Open AccessDevelopment of a nano-gold immunodiagnostic assay for rapid on-site detection of invasive aspergillosis
Author(s) -
Komal M. Raval,
Vandana Ghormade,
P. R. Rajamohanan,
Hansraj Choudhary,
Shivaprakash M. Rudramurthy,
Arunaloke Chakrabarti,
Kishore M. Paknikar
Publication year - 2019
Publication title -
journal of medical microbiology/journal of medical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.91
H-Index - 117
eISSN - 1473-5644
pISSN - 0022-2615
DOI - 10.1099/jmm.0.001040
Subject(s) - immunoassay , aspergillus fumigatus , galactomannan , aspergillosis , aspergillus , aspergillus flavus , dot blot , polyclonal antibodies , detection limit , microbiology and biotechnology , biology , allergic bronchopulmonary aspergillosis , antibody , chemistry , immunoglobulin e , immunology , chromatography , biochemistry , dna
. 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.