scholarly journals Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures.

1988 ◽  
Vol 107 (1) ◽  
pp. 163-175 ◽  
Author(s):  
D J Meyer ◽  
C L Afonso ◽  
D W Galbraith
Keyword(s):  
Monoclonal Antibody ◽  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Solid Phase ◽  
Culture Media ◽  
Gradient Centrifugation ◽  
Cell Suspension Cultures ◽  
Isolation And Characterization

Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.

scholarly journals Possible Future Monoclonal Antibody (mAb)-Based Therapy against Arbovirus Infections

2013 ◽  
Vol 2013 ◽  
pp. 1-21 ◽  
Author(s):  
Giuseppe Sautto ◽  
Nicasio Mancini ◽  
Giacomo Gorini ◽  
Massimo Clementi ◽  
Roberto Burioni
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Side Effects ◽  
Antiviral Activity ◽  
The Other ◽  
Viral Escape ◽  
Passive Immunotherapy ◽  
Medical Need

More than 150 arboviruses belonging to different families are known to infect humans, causing endemic infections as well as epidemic outbreaks. Effective vaccines to limit the occurrence of some of these infections have been licensed, while for the others several new immunogens are under development mostly for their improvements concerning safety and effectiveness profiles. On the other hand, specific and effective antiviral drugs are not yet available, posing an urgent medical need in particular for emergency cases. Neutralizing monoclonal antibodies (mAbs) have been demonstrated to be effective in the treatment of several infectious diseases as well as in preliminaryin vitroandin vivomodels of arbovirus-related infections. Given their specific antiviral activity as well-tolerated molecules with limited side effects, mAbs could represent a new therapeutic approach for the development of an effective treatment, as well as useful tools in the study of the host-virus interplay and in the development of more effective immunogens. However, before their use as candidate therapeutics, possible hurdles (e.g., Ab-dependent enhancement of infection, occurrence of viral escape variants) must be carefully evaluated. In this review are described the main arboviruses infecting humans and candidate mAbs to be possibly used in a future passive immunotherapy.

The isolation of plasma membrane from protoplasts of soybean suspension cultures

1977 ◽  
Vol 24 (1) ◽  
pp. 295-310
Author(s):  
D.W. Galbraith ◽  
D.H. Northcote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Membrane Systems ◽  
Enzymic Digestion ◽  
Nadh Cytochrome

A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1–14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

Immunotherapy of Mouse Leukemia with Monoclonal Antibodies Directed against Type-C Virus Structural Proteins

1984 ◽  
Vol 70 (1) ◽  
pp. 9-16
Author(s):  
Mauro Boiocchi ◽  
Piera Mondellini
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Therapeutic Effect ◽  
Structural Proteins ◽  
Leukemia Cells ◽  
Envelope Glycoproteins ◽  
Mouse Leukemia ◽  
Type C

The monoclonal antibody A6, isolated during a study on the natural immunoresponse of BALB/c mice against leukemia cells (4), reacts with the envelope glycoproteins gp70 of the MuLV and with the cell surface of the SL2 AKR leukemia. In the present paper, we describe the in vivo immunotherapeutic effect exerted by the A6 monoclonal antibody on the growth of the transplanted leukemia SL2. The greater therapeutic effect observed when the A6 was used with exogenous complement cooperation suggests that the immunotherapeutic activity is mediated by C'-dependent cytotoxicity.

Use of monoclonal antibodies to detect human placental alkaline phosphatase.

1983 ◽  
Vol 29 (1) ◽  
pp. 115-119 ◽  
Author(s):  
G De Groote ◽  
P De Waele ◽  
A Van de Voorde ◽  
M De Broe ◽  
W Fiers
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Solid Phase ◽  
Enzymatic Reactions ◽  
Placental Alkaline Phosphatase ◽  
Human Placental Alkaline Phosphatase ◽  
Human Sera ◽  
Starch Gel ◽  
Alkaline Phosphatase Isoenzyme

Abstract Convenient, sensitive, and specific solid-phase immunoassays involving monoclonal antibody are described for the determination of human placental alkaline phosphatase (hPLAP). An endogenous enzyme immunoassay combined the specificity of the immunological and the enzymatic reactions. Alternatively, a solid-phase "sandwich" radioimmunoassay involving immobilized polyclonal rabbit anti-hPLAP in combination with iodinated monoclonal antibody provided some additional advantages. Both tests can be used to detect hPLAP from various sources, e.g., in human sera during pregnancy or as a tumor marker. The radioimmunoassay detected an increase in hPLAP at nine weeks of gestation. We discuss the use of monoclonal antibodies for the differentiation of different alkaline phosphatase isoenzyme types by electrophoresis on starch gel.

Modulation of the biological activity of thyrotrophin by anti-human thyrotrophin monoclonal antibodies

1990 ◽  
Vol 126 (2) ◽  
pp. 333-340 ◽  
Author(s):  
S. R. Page ◽  
A. H. Taylor ◽  
W. Driscoll ◽  
M. Baines ◽  
R. Thorpe ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Biological Activity ◽  
Monoclonal Antibodies ◽  
Cyclic Amp ◽  
Receptor Activation ◽  
Camp Production ◽  
Dose Dependent ◽  
Bovine Tsh

ABSTRACT The mechanism by which monoclonal antibodies enhance the biological activity of a number of hormones is poorly understood. One such antibody (GC73), which binds to human but not bovine TSH, enhances the bioactivity of human TSH in vivo. We have investigated whether GC73 enhancement of TSH bioactivity involves potentiation of hormone-receptor activation assessed by the cyclic AMP (cAMP) responses of both primary human thyrocyte cultures and a TSH-responsive human thyrocyte cell line (SGHTL-45). GC73 had no effect on basal cAMP production. In contrast to its enhancement of the bioactivity of human TSH in vivo, it markedly inhibited the cAMP response to 1 and 10 mU human TSH/ml in primary thyrocytes. This effect was dose-dependent with neutralization of the bioactivity of TSH occurring at 2 mg GC73/ml. GC73 had no effect on the bioactivity of bovine TSH. In contrast, a second anti-TSH monoclonal antibody (TC12), which binds to both human and bovine TSH, inhibited the bioactivity of both species of TSH. Similar results were obtained using SGHTL-45 cells, although the peak concentrations of cAMP were lower. We conclude that binding of GC73 to human TSH resulted in inhibition rather than enhancement of the in-vitro biological activity of human TSH. We suggest that GC73 enhancement of human TSH bioactivity seen in vivo does not result from a mechanism involving potentiation of receptor activation by human TSH. Journal of Endocrinology (1990) 126, 333–340

scholarly journals Tubulin-tyrosine ligase has a binding site on beta-tubulin: a two-domain structure of the enzyme

1987 ◽  
Vol 104 (4) ◽  
pp. 1059-1067 ◽  
Author(s):  
J Wehland ◽  
K Weber
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Enzymatic Activity ◽  
Binding Site ◽  
Gradient Centrifugation ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Alpha Beta ◽  
Tubulin Tyrosine Ligase ◽  
Carboxy Terminal

Tubulin-tyrosine ligase and alpha beta-tubulin form a tight complex which is conveniently monitored by glycerol gradient centrifugation. Using two distinct ligase monoclonal antibodies, several subunit-specific tubulin monoclonal antibodies, and chemical cross-linking, a ligase-binding site was identified on beta-tubulin. This site is retained when the carboxy-terminal domains of both tubulin subunits are removed by subtilisin treatment. The ligase-tubulin complex is also formed when ligase is added to alpha beta-tubulin carrying the monoclonal antibody YL 1/2 which binds only to the carboxyl end of tyrosinated alpha-tubulin. The beta-tubulin-binding site described here explains the extreme substrate specificity of ligase, which does not act on other cellular proteins or carboxy-terminal peptides derived from detyrosinated alpha-tubulin. Differential accessibility of this site in tubulin and in microtubules seems to explain why ligase acts preferentially on unpolymerized tubulin. Ligase exposed to V8-protease is converted to a nicked derivative. This is devoid of enzymatic activity but still forms the complex with tubulin. Gel electrophoresis documents both 30- and a 14-kD domains, each which is immunologically and biochemically distinct and seems to cover the entire molecule. The two domains interact tightly under physiological conditions. The 30-kD domain carries the binding sites for beta-tubulin and ATP. The 14-kD domain can possibly form an additional part of the catalytic site as it harbors the epitope for the monoclonal antibody ID3 which inhibits enzymatic activity but not the formation of the ligase-tubulin complex.

Subcellular location of phage infection protein (Pip) inLactococcus lactis

2006 ◽  
Vol 52 (7) ◽  
pp. 664-672 ◽  
Author(s):  
Duane T Mooney ◽  
Monica Jann ◽  
Bruce L Geller
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Lactococcus Lactis ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Subcellular Location ◽  
Recognition Site ◽  
Phage Infection ◽  
Sucrose Density Gradient Centrifugation ◽  
Membrane Spanning

The amino acid sequence of the phage infection protein (Pip) of Lactococcus lactis predicts a multiple-membrane-spanning region, suggesting that Pip may be anchored to the plasma membrane. However, a near-consensus sortase recognition site and a cell wall anchoring motif may also be present near the carboxy terminus. If functional, this recognition site could lead to covalent linkage of Pip to the cell wall. Pip was detected in both plasma membranes and envelopes (plasma membrane plus peptidoglycan) isolated from the wild-type Pip strain LM2301. Pip was firmly attached to membrane and envelope preparations and was solubilized only by treatment with detergent. Three mutant Pip proteins were separately made in which the multiple-membrane-spanning region was deleted (Pip-Δmmsr), the sortase recognition site was converted to the consensus (Pip-H841G), or the sortase recognition site was deleted (Pip-Δ6). All three mutant Pip proteins co-purified with membranes and could not be solubilized except with detergent. When membranes containing Pip-Δmmsr were sonicated and re-isolated by sucrose density gradient centrifugation, Pip-Δmmsr remained associated with the membranes. Strains that expressed Pip-H841G or Pip-Δ6 formed plaques with near unit efficiency, whereas the strain that expressed Pip-Δmmsr did not form plaques of phage c2. Both membranes and cell-free culture supernatant from the strain expressing Pip-Δmmsr inactivated phage c2. These results suggest that Pip is an integral membrane protein that is not anchored to the cell wall and that the multiple-membrane-spanning region is required for productive phage infection but not phage inactivation.Key words: phage infection protein, Pip, Lactococcus lactis, subcellular location.

scholarly journals Unique role of the complement receptor CR1 in the degradation of C3b associated with immune complexes.

1982 ◽  
Vol 156 (6) ◽  
pp. 1739-1754 ◽  
Author(s):  
M E Medof ◽  
K Iida ◽  
C Mold ◽  
V Nussenzweig
Keyword(s):  
Monoclonal Antibodies ◽  
Immune Complexes ◽  
Solid Phase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alpha Chain ◽  
Sds Page ◽  
High Concentrations ◽  
Polypeptide Chains

The main finding of this paper is that CR1, the membrane receptor for C3b and C4b, together with C3b/C4b-inactivator (I), degrades C3b bound to immune complexes (C3b*). Two fragments are generated: C3c, which is released from the immune complexes, and C3d*. The C3c fragment released from the cell intermediate EAC1423b prepared with 125I-C3 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. It has a 135,000 mol wt and contains disulfide bonded labeled polypeptide chains of 75,000 and 31,000 mol wt, which presumably represent the beta and a fragment of the alpha-chain of C3b*. Silver staining of the SDS-PAGE gels revealed other C3-derived bands with 39-42,000 mol wt. Human erythrocytes + I also cleave C3b* into C3c and C3d*. The activity of the erythrocytes is CR1 mediated because it can be totally inhibited by monoclonal antibodies to CR1. In contrast with these results, I together with the serum protein beta 1H (H) transform EAC1423b into hemolytically inactive EAC1423bi and cleave the alpha' chain of C3b* into fragments of 70,000 and 40,000 mol wt. Small amounts of C3c are also released at relatively high concentrations of H. On a molar basis, the efficiency of CR1 in the generation of C3c and C3d is 10(4)-10(5) greater than H. An additional observation was that C3c could be released by treating EAC1423bi with CR1 + I and that this reaction was also inhibited by monoclonal antibodies to CR1. Therefore, it is likely that CR1 has binding affinity for iC3b and that the degradation of C3b* proceeds as follows: C3b (formula, see text) C3c + C3d*. Taken together, our findings argue that the processing of C3b* in vivo occurs in solid phase, that is, on the surface of cells bearing CR1.

scholarly journals Panitumumab and cetuximab affect differently miRNA expression in colorectal cancer cells

2021 ◽  
Author(s):  
Romain Chautard ◽  
Laetitia Corset ◽  
Sajida Ibrahim ◽  
Céline Desvignes ◽  
Gilles Paintaud ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cancer Cells ◽  
Two Phase ◽  
Colorectal Cancer Cells ◽  
Significant Expression ◽  
Antibody Resistance ◽  
Phase Process

Structured abstract Background & aim: Resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer (CRC) is frequent and prognostic biomarkers are lacking. MicroRNAs (miR) are good candidates in this context. We aimed to characterize cetuximab and panitumumab exposure influence on miR expression in colorectal cancer cells to identify those regulating the EGFR pathway and implicated in resistance to treatment. Finally, we aimed to identify miR expression in serum of patients with advanced CRC treated with cetuximab or panitumumab. Results: Cetuximab and panitumumab exposure induced significant expression variations of 17 miR out of a miRnome panel of 752. Six of those miR interacted with at least one downstream element of the EGFR pathway. Conclusion: After the bioinformatics two-phase process, 5 miR rarely described before could be potential actors of anti-EGFR monoclonal antibody resistance: miR-95-3p, miR-139-5p, miR-145-5p, miR-429 and miR-1247-5p. In vivo, we detected the expression of miR-139-5p and miR-145-5p in serum of patients with metastatic CRC.

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