CLINICAL AND VACCINE IMMUNOLOGY, July 2008, p. 1095–1105
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 15, No. 7
Development of an Immunochromatographic Lateral-Flow Device for
Rapid Serodiagnosis of Invasive Aspergillosis?
Christopher R. Thornton*
Hybridoma Laboratory, School of Biosciences, Geoffrey Pope Building, University of Exeter, Stocker Road,
Exeter, Devon EX4 4QD, United Kingdom
Received 22 February 2008/Returned for modification 14 March 2008/Accepted 29 April 2008
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 (135)-?-D-galactofurano-
side, the immunodominant epitope in GM. The potential cross-reactivity of MAb EB-A2 with non-
Aspergillus fungi, with contaminating GM in ?-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, Pseud-
allescheria 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 (133)-?-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.
The dramatic increase in the numbers of opportunistic in-
fections of humans caused by Aspergillus species over the last
decade is associated with a rise in the numbers of solid-organ
transplants and the use of aggressive cancer therapies and
other immunomodulating treatments (4, 15). The rate of mor-
tality due to invasive aspergillosis (IA) has increased by 357%
over the last 25 years; and IA has become one of the leading
causes of death in immunocompromised patients, with mortal-
ity rates ranging from 60 to 90% (21), even following the recent
introduction of new broad-spectrum antifungal agents. The
most common species of Aspergillus causing invasive disease
include Aspergillus fumigatus, A. flavus, A. niger, A. terreus, and
A. nidulans (7, 25). Other less common species can also cause
the disease, but A. fumigatus accounts for ?90% of all cases of
In the absence of a single “gold standard” test for diag-
nosis of the disease, the definitive diagnosis of IA encom-
passes data from clinical, radiological, serological, molecu-
lar biological, mycological, and histopathological sources. It
is imperative that a diagnosis be made without delay, since
the prognosis worsens significantly in the absence of recog-
nition and effective treatment. The rapid detection of IA by
immunodiagnostic methods has centered around the detec-
tion of fungal galactomannan (GM) (16, 24, 25). Monoclo-
nal antibodies (MAbs) have successfully been used to detect
GM, and they form the basis of commercial laboratory-
based tests, such as the Platelia Aspergillus enzyme-linked
immunosorbent assay (ELISA) kit that incorporates a rat
MAb (MAb EB-A2) directed against tetra (135)-?-D-ga-
lactofuranoside, the immunodominant epitope in the anti-
gen (23, 31, 32). Immunoassays for GM detection are a
significant asset for the management of patients at risk of IA
because of detection of the antigen in the early stages of
disease progression. Despite their widespread use, recent
studies have revealed significant variation in performance.
While the specificity of the GM assay is consistently ?85%,
the sensitivity of the assay can vary considerably from 29%
to 100% and the rate of false-positive reactivity can vary
from 5% in adults to 83% in newborn babies (39). False-
positive results have been attributed to the cross-reaction of
MAb EB-A2 with GM from non-Aspergillus fungi (8, 12, 25,
34, 39); with galactoxylomannan from Cryptococcus neofor-
mans (5, 6); with lipoteichoic acid from intestinal bifidobac-
teria in the gastrointestinal microbiota of neonates (22);
with the cancer prodrug cyclophosphamide (10); and with
the GM in food, drink, and infant milk formulas (1). Con-
tamination of ?-lactam antibiotics with Penicillium GM may
account for the serum reactivity of patients receiving pip-
eracillin-tazobactam or amoxicillin-clavulanic acid (2, 20,
39, 40), although these reports have been disputed (46).
There is therefore scope in IA immunodiagnostics for tests
that employ MAbs directed at epitopes other than those
present on GM. While a “panfungal” test that detects fungal
(133)-?-D-glucan has been used for the diagnosis of inva-
sive fungal infections (24, 25), its lack of specificity means
that it is unable to discriminate between Aspergillus species
* Mailing address: Hybridoma Laboratory, School of Biosciences,
Geoffrey Pope Building, University of Exeter, Stocker Road, Exeter,
Devon EX4 4QD, United Kingdom. Phone: 44 (0)1392 264653. Fax: 44
(0)1392 263434. E-mail: C.R.Thornton@ex.ac.uk.
?Published ahead of print on 7 May 2008.
and other opportunistic pathogens, which compromises the
ability to select the most appropriate antifungal agent. In
contrast, an ELISA used to detect the Afmp1p cell wall
antigen of A. fumigatus in a patient’s serum provides a high
degree of specificity but does not allow the detection of IA
caused by other Aspergillus species (45). Furthermore, com-
binations of antibody and antigen testing of serum samples
are required to provide serodiagnostic sensitivities for A.
fumigatus IA detection comparable to those of tests for GM.
The development of a noninvasive immunodiagnostic test
that is rapid, reliable, and relatively inexpensive and that
detects surrogate (non-GM and non-Afmp1p) markers for
IA would allow the routine testing of vulnerable patients
who have an elevated risk of infection, such as allogeneic
hematopoietic stem cell transplant recipients, patients with
hematological malignancies, and recipients of solid-organ
transplants, especially of the lung. The aim of this paper is
to report on the development of a mouse hybridoma cell line
secreting an Aspergillus protein-specific MAb (MAb JF5)
and its utilization in the development of a lateral-flow device
(LFD) for the rapid serodiagnosis of IA. The assay exploits
the lateral-flow technology that has been used to date in
tests for the detection of viruses, bacteria, and toxins (11, 13,
28–30) and, most famously, for the home pregnancy tests
first introduced by Unipath in 1988. While immunochro-
matographic assays have been developed for the identifica-
tion of Candida species (19) and for the detection of fungi in
soil (36, 37), this is the first time, to the best of the author’s
knowledge, that an LFD has been developed for the detec-
tion of Aspergillus antigens in human serum.
Current diagnostic tests for IA are confined to laboratories
equipped to perform tests for the detection of GM or ?-glucan
or nucleic acid-based diagnostic tests. The simplicity of the
LFD format allows it to be used with minimal training and
provides an additional diagnostic platform for the manage-
ment of IA in high-risk patient groups. The ability of the LFD
to detect Aspergillus antigens in clinical samples is demon-
strated with sera from IA patients.
MATERIALS AND METHODS
Fungal culture. All fungi were cultured on Sabouraud agar (SA) under a 16-h
fluorescent light regimen.
Development of MAb, preparation of immunogen, and immunization regimen.
Mice were immunized with lyophilized mycelium (LM) of A. fumigatus
AF293. Minimal medium [19 mM (NH4)2PO4, 0.5% (wt/vol) yeast extract, 7
mM sodium citrate, 2 mM MgSO4? 7H2O, 0.5 mM CaCl2? 2H2O, and 50 mM
glucose adjusted to pH 5.5 with 1 N HCl] was sterilized by autoclaving at
121°C for 15 min. Three-week-old SA petri dish cultures of the fungus were
flooded with 20 ml distilled H2O (dH2O), and the conidia were suspended by
gentle agitation with an inoculation loop. Spore suspensions were filtered
through Miracloth to remove the mycelium, and the filtrate containing the
conidia was transferred to 1.5-ml microcentrifuge tubes. The conidia were
washed three times with dH2O by repeated vortexing and centrifugation at
12,000 ? g for 5 min and were finally suspended in dH2O to give a concen-
tration of 106conidia/ml solution. Flasks containing 150 ml of medium were
inoculated with 200 ?l of the conidial suspension and incubated with shaking
(150 rpm) for 24 h at 37°C. The mycelium was collected by filtering the
contents of each flask through Miracloth, snap frozen in liquid N2, and
One milligram of LM was suspended in 1 ml of phosphate-buffered saline
(PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4[pH 7.2]).
Six-week-old BALB/c white female mice were given four intraperitoneal injec-
tions (300 ?l per injection) of immunogen at 2-week intervals and a single
booster injection 5 days before fusion.
Production and screening of hybridomas and determination of antibody spec-
ificity. Hybridoma cells were produced by the method described elsewhere (35),
and the supernatants were screened by ELISA against soluble antigens extracted
from LM in PBS and immobilized to the wells of Maxisorp microtiter plates (50
?l per well). For antibody specificity tests, fungi were grown on SA and surface
washings prepared in PBS as described by Thornton (35). Protein concentra-
tions, determined spectrophotometrically at 280 nm (Nanodrop; Agilent Tech-
nologies Limited, Berkshire, United Kingdom), were adjusted to 64 ?g/ml buffer,
and 50-?l volumes were used to coat the wells of microtiter plates. After coating
of the plates overnight at 4°C, the wells were washed four times with PBS
containing 0.05% (vol/vol) Tween 20 (PBST) and once each with PBS and dH2O
and air dried at 23°C in a laminar-flow hood. The plates were stored in sealed
plastic bags at 4°C in preparation for screening of the hybridoma supernatants by
ELISA, as described below.
ELISA. Wells containing immobilized antigens were successively incubated
with hybridoma supernatant for 1 h, followed by goat anti-mouse polyvalent
(immunoglobulin G [IgG], IgA, and IgM classes) peroxidase conjugate (Sigma
Chemical Company, Poole, United Kingdom) diluted 1 in 1,000 in PBST for a
further hour. Bound antibody was visualized by incubation of the wells with
tetramethylbenzidine substrate solution for 30 min, and the reactions were
stopped by the addition of 3 M H2SO4. Absorbance values were determined at
450 nm with an MRX automated microplate reader (Dynex Technologies, Bill-
ingshurst, United Kingdom). The wells were given four 5-min rinses with PBST
between incubations. Working volumes were 50 ?l per well, and control wells
were incubated with tissue culture medium (TCM) containing 10% (vol/vol) fetal
calf serum. All incubation steps were performed at 23°C in sealed plastic bags.
The threshold for the detection of antigen by ELISA was determined from the
control means (2? TCM absorbance values) (33). These values were consistently
in a range from 0.050 to 0.100. Consequently, absorbance values ?0.100 were
considered positive for the detection of antigen.
Determination of Ig subclass and cloning procedure. The Ig class of the MAbs
was determined with a commercial mouse MAb isotyping kit (ISO-1), according
to the manufacturer’s instructions (Sigma). Hybridoma cell lines were cloned by
limiting dilution; and the cell lines were grown in bulk in a nonselective medium,
preserved by slowly freezing them in fetal bovine serum-dimethyl sulfoxide (92:8
[vol/vol]), and stored in liquid nitrogen.
Epitope characterization by protease digestion. Microtiter wells containing
immobilized antigens were incubated with pronase (0.25 U per well; Protease
XIV; Sigma) or trypsin (Sigma) solution (1 mg/ml in PBS) at 37°C or 4°C for
5 h and washed three times with PBS. Wells incubated with trypsin were
treated for 10 min with a 0.1-mg/ml solution of trypsin inhibitor (Sigma) and
given three more washes with PBS. Controls received PBS without pronase or
trypsin and inhibitor but were otherwise treated similarly. The wells were
assayed by ELISA with MAb JF5 as described above. There were six repli-
cates for each treatment.
Epitope characterization by periodate oxidation. The immobilized antigens
were treated with sodium meta-periodate (20 mM NaIO4in 50 mM sodium
acetate buffer [pH 4.5]), whereas the control wells received only buffer. After
incubation for the appropriate time period in darkness at 4°C, the wells were
washed three times with PBS and assayed by ELISA with MAb JF5 as described
above. There were four replicates for each treatment.
Antigen purification, PAGE, and Western blotting. Antigen was purified from
PBS extracts of LM by affinity chromatography with a Protein A IgG Plus
Orientation kit (Pierce Biotechnology, Rockford, IL) containing immobilized
MAb JF5. Ascitic fluid was prepared from JF5 hybridoma cells in female BALB/c
mice (Eurogentec s.a., Belgium). The mice were injected with 106hybridoma
cells washed in PBS, and after 3 weeks, approximately 5 ml of ascitic fluid was
recovered from each mouse and was stored at ?20°C prior to use. For prepa-
ration of the affinity column, 15 ?l of ascitic fluid was mixed with 2 ml of binding
buffer and the solution was applied to the protein A-agarose matrix. Crude PBS
antigen extract was then incubated with the immobilized antibody and bound
antigen was eluted with 0.1 M glycine-HCl (pH 2.8) buffer. Polyacrylamide gel
electrophoresis (PAGE) was carried out by using the system of Laemmli (14)
with 4 to 20% (wt/vol) gradient polyacrylamide gels (Bio-Rad Laboratories
Limited, Hemel Hempstead, United Kingdom) under denaturing conditions.
Purified antigen was mixed with Laemmli buffer and denatured by heating at
95°C for 10 min in the presence of ?-mercaptoethanol before it was loaded onto
the gel. The proteins were separated for 1.5 h at 23°C (165 V). Prestained,
broad-range markers (Bio-Rad) were used for molecular mass determinations.
For Western blotting, the separated proteins were transferred electrophoretically
to a polyvinylidene difluoride membrane (Bio-Rad). The membranes were
washed three times with PBS and then blocked for 16 h at 4°C with PBS
containing 1% (wt/vol) bovine serum albumin (BSA). The blocked membranes
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