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Journal of Clinical Microbiology, October 1998, p. 3079-3080, Vol. 36, No. 10
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Aspergillus Galactomannan:
Comparison of an Enzyme-Linked Immunoassay and a Europium-Linked
Time-Resolved Fluoroimmunoassay
A.
Paugam,1,*
J.
Sarfati,2
R.
Romieu,3
M.
Viguier,3
J.
Dupouy-Camet,1 and
J. P.
Latgé2
Laboratoire de
Mycologie1 and
INSERM
445,3 Hôpital Cochin, and
Laboratoire des Aspergillus, Institut
Pasteur,2 Paris, France
Received 4 May 1998/Returned for modification 8 June 1998/Accepted 7 July 1998
 |
ABSTRACT |
With a view to improving the sensitivity of serological detection
of Aspergillus galactomannan (GM), a
europium-linked time-resolved fluoroimmunoassay was developed.
This method was compared to an enzyme-linked immunosorbent assay using
a peroxidase-conjugated detector antibody. No increase in the
sensitivity of the detection of GM standards was seen with the
europium-based fluoroimmunoassay.
| |
TEXT |
Detection of
Aspergillus antigens is the most promising
approach to serological diagnosis of invasive aspergillosis (4, 8). Research has focused on the detection of the galactomannan antigen (GM), a major cell wall component and secreted molecule of Aspergillus species (1, 6). GM is composed of
linear 
-(1-2) -(1-6)-linked mannans with 
-(1-5)
galactofuranose containing side chains (6). A rat
antigalactofuran immunoglobulin M (IgM) monoclonal antibody (MAb) named
EB-A2 (14) has been used as a captor and detector in a
direct double-sandwich enzyme-linked immunosorbent assay (ELISA)
(commercially available as Platelia Aspergillus; Sanofi
Diagnostics Pasteur, Marnes-La-Coquette, France) (15). This
is currently the most sensitive technique for GM detection
(13), meaning that it gives the earliest serological diagnosis of invasive aspergillosis (9). Recently,
ultrasensitive bioanalytical assays with time-resolved
fluorescence detection have been developed (DELFIA [dissociation
enhancement lanthanide fluorescent immunoassay]; Wallac, Turku,
Finland), based on lanthanide fluorescence (europium, samarium,
or terbium). Immunoassays using time-resolved fluorometry have been
proposed as a more sensitive alternative to classical ELISAs
(2). To improve GM detection, we developed a
time-resolved fluoroimmunoassay in which EB-A2 was conjugated
with europium instead of peroxidase.
The sandwich immunoassay was carried out as described by Stynen et al.
(13). Briefly, GM was isolated from a culture of Aspergillus fumigatus (6). The captor MAb
EB-A2 was kindly provided by M. Tabouret (Sanofi Diagnostics
Pasteur, Steenvorde, France) and was produced and purified as
previously described (14). The detector was
peroxidase-conjugated EB-A2 (Sanofi Diagnostics Pasteur,
Marnes-La-Coquette, France) or europium-conjugated EB-A2 (Wallac labelling service, Turku, Finland). These two conjugates have been stored for at least 3 months at 4°C without any loss of
activity. Polystyrene microtiter plates (Nunc; Becton Dickinson) were
coated with 100 µl of EB-A2 at 1 µg per ml at +4°C in 0.1 M
carbonate buffer (pH 9.4). After overnight incubation at room temperature, the solution was removed by aspiration and the wells were
washed with washing solution and postcoated with 200 µl of 0.05%
phosphate-buffered saline(PBS)-Tween and 1% bovine serum albumin at
37°C for 1 h. After a wash with PBS-Tween, 50-µl
volumes of twofold serial dilutions of GM in distilled water (1.25 to 0.1 ng per ml) were added and plates were incubated for 1 h
at 37°C. After washing with PBS-Tween, 50 µl of
peroxidase-conjugated EB-A2 or europium-conjugated EB-A2 at 1 µg/ml was added and the mixture was incubated for 1 h at 37°C.
In the ELISA method, after extensive washings, optical density was read
at 490 nm after 30 min of incubation in the dark at room temperature
with 100 µl of revelation buffer containing
O-phenylenediamine. In the DELFIA method, after extensive
washings, Eu3+ was released from the chelate within a few
minutes by the low pH (
3) of the DELFIA enhancement
solution (Wallac). After plates had been agitated for 5 min on a plate
shaker, fluorescence was measured with a time-resolved fluorometer
(DELFIA Research Fluorometer; Wallac).
The results are shown in Fig. 1. The
sensitivity of the sandwich peroxidase-linked assay was consistent with
that found in previous studies (0.5 to 1 ng/ml) (13). The
background fluorescence seen in this time-resolved fluorometry
procedure was high (blank with no antigen, about 85,000 cps). The
nonspecific binding of the europium-bound conjugate to the first
antibody as well as the high labelling yield for the IgM MAb
(22.7 europium molecules per EB-A2 IgM molecule versus
about 8 europium molecules per IgG molecule [7]) may
be responsible for the high background fluorescence seen in the
time-resolved fluorimetry procedure.
No major improvement in sensitivity was obtained with the
fluorescence time-resolved immunoassay. The two methods
differed only by the substance to which the detector antibody was
conjugated (peroxidase or europium). Time-resolved fluorescence
immunoassays have been used successfully in virology, hormonology,
antitoxin, and cytokine research, etc., and have proved to be 6 to 40 times more sensitive than standard ELISA methods (3, 5, 10, 11); in another application, anti-human immunodeficiency
virus antibodies were detected 16 days earlier than by standard ELISA (12). To our knowledge, this is the first time that a
time-resolved fluorescence immunoassay has been used to detect a
polysaccharide (GM) rather than a polypeptide and the first time that
europium-labelled IgM has been used instead of IgG. The chemical nature
of the antigen and the IgM detector antibody may explain this lack of
increase in sensitivity.
| |
ACKNOWLEDGMENTS |
This work was supported by Assistance Publique-Hôpitaux de
Paris (CRC grant 950228).
We thank David Young for checking the English.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Mycologie, Hôpital Cochin, 75014 Paris, France. Phone: 1 42 34 14 97. Fax: 1 42 34 14 96. E-mail:
andre.paugam{at}cch.ap-hop-paris.fr.
| |
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Journal of Clinical Microbiology, October 1998, p. 3079-3080, Vol. 36, No. 10
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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