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  Contributed Papers: Posters
Diagnosis and Epidemiology

 

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

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