Immunoassay-based quantification of full-length peptidylglycine alpha-amidating monooxygenase in human plasma
Ilina, Yulia ; Kaufmann, Paul ; Melander, Olle LU
; Press, Michaela
; Thuene, Katrin
and Bergmann, Andreas
(2023)
In Scientific Reports
13(1).
- Abstract
A one-step sandwich chemiluminescence immunometric assay (LIA) was developed for the quantification of bifunctional peptidylglycine-α-amidating monooxygenase (PAM) in human plasma (PAM-LIA). PAM is responsible for the activation of more than half of known peptide hormones through C-terminal α-amidation. The assay employed antibodies targeting specific catalytic PAM-subunits, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), to ensure detection of full-length PAM. The PAM-LIA assay was calibrated with a human recombinant PAM enzyme and achieved a detection limit of 189 pg/mL and a quantification limit of 250 pg/mL. The assay demonstrated good inter-assay (6.7%) and... (More)
A one-step sandwich chemiluminescence immunometric assay (LIA) was developed for the quantification of bifunctional peptidylglycine-α-amidating monooxygenase (PAM) in human plasma (PAM-LIA). PAM is responsible for the activation of more than half of known peptide hormones through C-terminal α-amidation. The assay employed antibodies targeting specific catalytic PAM-subunits, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), to ensure detection of full-length PAM. The PAM-LIA assay was calibrated with a human recombinant PAM enzyme and achieved a detection limit of 189 pg/mL and a quantification limit of 250 pg/mL. The assay demonstrated good inter-assay (6.7%) and intra-assay (2.2%) variabilities. It exhibited linearity when accessed by gradual dilution or random mixing of plasma samples. The accuracy of the PAM-LIA was determined to be 94.7% through spiking recovery experiments, and the signal recovery after substance interference was 94–96%. The analyte showed 96% stability after six freeze–thaw cycles. The assay showed strong correlation with matched EDTA and serum samples, as well as matched EDTA and Li-Heparin samples. Additionally, a high correlation was observed between α-amidating activity and PAM-LIA. Finally, the PAM-LIA assay was successfully applied to a sub-cohort of a Swedish population-based study, comprising 4850 individuals, confirming its suitability for routine high throughput screening.
(Less)
- author
- Ilina, Yulia
; Kaufmann, Paul
; Melander, Olle
LU
; Press, Michaela
; Thuene, Katrin
and Bergmann, Andreas
- organization
- publishing date
- 2023
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Scientific Reports
- volume
- 13
- issue
- 1
- article number
- 10827
- publisher
- Nature Publishing Group
- external identifiers
-
- pmid:37402878
- scopus:85164246398
- ISSN
- 2045-2322
- DOI
- 10.1038/s41598-023-37976-3
- language
- English
- LU publication?
- yes
- id
- cb205f05-8cdb-4a79-8132-308b8bf0f5f1
- date added to LUP
- 2023-08-24 14:54:21
- date last changed
- 2024-04-20 01:36:02
@article{cb205f05-8cdb-4a79-8132-308b8bf0f5f1,
abstract = {{<p>A one-step sandwich chemiluminescence immunometric assay (LIA) was developed for the quantification of bifunctional peptidylglycine-α-amidating monooxygenase (PAM) in human plasma (PAM-LIA). PAM is responsible for the activation of more than half of known peptide hormones through C-terminal α-amidation. The assay employed antibodies targeting specific catalytic PAM-subunits, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), to ensure detection of full-length PAM. The PAM-LIA assay was calibrated with a human recombinant PAM enzyme and achieved a detection limit of 189 pg/mL and a quantification limit of 250 pg/mL. The assay demonstrated good inter-assay (6.7%) and intra-assay (2.2%) variabilities. It exhibited linearity when accessed by gradual dilution or random mixing of plasma samples. The accuracy of the PAM-LIA was determined to be 94.7% through spiking recovery experiments, and the signal recovery after substance interference was 94–96%. The analyte showed 96% stability after six freeze–thaw cycles. The assay showed strong correlation with matched EDTA and serum samples, as well as matched EDTA and Li-Heparin samples. Additionally, a high correlation was observed between α-amidating activity and PAM-LIA. Finally, the PAM-LIA assay was successfully applied to a sub-cohort of a Swedish population-based study, comprising 4850 individuals, confirming its suitability for routine high throughput screening.</p>}},
author = {{Ilina, Yulia and Kaufmann, Paul and Melander, Olle and Press, Michaela and Thuene, Katrin and Bergmann, Andreas}},
issn = {{2045-2322}},
language = {{eng}},
number = {{1}},
publisher = {{Nature Publishing Group}},
series = {{Scientific Reports}},
title = {{Immunoassay-based quantification of full-length peptidylglycine alpha-amidating monooxygenase in human plasma}},
url = {{http://dx.doi.org/10.1038/s41598-023-37976-3}},
doi = {{10.1038/s41598-023-37976-3}},
volume = {{13}},
year = {{2023}},
}