@article {46254, title = { Development of a nano-gold immunodiagnostic assay for rapid on-site detection of invasive aspergillosis }, journal = {Journal of Medical Microbiology}, volume = {68}, year = {2019}, month = {SEP}, pages = {1341-1352}, type = {Article}, abstract = {

Introduction. Timely detection of invasive aspergillosis (IA) caused by fungal pathogens, i.e. Aspergillus fumigatus and Aspergillus flavus, in immunocompromised patients is crucial in preventing high mortality.

Aim. To develop a simple immunoassay for the detection of galactomannan (GM), an IA biomarker.

Methodology. GM from A. fumigatus and A. flavus clinical strains was purified and characterized by X-ray diffraction, IR spectroscopy and C-13/H-1 nuclear magnetic resonance (NMR) for polyclonal antibody (pAb) production in rabbits. An enzyme-linked immunosorbent assay (ELISA) was standardized using concanavalin A to capture Aspergillus GM and pAbs to detect it. Gold nanoparticles (AuNPs) were synthesized and conjugated to pAbs for the development of a dot-blot immunoassay. The developed dot-blot was evaluated with 109 clinical serum and bronchoalveolar lavage samples.

Results. Spectroscopy studies characterized the D-galactofuranosyl groups of GM responsible for the immune response and generation of pAbs. The ELISA employing pAbs showed a sensitivity of 1 ng ml(-1) for Aspergillus GM. Furthermore, a sensitive, visual, rapid dot-blot assay developed by the conjugation of pAbs to AuNPs (similar to 24 +/- 5 nm size, -36 +/- 2 mV zeta potential) had a detection limit of 1 pg ml(-1) in serum. The pAbs interacted with Aspergillus spp. but did not cross-react with other fungal pathogen genera such as Penicillium and Candida. Evaluation of the dot-blot with 109 clinical samples showed high sensitivity (80\%) and specificity (93.2 \%), with an overall assay accuracy of 89\%.

Conclusion. The developed nano-gold immunodiagnostic assay has immense potential for practical use in rapid, specific and sensitive on-site diagnosis of IA, even under resource-limited settings.

}, doi = {10.1099/jmm.0.001040}, author = {Raval, Komal M. and Ghormade, Vandana and Rajamohanan, P. R. and Choudhary, Hansraj and Rudramurthy, Shivaprakash M. and Chakrabarti, Arunaloke and Paknikar, Kishore} }