Evaluation of
a rapid screening assay for Cryptosporidium identification
(DOT-ELISA) using monoclonal antibody
Bozzoli, L.M1, Araújo,A.J
.U.S2, De Gaspari, E.N.1 Seção de Imunologia,
Instituto Adolfo Lutz, Laboratório de Parasitologia2,
UNITAU,São Paulo,SP. egaspari@ial.sp.gov.br
A rapid screening
assay for Cryptosporidium parvum was evaluated using
monoclonal antibody with the objective of standardizing
a Dot Enzyme-Linked Immunosorbent Assay (Dot-ELISA)
to detect antigens of fecal parasite samples with
different concentrations of this parasite, and its
efficacy.. The immunoenzymatic Dot-ELISA test, derived
from the Dot-Immunobinding Assay with a modification
of the Dot-hybridization to test monoclonal antibodies,
offers extensive applicability in laboratory diagnosis.
The efficiency of the Dot-ELISA was evaluated by using
oocyst concentrations of 25, 50, 102,103,104 and 105
cysts. As a support for the technique, a nitrocellulose
membrane 0.22mc pore size was cut into 7 × 0.5
cm strips and carefully placed in the canals of an
acrylic tray. Prior to this, areas of 0.5 ×
0.5 cm were marked off on the strips and onto each
of these squares, samples obtained from fecal material
antigen or pure parasites were applied with a micropipette
(1ml). The antigen was then fixed in oven at 37°C
for 15 minutes. This was achieved by adding a blocking
solution selected through pre-testing: (gelatin at
5% in Tris-buffered-saline, pH 7.5) for 60 minutes
at 37°C under constant shaking, with mild movements
on a stirring plate. The nitrocellulose strips in
the acrylic tray were washed 3 times with Tris-buffered-saline
(TBS pH 7.5) for 10 minutes under constant shaking.
The diagnosis of intestinal parasites was performed
by microscopic examination of the stool, which is
recognized as the gold standard method. Confirmation
of the presence of C. parvum in feces samples is labor-intensive,
time-consuming, costly, and often difficult, highly
dependent on training and expert knowledge in morphologic
differentiation. The Dot-ELISA is simple and rapid
to use, and offers a less subjective method than microscopy
for detecting the protozoan in fecal samples submitted
to a busy diagnostic laboratory. It is a highly sensitive
and specific technique, and it is useful for screening
large numbers of specimens in a short period of time.
Also, it does not really need special microscopy skills.
Overall, sensitivity of the immunoenzymatic test was
100%, with a specificity of 98%. In conclusion, our
study showed that Dot-ELISA is clearly a reliable
and rapid method for the detection of Cryptosporidium
in fecal samples.
Supported by FAPESP