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. 2008 Jul;15(7):1095-105.
doi: 10.1128/CVI.00068-08. Epub 2008 May 7.

Development of an immunochromatographic lateral-flow device for rapid serodiagnosis of invasive aspergillosis

Christopher R Thornton  1
Affiliations

Development of an immunochromatographic lateral-flow device for rapid serodiagnosis of invasive aspergillosis

Christopher R Thornton. Clin Vaccine Immunol. 2008 Jul.

Abstract

Aspergillus fumigatus is a cosmopolitan saprotrophic fungus that is second only to Candida species as a cause of invasive fungal infections in immunocompromised humans. Current immunodiagnostic tests for invasive aspergillosis (IA) are based on the detection of circulating galactomannan (GM) in a patient's serum by using a rat monoclonal antibody (MAb), EB-A2, that binds to tetra (1-->5)-beta-D-galactofuranoside, the immunodominant epitope in GM. The potential cross-reactivity of MAb EB-A2 with non-Aspergillus fungi, with contaminating GM in beta-lactam antibiotics and foodstuffs, and with bacterial lipoteichoic acids has prompted efforts to discover non-GM antigens that can act as surrogate markers for the diagnosis of IA. This paper describes the development of a mouse MAb, JF5, that binds to a protein epitope present on an extracellular glycoprotein antigen secreted constitutively during the active growth of A. fumigatus. The MAb was used to develop an immunochromatographic lateral-flow device (LFD) for the rapid (15-min) detection of Aspergillus antigens in human serum. The test is highly specific, reacting with antigens from Aspergillus species but not with antigens from a large number of clinically important fungi, including Candida species, Cryptococcus neoformans, Fusarium solani, Penicillium marneffei, Pseudallescheria boydii, and Rhizopus oryzae. The LFD was able to detect circulating antigen in serum samples from patients suspected of having or shown to have IA on the basis of their clinical symptoms and results from tests for GM and fungal (1-->3)-beta-D-glucan. The ease of use of the LFD provides a diagnostic platform for the routine testing of vulnerable patients who have an elevated risk of IA.

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Figures

FIG. 1.
FIG. 1.
Analysis of affinity-purified antigen by PAGE and Western blotting. Lane Mr, molecular mass marker; lane A, Western immunoblot with MAb JF5 after separation of purified antigen by sodium dodecyl sulfate-PAGE under reducing conditions; the well was loaded with 0.2 μg of protein; lane B, Western immunoblot with MAb JF5 after treatment of purified antigen with PNGase and separation by sodium dodecyl sulfate-PAGE under denaturing conditions; the well was loaded with 0.2 μg of protein.
FIG. 2.
FIG. 2.
Photomicrographs of A. fumigatus AF293 cells immunostained with MAb JF5 and anti-mouse polyvalent Ig fluorescein isothiocyanate. (A) Germlings examined under a bright-field microscope. (B) Same slide shown in panel A but examined under epifluorescence. Note the intense staining of the cell walls of the germ tubes but the lack of staining in ungerminated conidia (arrows). (C) Hypha examined under a bright-field microscope. (D) Same slide shown in panel C but examined under epifluorescence. Note the intense staining of the cell wall and secretion of the antigen at the growing tip (arrow). Bars, 6 μm.
FIG. 3.
FIG. 3.
Immunogold localization of the JF5 antigen in cells of A. fumigatus AF293. A longitudinal section of a germling grown in human serum shows the localization of the antigen in the cell walls of the germ tube (GT) and swollen conidium, in the septum (S), and in a surrounding capsular-like layer (C). Bar, 0.5 μm.
FIG. 4.
FIG. 4.
Serum LFD tests. (A) LFD tests with normal human serum following inoculation with fungi and incubation for 48 h at 37°C. Negative reactions (single control line only) exhibited by Candida albicans (LFD 1), Pseudallescheria boydii (LFD 2), Rhizopus oryzae (LFD 3), and Fusarium solani (LFD 4) and a positive reaction (two lines) exhibited by Aspergillus fumigatus (LFD 5) are shown. (Β) LFD tests of normal human serum (LFD 1) and serum spiked with affinity-purified antigen (LFD 2) at a concentration of 2.5 μg protein/ml. (C) Examples of results from LFD tests with serum samples from healthy individuals or patients with confirmed IA. Negative, weak, and strong reactions exhibited by specimens 9OHD (LFD 1), 1657 (LFD 2), and 1131 (LFD 3), respectively, are shown. Specimen numbers relate to those shown in Table 4.
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