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