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.
Link to the article here.
