolate VA of O. ulmi, were treated with QP1 antibodies and protein A – gold. The gold label was localized in thin sections over conidial and hyphal cell walls of the nonaggressive isolate. The aggressive isolate was nonreactive. Mycelia from nonaggressive isolates Q412 and Q311 and aggressive isolates VA and CESS16K of O. ulmi were grown on solid medium, treated with QP1 antibodies, labelled with protein A – gold, and prepared for scanning electron microscopy. The gold-labelled QP1 polypeptide was detected on the leading edge of a small number of hyphae from nonaggressive isolates Q412 and Q311. Key words: immunogold labelling, Ophiostoma ulmi, soluble proteins.

Applications of post-embedding immunogold labelling in the study of human tumors

1989 ◽  
Vol 47 ◽  
pp. 876-877
Author(s):  
Stephen Hearn ◽  
Theodosios Kontozoglou ◽  
Harvey Cramer
Keyword(s):  
Cell Carcinoma ◽  
Tumor Cells ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Immunogold Labelling ◽  
Needle Aspiration ◽  
Peripheral Blood Cell ◽  
Specimen Preparation ◽  
Thin Sections ◽  
Molecular Features

Post-embedding immunogold labelling (IEM) can provide an exact correlation between the ultrastructure of tumor cells and the location of specific antigens. This technology links the molecular features of tumor cells derived from immunohistochemistry with our knowledge of tumor ultrastructure from electron microscopy. Criteria for the use of IEM in the study of human tissues include: the ability to localize many different antigens with commercially available antibodies; specimen preparation that is rapid, standardized and effective for different biopsies and tissue culture; results should compare with immunoperoxidase labelling on formalin-fixed, paraffin-embedded tissue.The tumors examined in this study included: squamous carcinomas, melanomas, multiple myelomas, schwannomas, medullary carcinomas of the thyroid, adenocarcinomas, leiomyomas, germ-cell carcinoma, myeloid leukemia, lymphomas, merkel cell carcinoma, and carcinoid tumors. Fine-needle aspiration biopsies (FNAB), core biopsies of bone marrow, peripheral blood, cell culture monolayers and surgically removed tissues were fixed in 1.6% glutaraldehyde. Samples were dehydrated, infiltrated in Lowicryl K4M and UV polymerized (total preparation time: 3 days). Technique for FNAB samples used mesh filtration to purify tumor cells which were then entrapped in albumin prior to embedding to prevent loss of cells. Glass coverslips (4x2mm) bearing cell monolayers were fixed in situ, infiltrated, placed in gelatin capsules and the cells separated from the coverslips after polymerization for en face sectioning. Thin sections were immunolabelled with readily available monoclonal and polyclonal antibodies using the protein A-gold technique. Samples of normal tissues were labelled for each antigen as positive controls and the negative controls used had appropriate low levels of non-specific labelling.

scholarly journals Isolation from human chronic-lymphocytic-leukaemia cells of membrane glycoproteins associated with Fc-receptor functions. Physical parameters and production of polyclonal antibodies

1987 ◽  
Vol 245 (1) ◽  
pp. 75-83 ◽  
Author(s):  
G Gorini ◽  
G A Medgyesi ◽  
M Garavini ◽  
K J Dorrington ◽  
J Down
Keyword(s):  
Chronic Lymphocytic Leukaemia ◽  
Gel Electrophoresis ◽  
Lymphocytic Leukaemia ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fc Receptor ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Membrane Glycoproteins

Two membrane glycoproteins that bound immune complexes and inhibited Fc-receptor- (FcR-)mediated functions in vitro were purified from human FcR+ chronic-lymphocytic-leukaemia cells. A multi-step purification was developed, consisting essentially in: (i) Tween 40 extraction of crude cell membranes; (ii) solubilization of membrane fragments by Renex-30; (iii) isolation of glycoproteins by affinity chromatography on Lens culinaris haemagglutinin-Sepharose; (iv) papain treatment of the eluted glycoproteins followed by gel-filtration chromatography; (v) purification by polyacrylamide-gel electrophoresis of two molecular species from the protein-size fraction enriched for immune-complex-binding activity. The two electrophoretically isolated components displayed apparent molecular masses of 70 and 45 kDa by SDS/polyacrylamide-gel electrophoresis and restricted charge heterogeneity by two-dimensional analysis. Two-dimensional peptide mapping revealed the presence of many peptides in common between the two proteins and the absence of a number of peptides in the 45 kDa component. These two polypeptides were used as immunogens to produce polyclonal antibodies that cross-reacted with both proteins and specifically inhibited FcR-mediated reactions in vitro. Furthermore, FcR-related components from detergent-extracted lysates of the human K562 and U937 cell lines or human placental membranes were revealed by the putative anti-FcR antibodies adsorbed on Protein A-Sepharose.

Immunological Relationship Between the Fast-Acting Plasminogen Activator Inhibitors from Plasma, Blood Platelets and Endothelial Cells Demonstrated with a Monoclonal Antibody Against an Inhibitor from Placenta

1986 ◽  
Vol 55 (02) ◽  
pp. 213-217 ◽  
Author(s):  
M Philips ◽  
Anne-Grethe Juul ◽  
S Thorsen ◽  
J Selmer ◽  
J Zeuthen
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitors ◽  
Fast Acting

SummaryTwo plasminogen activator inhibitors (I and II) were demonstrated in human placenta. The complex between inhibitor I and tissue-type plasminogen activator was purified by immunoadsorption to solid-phase anti-activator antibodies. The purified complex (Mr 95.000) was used for immunization of mice and subsequent production of monoclonal antibodies. One antibody (F37), which reacted with both free and complex-bound inhibitor I, was used for further study by a method involving binding of the antibody to protein A-Sepharose, immunoadsorp-tion of antigen and analysis of the resulting supernatant by SDS-polyacrylamide gel electrophoresis and enzymography. The analysis showed that F37 reacted with the fast-acting plasminogen activator inhibitors recently demonstrated in plasma, blood platelets and endothelial cells, indicating that these inhibitors and inhibitor I share a common epitope. Inhibitor II did not react with F37. Inhibitor II is identical to the placenta inhibitor previously described by others. It reacted selectively with polyclonal antibodies against that inhibitor.

scholarly journals Ultrastructural localization of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 929-938 ◽  
Author(s):  
JD Wencel-Drake ◽  
RG Painter ◽  
TS Zimmerman ◽  
MH Ginsberg
Keyword(s):  
Von Willebrand Factor ◽  
Plasma Membranes ◽  
Polyclonal Antibodies ◽  
Spherical Shape ◽  
Ultrastructural Localization ◽  
Resting Cells ◽  
Western Blots ◽  
Thin Sections ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We have investigated the localization of thrombospondin (TSP), fibrinogen, fibronectin, and von Willebrand factor in human platelets by transmission electron microscopy of antibody-stained ultrathin frozen sections. In negatively stained thin sections, alpha granules were identified on the basis of their smooth, roughly spherical shape, size, single limiting electron-lucent 100 A membrane, and frequent presence of electron-dense nucleoid. In contrast, mitochondria exhibited characteristic double membranes and cristae. Sections were separately stained with affinity-purified polyclonal antibodies to these proteins as well as with three monoclonal anti-TSP antibodies. Antibody specificity was documented in radioimmunoassays, by immunofluorescent cross-blocking, and by staining of bands of appropriate mobility in Western blots of whole platelets. Bound antibody was visualized using a 5-nm colloidal gold-avidin conjugate. In resting cells, staining of virtually all alpha granules was observed for all four proteins. In contrast, consistent staining was absent from other organelles, including plasma membranes, mitochondria, and vacuolar structures that may represent the open canalicular system.

scholarly journals Immunoelectron microscopic labeling of immunoglobulin in plasma cells after osmium fixation and epoxy embedding.

1985 ◽  
Vol 33 (12) ◽  
pp. 1212-1218 ◽  
Author(s):  
S A Hearn ◽  
M M Silver ◽  
J A Sholdice
Keyword(s):  
Epoxy Resin ◽  
Osmium Tetroxide ◽  
Secretory Pathway ◽  
Plasma Cells ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Cell Organelles ◽  
Human Tonsil ◽  
Sodium Metaperiodate ◽  
Gold Label

Previous studies have found that immunoglobulin cannot be immunolabeled in tissues prepared for electron microscopy by usual methods. To test this conclusion, we used a protein A-gold postembedding immunolabeling method on tissues that were fixed in glutaraldehyde, post-fixed in osmium tetroxide, and embedded in epoxy resin; sections were pretreated with sodium metaperiodate. A variety of common fixation protocols were also used and the most suitable conditions for immunolabeling were determined. This technique permitted the ultrastructural localization of immunoglobulin light chains in optimally preserved and contrasted plasma cells from human tonsil, lymph nodes, plasmacytomas, and a renal biopsy. We were able to demonstrate multiple antigens in the same tissue and label antigens in tissues that had been stored for many years in epoxy resin. The technique allows quantitation of the gold label over plasma cell organelles and therefore gives information about the immunoglobulin secretory pathway in these cells. We found that the protein A-gold procedure compares favorably in technical ease with the immunoperoxidase, avidin-biotin peroxidase, and immunoglobulin-colloidal gold immunolabeling methods, and has added advantages in allowing precise localization and quantitation of the labeled antigen.

scholarly journals Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique.

1983 ◽  
Vol 31 (1) ◽  
pp. 101-109 ◽  
Author(s):  
M Bendayan ◽  
M Zollinger
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Liver Mitochondria ◽  
Secretory Proteins ◽  
Additional Advantage ◽  
Sodium Metaperiodate ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Structural Preservation

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.

scholarly journals Ultrastructural localization of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 929-938 ◽  
Author(s):  
JD Wencel-Drake ◽  
RG Painter ◽  
TS Zimmerman ◽  
MH Ginsberg
Keyword(s):  
Von Willebrand Factor ◽  
Plasma Membranes ◽  
Polyclonal Antibodies ◽  
Spherical Shape ◽  
Ultrastructural Localization ◽  
Resting Cells ◽  
Western Blots ◽  
Thin Sections ◽  
Von Willebrand ◽  
Willebrand Factor

We have investigated the localization of thrombospondin (TSP), fibrinogen, fibronectin, and von Willebrand factor in human platelets by transmission electron microscopy of antibody-stained ultrathin frozen sections. In negatively stained thin sections, alpha granules were identified on the basis of their smooth, roughly spherical shape, size, single limiting electron-lucent 100 A membrane, and frequent presence of electron-dense nucleoid. In contrast, mitochondria exhibited characteristic double membranes and cristae. Sections were separately stained with affinity-purified polyclonal antibodies to these proteins as well as with three monoclonal anti-TSP antibodies. Antibody specificity was documented in radioimmunoassays, by immunofluorescent cross-blocking, and by staining of bands of appropriate mobility in Western blots of whole platelets. Bound antibody was visualized using a 5-nm colloidal gold-avidin conjugate. In resting cells, staining of virtually all alpha granules was observed for all four proteins. In contrast, consistent staining was absent from other organelles, including plasma membranes, mitochondria, and vacuolar structures that may represent the open canalicular system.

Ultrastructural localization in host tissues of a toxic glycopeptide produced by Ophiostoma ulmi, using monoclonal antibodies

1985 ◽  
Vol 63 (7) ◽  
pp. 1185-1195 ◽  
Author(s):  
Nicole Benhamou ◽  
J. G. Lafontaine ◽  
J. R. Joly ◽  
G. B. Ouellette
Keyword(s):  
Monoclonal Antibodies ◽  
Protein A ◽  
Middle Lamella ◽  
Protective Layer ◽  
Ultrastructural Localization ◽  
Wall Layer ◽  
Dutch Elm Disease ◽  
Ophiostoma Ulmi

Monoclonal antibodies against a phytotoxic glycopeptide from Ophiostoma ulmi (Buism.) Nannf., the Dutch elm disease pathogen, were used in a postembedding protein A – gold labeling technique to localize this toxin in experimentally infected tissues of young elm seedlings. Significant labeling was observed over pit membranes, over the innermost wall layer (protective layer) of paratracheal parenchyma cells, and over the intercellular spaces and, adjacently, the middle lamella. Host secondary walls, cytoplasm, and various organelles, except regions of amyloplastids, were free of labeling. From day 1 to day 4 after inoculation an intensification of the labeling reaction was noted that corresponded to an increase in the disease symptoms. This specific and sensitive technique has thus proved to be highly suitable for the in situ identification of antigenic macromolecules in diseased elm tissues. The classification of the toxic glycopeptide among the group of vivotoxins is discussed.

scholarly journals Ultrastructural localization of intracellular antigens by the use of protein A-gold complex.

1978 ◽  
Vol 26 (12) ◽  
pp. 1074-1081 ◽  
Author(s):  
J Roth ◽  
M Bendayan ◽  
L Orci
Keyword(s):  
Endocrine Pancreas ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Gold Complex ◽  
Secretory Proteins ◽  
Staphylococcal Protein A ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Step Procedure ◽  
General Method

An immunocytochemical technique for the demonstration of intracellular antigens (secretory proteins) on thin sections is reported. Staphylococcal protein A which reacts with the Fc fragment of IgG molecules was labeled with colloidal gold as a marker. The antigenic sites were visualized on aldehyde-fixed and Epon-embedded tissue in a two step procedure. The specific antisera were applied to thin sections for binding to the antigens and then visualized by the protein A-gold complex. By using this technique different secretory proteins of the exocrine and endocrine pancreas were localized. The protein A-gold technique is proposed as a general method for visualization of antigenic sites on thin sections.

scholarly journals Purification and characterization of glucosidase I involved in N-linked glycoprotein processing in bovine mammary gland

1987 ◽  
Vol 247 (3) ◽  
pp. 555-562 ◽  
Author(s):  
K Shailubhai ◽  
M A Pratta ◽  
I K Vijay
Keyword(s):  
Mammary Gland ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Gel Filtration ◽  
Mammary Tissue ◽  
Ph Optimum ◽  
Reducing Conditions ◽  
Solubilized Enzyme ◽  
Biological Substrates

Glucosidase I, the first enzyme involved in the post-translational processing of N-linked glycoproteins, was purified to homogeneity from the lactating bovine mammary tissue. The enzyme was extracted by differential treatment of the microsomal fraction with Triton X-100 and Lubrol PX. The solubilized enzyme was subjected to affinity chromatography on Affi-Gel 102 with N-5-carboxypentyldeoxynojirimycin as ligand and DEAE-Sepharose CL-6B chromatography. Purified glucosidase I shows a molecular mass of 320-330 kDa by gel filtration on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis under reducing conditions indicates a single band of approx. 85 kDa, indicating that the native enzyme is probably a tetrameric protein. Several criteria, including pH optimum of 6.6-7.0, specific hydrolytic action towards Glc3Man9GlcNAc2, to release the terminally alpha-1,2-linked glucosyl residue, and total lack of activity towards Glc1Man9GlcNAc2 and Glc2Man9GlcNAc2 saccharides, which are the biological substrates for processing glucosidase II, and 4-methylumbelliferyl alpha-D-glucopyranoside show the non-lysosomal origin and the processing-specific role of the purified enzyme. The enzyme does not require any metal ions for its activity. Hg2+, Ag+ and Cu2+ are potent inhibitors of the enzyme; this inhibition can be reversed by adding an excess of dithiothreitol. Among the saccharides tested, kojibiose (Glc alpha 1----2Glc) was inhibitory to the enzyme. Polyclonal antibodies raised against the enzyme in rabbit were found to be specific for glucosidase I, as revealed by Western-blot analysis and by immunoadsorption with Protein A-Sepharose. Anti-(glucosidase I) antibodies were cross-reactive towards a similar antigen in solubilized microsomal preparations from liver, mammary gland and heart from the bovine, guinea pig, rat and mouse.

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