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A novel mouse monoclonal antibody c42 against c-terminal peptide of alpha-1-antitrypsin

Tumpara, Srinu ; Korenbaum, Elena ; Kühnel, Mark ; Jonigk, Danny ; Olejnicka, Beata LU ; Davids, Michael ; Welte, Tobias ; Martinez-Delgado, Beatriz and Janciauskiene, Sabina (2021) In International Journal of Molecular Sciences 22(4).
Abstract

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 mono-clonal 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... (More)

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 mono-clonal 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.

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author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Alpha1-antitrypsin, C-terminal peptide, C42 clone, Immunoassays, Molecular forms, Monoclonal antibody
in
International Journal of Molecular Sciences
volume
22
issue
4
article number
2141
pages
16 pages
publisher
MDPI AG
external identifiers
  • pmid:33670003
  • scopus:85100940407
ISSN
1661-6596
DOI
10.3390/ijms22042141
language
English
LU publication?
yes
id
c8f0d279-6ff0-4509-88bd-46acbce6ed76
date added to LUP
2021-03-02 07:57:47
date last changed
2024-08-08 13:16:55
@article{c8f0d279-6ff0-4509-88bd-46acbce6ed76,
  abstract     = {{<p>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 mono-clonal 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.</p>}},
  author       = {{Tumpara, Srinu and Korenbaum, Elena and Kühnel, Mark and Jonigk, Danny and Olejnicka, Beata and Davids, Michael and Welte, Tobias and Martinez-Delgado, Beatriz and Janciauskiene, Sabina}},
  issn         = {{1661-6596}},
  keywords     = {{Alpha1-antitrypsin; C-terminal peptide; C42 clone; Immunoassays; Molecular forms; Monoclonal antibody}},
  language     = {{eng}},
  number       = {{4}},
  publisher    = {{MDPI AG}},
  series       = {{International Journal of Molecular Sciences}},
  title        = {{A novel mouse monoclonal antibody c42 against c-terminal peptide of alpha-1-antitrypsin}},
  url          = {{http://dx.doi.org/10.3390/ijms22042141}},
  doi          = {{10.3390/ijms22042141}},
  volume       = {{22}},
  year         = {{2021}},
}