@article{mbs:/content/journal/jmm/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", title = "Development of a nano-gold immunodiagnostic assay for rapid on-site detection of invasive aspergillosis", journal= "Journal of Medical Microbiology", year = "2019", volume = "68", number = "9", pages = "1341-1352", doi = "https://doi.org/10.1099/jmm.0.001040", url = "https://www.microbiologyresearch.org/content/journal/jmm/10.1099/jmm.0.001040", publisher = "Microbiology Society", issn = "1473-5644", type = "Journal Article", keywords = "gold nanoparticles", keywords = "invasive aspergillosis", keywords = "galactomannan", keywords = "Aspergillus", keywords = "dot blot assay", 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 13C/1H 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 (~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.", }