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
1096THORNTONCLIN. VACCINE IMMUNOL.
TABLE 1. Details of organisms and results of ELISA specificity tests
Eurotium amstelodami34 CRT 0.866
Aspergillus restrictus 116.50CBS0.938
Neosartorya fischeri var. fischeri
A. ornatus (Hemicarpenteles ornatus) 184CRT 1.381
Aspergillus clavatus514.65 CBS1.307
Aspergillus nidulans (Emericella nidulans var. nidulans)
Aspergillus versicolor599.65CBS 1.120
Aspergillus ustus209.92CBS 0.510
Aspergillus terreus var. terreus601.65 CBS1.186
Aspergillus niveus (Fennelia nivea) 261.73CBS1.085
Aspergillus wentii 229.67CBS 0.000
Continued on following page
VOL. 15, 2008ASPERGILLUS LATERAL-FLOW DEVICE1097
1.066 Aspergillus niger 553.65CBS
Aspergillus ochraceous625.78 CBS1.249
Aspergillus candidus266.81CBS 0.541
Fusarium oxysporum f. sp. melonis
Fusarium oxysporum f. sp. pisi
Fusarium solani var. petrophilum
Mucor hiemalis var. silvaticus
Rhizopus microsporus var. rhizopodiformis
Rhizopus sexualis var. sexualis
aCBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; FGSC, Fungal Genetics Stock Centre, University of Missouri, Kansas City; CRT, C. R.
Thornton; IMI, International Mycological Institute, Egham, England; SB, S. Bates, School of Biosciences, University of Exeter; SV, S. Krappman, Institute of
Microbiology and Genetics, Department of Molecular Microbiology and Genetics, Georg-August-University, Gottingen, Germany.
bEach value represents the mean of replicate values. Threshold absorbance value for detection of antigen, ?0.100.
1098THORNTONCLIN. VACCINE IMMUNOL.
were incubated with the MAb JF5 supernatant diluted 1 in 2 with PBS containing
0.5% (wt/vol) BSA (PBSA) for 2 h at 23°C. After the membranes were washed
three times with PBS, they were incubated for 1 h with goat anti-mouse IgG
(whole molecule) alkaline phosphatase conjugate (Sigma) diluted 1 in 15,000 in
PBSA. The membranes were washed twice with PBS and once with PBST, and
the bound antibody was visualized by incubation in substrate solution. The
reactions were stopped by immersion in dH2O and air dried between sheets of
Whatman filter paper. Modification of the JF5 antigen with peptide-N-glycosi-
dase (PNGase) was carried out prior to electrophoresis and Western blotting,
according to procedures described elsewhere (3).
Immunofluorescence and immunogold electron microscopy of A. fumigatus
conidia and germlings. Immunogold labeling was performed with germlings of A.
fumigatus AF293. Germlings were prepared by incubating washed conidia in
normal human serum (Biosera, Ringmer, United Kingdom) or in sterile filtered
(pore size, 0.2 ?m) 1% (wt/vol) glucose solution for 16 h at 37°C with gentle
mixing. The germlings were pelleted by centrifugation, and low-temperature
embedding of the material was carried out as described elsewhere (38). Immu-
nolabeling was carried out with MAb JF5 and goat anti-mouse 20-nm-diameter
gold conjugate (British Biocell International, Cardiff, Wales) as the secondary
reporter molecule. Control grids were incubated with TCM instead of the MAb
supernatant but were otherwise treated the same. For the immunofluorescence
studies, the washed conidia were suspended in glucose solution and transferred
to the wells of multiwell slides. After incubation at 37°C for 16 h, the slides were
air dried and fixed as described by Thornton (35). The wells were incubated for
1 h with 50 ?l of the MAb JF5 supernatant or TCM only. The slides were washed
three times with PBS with gentle agitation and incubated for a further 30 min
with goat anti-mouse polyvalent fluorescein isothiocyanate conjugate (Sigma)
diluted 1 in 40 in PBS. The slides were given three 5-min rinses with PBS, and the
wells were overlaid with coverslips mounted in PBS-glycerol mounting medium
(Sigma). The slides were examined with a Zeiss Axiophot microscope fitted with
epifluorescence by using a UV excitation filter of 365 nm and an absorption filter
of 420 nm. All incubation steps were performed at 23°C in a moist environment,
and the slides were stored at 4°C in the dark in petri dishes containing moistened
Whatman no. 1 filter paper.
Configuration of the LFD. The LFD consisted of a G&L Diecut 1734 backing
card; Whatman 17chr and 1281 top and sample pads, respectively; and a What-
man Immunopore 5 ?M nitocellulose membrane. MAb JF5 was conjugated to
40-nm-diameter gold particles, applied to the release pad at 100 units of conju-
gate/cm, and dried for 16 h at 37°C. The test line antibody consisted of MAb JF5
at 0.5 mg protein/ml of PBS containing 1% (wt/vol) BSA, while a commercial
rabbit anti-mouse Ig acted as the control line.
Sensitivity and specificity of the LFD. Affinity-purified antigen (protein con-
centrations were determined as described above) was diluted into normal human
serum or PBS, and 100-?l samples were applied to the LFD. Unspiked serum
and PBS acted as the negative controls. After 15 min, the results were recorded
as positive for the presence of Aspergillus antigen (two lines) or negative (a single
control line only). The specificity of the LFD was determined by growing fungi
in normal human serum. Replicate 1-ml serum samples contained in 1.5-ml
Eppendorf tubes were inoculated with 104washed conidia from filamentous
fungi (Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Fusarium solani, Pseud-
allescheria boydii, and Rhizopus oryzae) or an equivalent number of washed yeast
cells (Candida albicans and Cryptococcus neoformans). The tubes were incubated
at 37°C with shaking (100 rpm) for 48 h, and fungal propagules were precipitated
by centrifugation. One hundred-microliter samples of neat, cell-free superna-
tants were applied to LFD devices and the results were recorded as described
above. The growth of the filamentous fungi and the yeast Candida albicans was
determined by visual appraisal of hyphal development or by increases in the
turbidity of serum samples (C. neoformans). Unspiked serum incubated under
the same conditions acted as the negative control.
Further tests of the specificity of the LFD were conducted with serum con-
taining the ?-lactam antibiotics penicillin G (Melford Laboratories Limited,
Ipswich, United Kingdom), amoxicillin (Fluka), and piperacillin (Sigma); the
?-lactamase inhibitor tazobactam (Sigma); the cancer prodrug cyclophospha-
mide (Sigma); and lipoteichoic acids from the bacteria Enterococcus faecalis and
Staphylococcus aureus (both from Sigma). Following reconstitution, 100-?l vol-
umes of solutions containing 5 mg of solid/ml serum (lipoteichoic acids) or 50 mg
solid/ml serum (antibiotics, tazobactam, and cyclophosphamide) were applied to
the LFDs and the results were recorded as described above. Unspiked serum
acted as the negative control, while serum samples containing purified antigen
and test chemicals acted as positive controls. Three replicates were performed
for each test.
LFD detection of antigen in sera from patients with IA. The ability of the LFD
to detect circulating antigen in humans with IA was tested with sera collected
from patients with known or suspected IA and from healthy controls. The
samples were kindly provided during a blind assessment of assay sensitivity and
specificity conducted in collaboration with Elizabeth Johnson (Bristol Health
Protection Agency). The samples had previously been tested by using the Platelia
GM enzyme immunoassay (EIA) and a panfungal ?-glucan test (Fungitell). One
hundred-microliter samples of undiluted serum or serum diluted 1 in 10 in
normal human serum were applied to the LFDs, and the results were recorded
as described above. Three replicates were performed for each sample.
Production of hybridoma cell lines and isotyping of MAbs.
A single fusion was performed. Cell lines were selected for
further study on the basis of the strength of the MAb reaction
in the ELISA. The JF5 cell line was selected and was subcloned
three times. The MAb from the subcloned JF5 cell line be-
longed to the IgG3 class.
MAb specificity tests. MAb JF5 was tested for specificity
against a wide range of related and unrelated fungi (Table 1).
It reacted with antigens from Aspergillus species and related
fungi from the teleomorphic genera Emericella, Eurotium, and
Neosartorya. It cross-reacted with antigens from certain Peni-
cillium species but not with Penicillium species in the subgenus
Biverticillium or teleomorphic Talaromyces species whose Pen-
icillium anamorphs belong to this subgenus. It cross-reacted
weakly with antigens from the closely related fungus Paecilo-
myces variotii but did not react with antigens from a wide range
of unrelated fungi, including the well-documented invasive
pathogens Candida albicans; Cryptococcus neoformans; or the
emerging pathogens Fusarium solani, Pseudallescheria boydii,
and Rhizopus oryzae (9, 26, 41, 42).
Characterization of antigen and effects of protease and pe-
riodate. A reduction in MAb binding in the ELISA following
treatment with pronase shows that its epitope consists of pro-
tein. Consequently, the reductions in MAb JF5 binding follow-
ing the digestion of immobilized antigen with pronase showed
that the antibody binds to a protein epitope (Table 2). More
specifically, the sensitivity of the epitope to trypsin indicated
that JF5 binds to a protein epitope containing positively
charged lysine and arginine side chains. Reductions in anti-
body binding following chemical digestion of an antigen with
periodate shows that its epitope is carbohydrate. Conse-
quently, the lack of reduction of JF5 binding in the ELISA
following periodate treatment of immobilized antigen (Table
3) showed that its epitope does not contain carbohydrate moi-
PAGE and Western blotting. The affinity-purified antigen
eluted from the column as a single peak containing 0.340 mg
protein/ml of buffer. The diffuse binding pattern in Western
blotting studies showed that the antigen bound by MAb JF5 is
glycosylated and is a pattern consistent with the binding of
TABLE 2. Absorbance values from ELISAs with protease-treated
antigens by using MAb JF5 of IgG3
Absorbance (450 nm)a
0.559 ? 0.022
0.399 ? 0.006
1.134 ? 0.048
1.088 ? 0.025
1.097 ? 0.002
0.701 ? 0.003
1.217 ? 0.046
1.181 ? 0.050
aEach value represents the mean of replicated values ? standard error.
VOL. 15, 2008 ASPERGILLUS LATERAL-FLOW DEVICE 1099
MAbs to extracellular glycoproteins in A. fumigatus (32). De-
glycosylation of the antigen with the enzyme PNGase showed
that the protein moiety of the glycoprotein bound by MAb JF5
has an approximate molecular mass of 40 kDa and has an
N-glycosylated component (Fig. 1, lane B).
Immunofluorescence and immunogold electron microscopy
of conidia and germlings. Immunofluorescence studies showed
that the antigen was absent from the surface of ungerminated
spores but was present on the hyphal surface of germlings and
was secreted from the hyphal tip (Fig. 2). Immunogold elec-
tron microscopy showed that the antigen was present in the
hyphal cell wall, in septa, and in a capsule-like layer surround-
ing cells (Fig. 3).
Sensitivity and specificity of the LFD. There was strong
detection of the affinity-purified antigen in the LFD tests (Fig.
4), with an assay sensitivity of 37 ng protein per ml of serum. In
PBS only, the sensitivity of the assay was 1.25 ng protein per
ml. After 48 h of growth of the fungi in human serum, there
was strong detection of the antigen in serum spiked with 104
conidia of A. fumigatus AF293 (Fig. 4) and with other Aspergil-
lus species (results not shown). No antigen was detected in
serum inoculated with the other fungi tested (Fig. 4), despite
prolific growth. No false-positive reactions were exhibited with
the ?-lactam antibiotics tested or with tazobactam, cyclophos-
phamide, or bacterial lipoteichoic acids. The chemicals did not
inhibit the detection of the purified antigen (results not
Detection of antigen in IA sera. The JF5 antigen was de-
tected in sera from patients with known or probable IA infec-
tion (Table 4). No false-negative results were found with sera
from healthy individuals. LFD test results were similar to those
for GM detection by the Platelia EIA. However, three of the
samples (samples 1655, 1665, and 1667) from patients diag-
nosed with IA on the basis of clinical symptoms gave positive
reactions with the LFD but were negative by the GM test. One
of these samples (sample 1655) and two others (samples 1537
and 1538) gave negative LFD reactions when they were used
undiluted but gave positive reactions when they were diluted
10-fold in normal serum. This was likely due to a high-dose
hook effect in which the high serum antigen concentrations
impaired antigen-antibody binding. The results for all other
samples were the same when they were used neat or diluted.
Examples of negative and positive reactions with sera are
shown in Fig. 4.
This paper describes the development of an LFD for the
rapid serodiagnosis of IA. The LFD incorporates a murine
MAb, MAb JF5, raised against a protein epitope on an
N-linked glycoprotein antigen present in the hyphal cell wall
and septa of A. fumigatus and that is secreted constitutively
at the hyphal apex. Specificity tests showed that MAb JF5
reacted strongly with antigens from species of fungi in the
genus Aspergillus and the closely related species Eurotium
Emericella nidulans (teleomorph of Aspergillus nidulans),
and Neosartorya fischeri (teleomorph of Aspergillus fischeri).
It cross-reacted weakly with antigens from the closely re-
lated fungus Paecilomyces variotii. Cross-reaction with anti-
gens from a number of Penicillium species was also exhib-
ited, but not with antigens from Penicillium islandicum,
Penicillium purpurogenum, or Penicillium variabile or with
the endemic human pathogen Penicillium marneffei, a spe-
cies of Penicillium belonging to the subgenus Biverticillium
(17). This was confirmed by the absence of cross-reactivity
with Talaromyces species (Talaromyces flavus and Talaromy-
ces stipitatus) whose Penicillium anamorphs belong to this
subgenus (17, 43). Recently, Schmechel et al. (27) showed
that mouse MAbs raised against spores of Aspergillus versi-
color also cross-reacted with Penicillium antigens but, simi-
larly, did not cross-react with antigens from these three
species. Analogous cross-reactivity of the antigalactoman-
nan rat MAb EB-A2 with Penicillium and Paecilomyces spe-
cies has also been shown (32, 34). However, unlike MAb
EB-A2, the MAb reported on here, MAb JF5, does not
cross-react with Acremonium, Alternaria, Botrytis, Cladospo-
rium, Fusarium, Geotrichum, Wangiella, or Wallemia species,
FIG. 1. Analysis of affinity-purified antigen by PAGE and Western
blotting. Lane Mr, molecular mass marker; lane A, Western immuno-
blot with MAb JF5 after separation of purified antigen by sodium
dodecyl sulfate-PAGE under reducing conditions; the well was loaded
with 0.2 ?g 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 ?g of protein.
TABLE 3. Absorbance values from ELISAs with periodate-treated
antigens by using MAb JF5 of IgG3
Absorbance (450 nm)a
1.212 ? 0.013
1.180 ? 0.010
1.178 ? 0.012
1.180 ? 0.014
1.205 ? 0.015
1.223 ? 0.013
1.245 ? 0.013
1.204 ? 0.010
1.219 ? 0.010
1.211 ? 0.014
1.234 ? 0.009
1.259 ? 0.008
aEach value represents the mean of replicated values ? standard errors.
1100THORNTONCLIN. VACCINE IMMUNOL.
fungi identified to be possible causes of false-positive re-
sponses in GM-based diagnostic tests (8, 12, 34). MAb JF5
therefore displays greater specificity than MAb EB-A2, and
while cross-reactivity with Penicillium remains an issue, it is
unlikely to represent a significant problem. With the excep-
tion of the endemic pathogen P. marneffei, there are very few
reports of Penicillium species as etiologic agents of invasive
diseases in humans (18, 41). Likewise, while invasive infec-
tions caused by Paecilomyces species have been reported,
they are also extremely rare (9, 41, 42).
Current immunodiagnostic tests for invasive aspergillosis are
based on the detection of circulating galactofuranose antigens
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 ?m.
VOL. 15, 2008 ASPERGILLUS LATERAL-FLOW DEVICE1101
in human serum. The Platelia Aspergillus sandwich ELISA,
which incorporates rat MAb EB-A2, is now commonly used to
monitor patients at high risk for IA and provides a valuable
tool for the early diagnosis of the disease. However, a number
of issues hamper the use of the assay. False-negative results
have been attributed to the heat pretreatment of serum sam-
ples that denatures protein but that may also eliminate pro-
tein-bound galactofuranose antigens, thereby leading to under-
estimation of serum reactivity (39). False-positive responses
have also been reported, and the reasons for these have al-
ready been discussed. Consequently, surrogate markers of IA
are desirable. Diagnostic tests that employ DNA detection by
PCR have been developed (44), but such technology is re-
stricted to laboratories equipped to perform such tests.
The recent observations by Morelle et al. (23) that circu-
lating Aspergillus antigen may consist not only of fungal
polysaccharide (GM) but also of glycoproteins raised the
possibility that the antigen bound by MAb JF5 might act as
a surrogate marker for the diagnosis of IA. Immunogold
labeling studies showed that in the presence of human se-
rum, the antigen was secreted into an extracellular capsule-
like layer surrounding developing propagules of the fungus,
reminiscent of the capsule induced in C. neoformans upon
exposure to serum. LFD tests of human sera, in which the
fungus and other angioinvasive species had been allowed to
proliferate, showed that the antigen was also detectable in
solution and that the test was specific for Aspergillus species.
A useful property of the LFD is its potential to discriminate
between active invasive growth of the fungus and quiescent
spore production. Immunofluorescence studies showed that
antigen production occurs at the growing tip of hyphae and
is absent from ungerminated conidia. The absence of false-
positive results with antibiotics and with bacterial lipotei-
choic acids and the ability to use non-heat-treated serum
samples provide additional benefits compared to tests based
on GM detection.
The analytical sensitivity of the LFD was determined in
the presence and the absence of serum proteins. The limit of
detection of the LFD in saline buffer was 1.25 ng protein per
ml, a level of sensitivity comparable to that of the Platelia
GM EIA (1 ng per ml). However, in the presence of serum
proteins, the sensitivity was reduced to 35 ng protein ml.
Comparisons of sensitivities between the LFD, the Platelia
GM EIA, and other assays such as the Afmp1p ELISA (45)
are problematic since each assay detects a different Aspergil-
lus antigen and each assay comprises different species of
antibody (a mouse MAb, a rat MAb, and rabbit and guinea
pig polyclonal antisera, respectively). Furthermore, the JF5
MAb binds to a protein epitope, whereas the rat MAb
EB-A2 used in the Platelia EIA binds to a carbohydrate
moiety, further complicating issues of assay sensitivity and
its clinical significance. In the absence of a source of purified
GM, a comparison of assay sensitivities cannot be made
here. Consequently, the results of GM and LFD tests with
clinical serum samples provide the most appropriate mea-
sure of accuracy of the LFD. A blind assessment of the
sensitivities and specificities was conducted with serum sam-
ples from patients with known or suspected IA and healthy
controls. The four control samples from healthy individuals
gave negative results for IA in the GM and LFD tests. Of
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 ?m.
1102 THORNTONCLIN. VACCINE IMMUNOL.
the 12 probable or proven cases of IA according to EORTC
criteria, 5 were determined to be IA positive according to
the GM test results, while 8 were determined to be IA
positive with the LFD. These results therefore suggest that
the LFD has a greater clinical sensitivity for the diagnosis of
disease, while it retains the specificity of the GM test. The
strongest parity between the two immunoassays was found
with the four samples deemed probable IA according to
EORTC criteria. In these cases, strong positive results were
recorded with both the GM and the LFD tests. Further
comparative testing of the assays with samples from a larger
cohort of patients is planned, but this work has shown the
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. (?) LFD tests of normal human serum
(LFD 1) and serum spiked with affinity-purified antigen (LFD 2) at a concentration of 2.5 ?g 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.
VOL. 15, 2008ASPERGILLUS LATERAL-FLOW DEVICE 1103
potential of an LFD that detects a surrogate marker for IA
to be a user-friendly diagnostic platform for the rapid and
specific detection of the disease.
I thank E. Johnson for the supply of serum samples.
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