Produção e avaliação de anticorpo policlonal anti-Leishmania para aplicação em diagnóstico
Begnami, Isabela dos Santos
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Leishmania is a protozoa genus with several species of intracellular parasites that cause diseases called leishmaniasis, divided into: visceral (VL), the most serious form that affects internal organs and can lead to death, or cutaneous (CL), attacking the skin or mucous membranes with ulcers. These diseases belong to the group of neglected diseases, because they mostly infect poorer populations and are not a priority for health services, with their specific methods of treatment and diagnosis not reaching the places with the highest number of cases. To solve the problem and increase specificity and sensitivity, we can use specific recombinant purifieds, such as the recombinant protein K39 (kinesin-like protein K39), in the tests. Therefore, the objective of the work was to produce anti-leishmania antibodies from K39 aiming at the development of an immunoenzymatic test. The polyclonal anti-leishmania antibody was produced, qualitatively evaluated by SDS-PAGE polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane for an Immunoblotting Assay and applied to an indirect ELISA immunoassay for sensitivity analysis. In the electrophoretic profile was possible to observe the presence of two bands, one short chain (25kDa) and one long chain (53kDa), in addition to albumin contamination. The 1: 1000 dilution sample was stronger than the 1: 5000 sample. Different dilutions were tested in Immunoblotting (1:25, 1:50, 1: 100, 1: 250 and 1: 500) and bands were seen in all of them, with the 1: 500 antigen being the most evident, which confirms the reactivity of the antibody produced. For being polyclonal, nonspecific bands were also found. The optical density of the ELISA was measured in duplicate and standardized for analysis, generating satisfactory results for the concentrations of 1: 5000 and 1: 1000, even with this having the last lower result. Consequantly, the K39 antigen obtained values that enable its use in diagnoses for leishmaniasis.
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