Expressão e avaliação de anticorpos de domínio único contra a enzima tripanotiona redutase de Leishmania braziliensis

Abstract

Leishmaniasis, caused by protozoa of the genus Leishmania, is endemic in 90 countries, affecting 12 million people, with up to 2 million new cases a year. Conventional treatment has high toxicity and growing parasite resistance, as well as difficulties in accessing drugs. Molecular targets, such as the trypanothione reductase enzyme, have therapeutic potential, protecting the parasite from oxidative stress. Single domain antibodies have emerged as a promising alternative due to their low immunogenicity and versatility, allowing them to be applied as intrabodies to intracellular targets. The present study aimed to express, purify, and characterize single-domain antibodies (VHHs), previously selected against the recombinant enzyme trypanothione reductase (rTRLb) from Leishmania braziliensis, with a view to their application as a therapeutic tool. For this purpose, the rTRLb enzyme and four anti-TRLb VHH clones were expressed in a microbial system in Terrific Broth (TB) medium, supplemented with 1% glucose and 100 µg/mL carbenicillin, and purified by affinity chromatography using a nickel resin. The purified clones were subjected to electrophoretic analysis on a 12.5% SDSPAGE gel, as well as Dot blot methodology and Enzyme-Linked Immunosorbent Assay (ELISA) verify their ability to recognize the enzyme. After verifying reactivity in the Dot blot, only 3RT02 proceeded to ELISA. ELISA confirmed the antigen recognition capacity observed in the Dot blot, and the clone appeared to demonstrate dose-dependent reactivity, with activity detected from 15.62 ng. The results indicate that single-domain antibodies have the potential to be used in disease diagnosis and, if their ability to inhibit trypanothione reductase is confirmed, they could be useful as a novel therapeutic approach. However, further studies are needed for a more detailed functional evaluation, including their inhibitory capacity (IC50) and crossreactivity with other enzymes, including those from other trypanosomatids.