Produção e caracterização de um anticorpo monoclonal contra os venenos das serpentes Bothrops alternatus e Bothrops neuwiedi

Abstract

In Brazil, the majority of registered snakebite accidents are caused by venomous snakes of the Bothrops genus. The conventional treatment for snakebite envenoming is the use of hyperimmune sera, which contains antibodies produced by horses. However, the production of these antivenoms is highly heterogeneous, and their use can cause several adverse effects. The production of monoclonal antibodies against components of snake venoms, which neutralize the toxic effects of venoms, is a therapeutic alternative to conventional antisera, to assist in the immunotherapy of patients and improve reproducibility between batches. In this work, a monoclonal antibody (mAb) was produced from the immunization of mice with a pool of venoms from Bothrops snakes (B. alternatus, B. jararaca, B. jararacussu, B. moojeni, and B. neuwiedi). The ELISA (Enzyme-Linked Immunosorbent Assay) and Western blot assays demonstrated that the mAb recognized the B. alternatus and B. neuwiedi venoms called mAb anti-Ba/Bn. The mAb anti-Ba/Bn inhibited the coagulant activity and the amidase activity on the synthetic substrate, BApNa, for serino proteases, caused by B. alternatus and B. neuwiedi venoms. In order to characterize the mAb anti-Ba/Bn, it was immobilized on a Sepharose column and the antigens from the B. neuwiedi and B. alternatus venoms were fractionated by immunoaffinity chromatography, thus obtaining the B. neuwiedi fractions (BnF) and B. alternatus (BaF). BnF showed proteolytic activity on the substrate, coupled to a FRET system (Abz-FLPRSFRQ-EDDnp), specific for serino proteases. BnF and BaF showed amidase activity on the BApNA substrate. These proteolytic and amidase activities were significantly inhibited by PMSF, a serino protease inhibitor. BnF degraded the human fibrinogen Aα subunit and partially the Bβ subunit. In the in vivo incoagulability assay, it was observed that the blood of animals that received 50 µg and 100 µg of BnF did not clot after 60 minutes of collection. Bottom-up proteomic analysis confirmed the prevalence of serine proteases in BnF. The amino acid sequences, of peptides generated by BnF tryptic digestion, showed high similarity/identity with the N-terminal portion of thrombin-like serino proteases. These results indicate that the protein recognized by the mAb is a serino protease thrombin-like.