scholarly journals Studies of mammalian ornithine decarboxylase using a monoclonal antibody

1984 ◽  
Vol 217 (1) ◽  
pp. 123-128 ◽  
Author(s):  
A E Pegg ◽  
J E Seely ◽  
L Persson ◽  
M Herlyn ◽  
K Ponsell ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Ornithine Decarboxylase ◽  
Mouse Kidney ◽  
Immunoglobulin M ◽  
Active Enzyme ◽  
Sodium Thiocyanate ◽  
Immunoaffinity Column ◽  
Rapid Turnover ◽  
Cytosolic Protein

A monoclonal antibody of the immunoglobulin M class was produced against mouse kidney ornithine decarboxylase. Screening for the antibody was carried out using alpha-difluoromethyl[5-3H]ornithine-labelled ornithine decarboxylase. The antibody reacted with this antigen and with native ornithine decarboxylase. The antibody attached to Sepharose could be used to form an immunoaffinity column that retained mammalian ornithine decarboxylase. The active enzyme could then be eluted in a highly purified form by 1.0M-sodium thiocyanate. The monoclonal antibody could also be used to precipitate labelled ornithine decarboxylase from homogenates of kidneys from androgen-treated mice given [35S]methionine. Only one band, corresponding to Mr of about 55000, was observed. The extensive labelling of this band is consistent with the rapid turnover of ornithine decarboxylase protein, since this enzyme represents only about 1 part in 10000 of the cytosolic protein.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

Purification and characterization of the plasminogen activator inhibitors PAI-1, PAI-2, and PN-1 from the human glioblastoma U138

1993 ◽  
Vol 71 (5-6) ◽  
pp. 248-254 ◽  
Author(s):  
Patricia G. Murphy ◽  
Steven P. Lenz ◽  
Mark Dobson ◽  
Allan D. Arndt ◽  
David A. Hart
Keyword(s):  
Monoclonal Antibody ◽  
Plasminogen Activator ◽  
Concanavalin A ◽  
Northern Blot ◽  
Northern Blot Analysis ◽  
Plasminogen Activator Inhibitor ◽  
Immunoaffinity Column ◽  
Positive Hybridization ◽  
Activator Inhibitor ◽  
Pai 1

This investigation presents data which indicate that the plasminogen activator inhibitor (PAI) activity secreted from U138 cells is composed of three separate PAIs: PAI-1, PAI-2, and PN-1. It was demonstrated that the U138 PAI-1-like protein had an apparent molecular mass of 50 kilodaltons (kDa) and was purified to apparent homogeneity by elution from an anti-PAI-1 immunoaffinity column. These fractions were also reactive with a second anti-PAI-1 monoclonal antibody using immunoblotting techniques. Northern blot analysis of RNA isolated from unstimulated U138 cells demonstrated positive hybridization with the cDNA specific for human PAI-1. The U138 PAI-2-like protein was adherent to an anti-PAI-2 immunoaffinity column and was demonstrated to be nonadherent to concanavalin A – agarose, heparin–Sepharose, and the anti-PAI-1 immunoaffinity column. The eluted U138 PAI-2-like protein was demonstrated to have an apparent molecular mass of 60 kDa and was also reactive with a second anti-PAI-2 monoclonal antibody using immunoblotting techniques. Further, the cDNA specific for PAI-2 was demonstrated to hybridize to a 2.5-kilobase message from RNA isolated from U138 cells. A third PAI was detected that was nonadherent to concanavalin A – agarose and both of the anti-PAI columns. This 50-kDa PAI was adherent to heparin–Sepharose and thrombin–agarose columns, and was not reactive with any antibodies for either PAI-1 or PAI-2. Northern blot analysis of U138 RNA demonstrated positive hybridization with an oligodeoxynucleotide specific for PN-1. This investigation demonstrates with biochemical, immunological, and molecular data that the U138 glioblastoma constitutively produces three PAIs.Key words: plasminogen activator inhibitor, U138 glioblastoma, PAI purification, human tumor cell line, proteinase inhibitors.

Heterogenous expression of ornithine decarboxylase gene in the proximal tubule of the mouse kidney following testosterone treatment

1993 ◽  
Vol 100 (5) ◽  
pp. 325-330 ◽  
Author(s):  
Noriyuki Koibuchi ◽  
Shigeru Matsuzaki ◽  
Mikako Sakai ◽  
Hideki Ohtake ◽  
Sadao Yamaoka
Keyword(s):  
Proximal Tubule ◽  
Ornithine Decarboxylase ◽  
Mouse Kidney ◽  
Testosterone Treatment

Investigation of structure and rate of synthesis of ornithine decarboxylase protein in mouse kidney

Biochemistry ◽  
1984 ◽  
Vol 23 (16) ◽  
pp. 3777-3783 ◽  
Author(s):  
Lo Persson ◽  
James E. Seely ◽  
Anthony E. Pegg
Keyword(s):  
Ornithine Decarboxylase ◽  
Mouse Kidney
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