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.

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.

scholarly journals Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens.

1991 ◽  
Vol 39 (9) ◽  
pp. 1267-1279 ◽  
Author(s):  
M van Lookeren Campagne ◽  
A B Oestreicher ◽  
T P van der Krift ◽  
W H Gispen ◽  
A J Verkleij
Keyword(s):  
Monoclonal Antibodies ◽  
Rat Brain ◽  
Anhydrous Methanol ◽  
Protein A ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Polyclonal Antiserum ◽  
Double Labeling ◽  
Ultrastructural Studies

We examined the suitability of freeze-substitution and Lowicryl HM20 embedding of aldehyde-fixed rat brain to localize several neural antigens at the ultrastructural level. The following rabbit polyclonal and mouse monoclonal antibodies were used: affinity-purified polyclonal immunoglobulins G raised to B-50/GAP43 (a membrane-anchored, growth-associated protein); affinity-purified polyclonal immunoglobulins G to human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a neuropeptide present in dense-core granules); a polyclonal antiserum raised to myelin basic protein (a protein present in compact myelin of the central nervous system); and mouse monoclonal antibodies to synaptophysin (an integral membrane protein of small synaptic vesicles). Rat mesencephalon was fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde, cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees C. Semi-thin Lowicryl sections were used for light microscopic visualization of B-50 in the ventromedial mesencephalic central gray substance. The procedure preserves well the ultrastructure of this region and the immunoreactivity of the selected antigens. This study shows that dehydration by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero temperature, provides a successful method of preparation of fixed brain tissue for ultrastructural studies, allowing immunogold localization of several neural antigens by double labeling in the same section and in serial sections.

scholarly journals Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold.

1985 ◽  
Vol 33 (10) ◽  
pp. 1015-1025 ◽  
Author(s):  
M Castel ◽  
J Morris ◽  
Y Ben-Barak ◽  
R Timberg ◽  
N Sivan ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Secretory Granules ◽  
Picric Acid ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Neurosecretory Granules ◽  
Sodium Metaperiodate ◽  
Background Staining ◽  
Axonal Varicosity

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.

Ultrastructural localization of β-(1 → 4)-D-glucans in two pathogenic fungi and in their host tissues by means of an exoglucanase–gold complex

1987 ◽  
Vol 33 (5) ◽  
pp. 405-417 ◽  
Author(s):  
Nicole Benhamou ◽  
Hélène Chamberland ◽  
G. B. Ouellette ◽  
F. J. Pauze
Keyword(s):  
Close Contact ◽  
Middle Lamella ◽  
Pathogenic Fungi ◽  
Ultrastructural Localization ◽  
Dutch Elm Disease ◽  
Gold Complex ◽  
Tomato Root ◽  
Gold Particles ◽  
Infected Tomato ◽  
Crown And Root Rot

An exoglucanase, purified from a cellulase produce by the fungus Trichoderma harzianum Rifai., was successfully bound to colloidal gold and used for ultrastructural detection of intracellular cellulosic β-(1 → 4) glucans. These saccharides were found to be present in great amount in the walls of Ophiostoma ulmi (Buism.) Nannf., the Dutch elm disease agent, whereas they were randomly distributed in the walls of Fusarium oxysporum Schlecht f. sp. radicis-lycopersici Jarvis and Shoemaker (FORL), the agent of tomato crown and root rot. In O. ulmi cell walls, the β-(1 → 4) glucans were predominantly concentrated over the central portion of the inner walls. In both colonized elm wood and infected tomato root tissues, the compound middle lamella and secondary walls of parenchyma cells, fibers (absent in tomato roots), and vessel members were always intensely labeled, but gold particles appeared somewhat irregularly distributed. In fibers having an S3 gelatinous layer, the latter was always preferentially labeled. Penetration of O. ulmi in elm wood tissues resulted in the digestion of host wall areas free of labeling. Such areas were not observed in infected tomato tissues; instead, an accumulation of gold particles was noted along the fungal portion that was in close contact with the host wall. Results of this study confirm the potential value of gold-labeled exoglucanase and provide some new information about wall topochemistry during host-pathogen interactions.

scholarly journals Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies.

1984 ◽  
Vol 98 (6) ◽  
pp. 2239-2244 ◽  
Author(s):  
R Sealock ◽  
B E Wray ◽  
S C Froehner
Keyword(s):  
Monoclonal Antibodies ◽  
Acetylcholine Receptor ◽  
Protein A ◽  
Spatial Relationship ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Postsynaptic Membrane ◽  
Specific Protein ◽  
Cytoplasmic Surface ◽  
Membrane Bound

Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana . These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level. Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold. The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane. The distributions of the receptor and the 43K protein along the membrane were found to be coextensive. Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 +/- 7 nm and 26 to 29 +/- 7 to 10 nm, respectively). These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane. They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule.

Use of monoclonal antibodies to detect a phytotoxic glycopeptide produced by Ophiostoma ulmi, the Dutch elm disease pathogen

1985 ◽  
Vol 63 (7) ◽  
pp. 1177-1184 ◽  
Author(s):  
Nicole Benhamou ◽  
G. B. Ouellette ◽  
J. G. Lafontaine ◽  
J. R. Joly
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Myeloma Cell ◽  
Dutch Elm Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ophiostoma Ulmi ◽  
Mouse Myeloma Cell Line ◽  
Immunocytochemical Techniques ◽  
Host Tissues

Two hybridomas that secrete antibodies specific for a phytotoxic glycopeptide from Ophiostoma ulmi (Buism.) Nannf. were produced by fusing spleen cells of mice immunized with the purified toxin and the Sp2-0/Ag14 mouse myeloma cell line. Specificity of these antibodies was first demonstrated by an enzyme-linked immunosorbent assay (ELISA), then by immunoblotting on nitrocellulose membrane after sodium dodecyl sulfate – polyacrylamide gel electrophoresis of the glycopeptide. Both clones produced antibodies of IgM class as determined by immunodiffusion. These monoclonal antibodies were utilized to detect and localize the toxic glycopeptide in pathogen cells and infected host tissues by immunohistochemical and immunocytochemical techniques.

scholarly journals Insight into purification of monoclonal antibodies in industrial columns via studies of Protein A binding capacity by in situ ATR-FTIR spectroscopy

The Analyst ◽  
2021 ◽  
Author(s):  
James Beattie ◽  
Ruth Rowland-Jones ◽  
Monika Farys ◽  
Richard Kucia-Tran ◽  
Sergei Kazarian ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Ftir Spectroscopy ◽  
Binding Capacity ◽  
Protein A ◽  
Therapeutic Monoclonal Antibodies ◽  
Insight Into ◽  
Effective Treatments

Therapeutic monoclonal antibodies (mAbs) are effective treatments for a range of cancers and other serious diseases, however mAb treatments cost on average ~$100,000 per year per patient, limiting their use....

Rapid quantification and in situ detection of nitrifying bacteria in biofilms by monoclonal antibody method

2000 ◽  
Vol 41 (4-5) ◽  
pp. 301-308 ◽  
Author(s):  
N. Noda ◽  
H. Ikuta ◽  
Y. Ebie ◽  
A. Hirata ◽  
S. Tsuneda ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescent Antibody ◽  
Nitrifying Bacteria ◽  
Fluorescent Antibody Technique ◽  
Antibody Technique ◽  
Antibody Method ◽  
In Situ Detection ◽  
Rapid Quantification

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. winogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. It was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis

2020 ◽  
Vol 7 (2) ◽  
pp. 121-133
Author(s):  
Ayesha Akhtar ◽  
Shivakumar Arumugam ◽  
Shoaib Alam
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Protein A ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
High Yield ◽  
Staphylococcal Protein A ◽  
Purification Step ◽  
Purity Analysis

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.

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