Development of a nano-gold immunodiagnostic assay for rapid on-site detection of invasive aspergillosis. Issue 9 (29th July 2019)

• Record Type: Journal Article
• Authors: Raval, Komal M.; Ghormade, Vandana; Rajamohanan, P. R.; Choudhary, Hansraj; Rudramurthy, Shivaprakash M.; Chakrabarti, Arunaloke; Paknikar, Kishore
• Is Part Of: Journal of medical microbiology. Volume 68:Issue 9(2019)
• Page Start: 1341
• Page End: 1352
• Publication Date: 2019-07-29
• Subjects: invasive aspergillosis -- Aspergillus -- galactomannan -- gold nanoparticles -- dot blot assay; Medical microbiology -- Periodicals; 616.9041
• DOI: 10.1099/jmm.0.001040
• Languages: English
• ISSNs: 0022-2615

Abstract

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 13 C/ 1 H 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.