Moloney, Nicola M. and Larkin, Annemarie and Xu, Linan and Fitzpatrick, David A. and Crean, Holly L. and Walshe, Kieran and Haas, Hubertus and Decristoforo, Clemens and Doyle, Sean
(2021)
Generation and characterisation of a semi-synthetic siderophore-immunogen conjugate and a derivative recombinant triacetylfusarinine C–specific monoclonal antibody with fungal diagnostic application.
Analytical Biochemistry, 632.
p. 114384.
ISSN 0003-2697
Abstract
Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinatorial strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2a monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production.
| Item Type: |
Article
|
| Keywords: |
Siderophore;
Immunogen;
Hapten;
Aspergillus fumigatus;
Conjugate vaccine;
Competitive ELISA;
Lateral flow test;
SARS-CoV-2;
COVID-19; |
| Academic Unit: |
Faculty of Science and Engineering > Biology |
| Item ID: |
17041 |
| Identification Number: |
https://doi.org/10.1016/j.ab.2021.114384 |
| Depositing User: |
Dr. Sean Doyle
|
| Date Deposited: |
20 Mar 2023 12:20 |
| Journal or Publication Title: |
Analytical Biochemistry |
| Publisher: |
Elsevier Masson |
| Refereed: |
Yes |
| URI: |
|
| Use Licence: |
This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available
here |
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