ASSESSMENT OF NEUTRALIZING CAPACITY OF ANTIBODIES RAISED IN MICE AGAINST A CROTALUS DURISSUS TERRIFICUS PHOSPHODIESTERASE

MARIA DEL ROSARIO ALONSO; LAURA C. LEIVA; GAY C.; FUSCO LUCIANO S

Mar del Plata

Congreso; Reunión anual de sociedades de biociencia; 2019

SAIC. SAFE. SAB. SAP

Phosphodiesterases (PDE) belong to asuper-family of enzymes that have multiple roles in themetabolism of nucleotides. These enzyme classes have playedsuch a critical role as tools in the field of molecular biology, buttheir function is little known today. Here, we evaluated thehumoral response induced by a PDE from Crotalus durissusterrificus (CDT) venom used as antigen. The PDE of the Crotalusdurissus terrificus venom (CDT-PDE) was purified by gel filtrationchromatography (Sephadex G-75) and ion exchangechromatography (HiTrap Q-FF). The CDT-PDE activity was testedby chromogenic reaction with sodium salt of bis (p-nitrophenyl)phosphate and the purity was monitored by SDS-PAGE. BALB /cmice (n= 5) were immunized subcutaneously with 0.3, 0.6 and 1.2 μg of enzyme (CDT-PDE) on days 1, 15 and 30, respectively.Freund´s complete adjuvant was used in the first immunizationand incomplete adjuvant in the booster. On day 50, mice weresacrificed and plasma antibody titers were evaluated by ELISAand cross-reactions with Bothrops alternatus and B. diporusvenoms were analyzed by dot blotting. Mice plasma showed aspecific response to CDT-PDE and antibodies gave titers around1/1600. About of dot blotting test, C.d.t venom and it?s PDEshowed strong spots were noted in each case, but partial andnegative reactions were noted on B. diporus and B. alternatus,respectively. The neutralization capacity of PDE- antibodies was40 and 20 % when different amounts of serum were analyzed(36.66 μl; 18.33 μl). These results demonstrated that CDT-PDEproduces a humoral response. PDE-CDT antibodies recognize thecrotalic PDE and, to a lesser extent, the PDE present in thevenom of B. diporus. These antibodies can be used as tools inthe study of the effect of PDE on platelet aggregation, as well asin the purification of PDE by affinity chromatography to improvethe performance in the isolation from CDT venom.