A novel mouse monoclonal antibody c42 against c-terminal peptide of alpha-1-antitrypsin
(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.
(Less)
- author
- Tumpara, Srinu ; Korenbaum, Elena ; Kühnel, Mark ; Jonigk, Danny ; Olejnicka, Beata LU ; Davids, Michael ; Welte, Tobias ; Martinez-Delgado, Beatriz and Janciauskiene, Sabina
- organization
- publishing date
- 2021
- 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
-
- scopus:85100940407
- pmid:33670003
- 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}}, }