Evaluation of an experimental botropic antivenom produced with venom blocked with Na2-EDTA

Snake envenoming is a serious medical problem in tropical developing countries and antivenoms are the only effective therapy. Antisera are produced by immunizing horses with snake venom and adjuvant, usually Freund´s adjuvant. Botropic venoms contain metalloproteinases (SVMPs), responsible for the local effects of envenoming, such as hemorrhage, edema and myotoxicity as well as systemic bleeding. SVMPs represent around 34,2 % of the protein composition from B. diporus venom and causing lesions during the immunization of animals. In search of new processes, focused on animal welfare for the production immunobiologicals, and taking into account that the toxic action of SVMPs is inhibited by Na2-EDTA, in the present work, the immune response of animals inoculated with B.diporus venom blocked with Na2-EDTA was evaluated. For this proposal, the B. diporus venom (1,9 mg/mL) was blocked by Na2-EDTA (200 mM, B.dV/ Na2-EDTA) and used as antigen. Previously to the inoculation, the excess of chelate was removed by molecular exclusion chromatography (Sephadex G-25). Likewise, the venom without inhibitor (B.dV) received the same treatment and in both cases, the effective neutralization of SVMPs using azocasein as substrate was determined. Group of 5 CF-1 mice were immunized subcutaneously on days 0, 15 and 30 with B.dV or B.dV/ Na2-EDTA (7-30μg or 14-60 μg) emulsified with complete Freund?s Adjuvant and incomplete (booster). On days 14, 29, 37 blood samples were collected from the tip of the animals tail and day 45 the final bleeding and the separation of the different sera were performed. These were destined for immunoassays and neutralization assays for proteolytic, indirect hemolytic, and coagulant activity. The results of the enzyme-linked immunosorbent assay showed that both anti-B.dV and anti-B.dV/EDTA serum had high antibody titers (1/74.850 ? 1/186.150) at the end of the immunization protocol. Regarding the Western Blot, the anti-B.dV/EDTA serum recognized the main bands, corresponding to the venom proteins, in a similar way as the anti-B.dV. Additionally, the experimental sera produced showed neutralizing capacity of the main toxic activities tested in vitro. This result shows that Na2-EDTA does not affect the immunogenicity of proteins since animals immunized with B.dV/ Na2-EDTA respond to B.diporus venom in a similar way to animals immunized with venom without inhibitor.Fil: López, Gisela Lumila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Leiva, Laura Cristina Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Fusco, Luciano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaXXIII Annual Meeting of the Argentinean Biology SocietyBuenos AiresArgentinaSociedad Argentina de Biologí

Neutralizing capacity of antisera obtained by immunization with Bothrops alternatus venom blocked in its metalloproteinases

Specific treatment for snake bite accidents with antivenoms, obtained from immunized animals, is the only recommended therapy. Bothropic intoxication is characterized by proteolytic, coagulant and hemorrhagic effects that induce local tissue damage and systemic alterations that could lead to death. Snake venom metalloproteinases, zinc- dependent, play a relevant role in the pathogenesis of intoxication. They provoke a disruption of the hemostatic system, restricting their use as immunogen in high doses. In previous work, it was demonstrated that is it possible to immunize mice with high doses of Bothrops alternatus venom (BaV) neutralized with disodium salt of ethylenediaminetetraacetic acid (Na2EDTA) avoid of hemorrhagic lesions and then animals reported higher antibodies titer. The present study was performed to test the neutralizing ability of sera obtained from animals treated whit venom-Na2EDTA, and evaluate the effect of this immunogen on pulmonary parenchyma. Groups of 5 Balb/c mice were immunized subcutaneously with BaV, or BaV/Na2EDTA emulsified with Freund?s Adjuvant (complete first and incomplete-booster). On day 50 serums were collected for neutralization assays: proteolytic activity on azocasein, phospholipase activity on erythrocytes and thrombine-like activity on citrated plasma. Animals were sacrificed and lungs removed for histological analysis (hematoxylin-eosin). The results showed that the capacity of neutralize each of one of the three enzyme activities assayed was significantly higher (p <0.05) by the serum obtained from animals immunized with BaV/Na2EDTA. Histological analysis showed that the pulmonary parenchyma from immunized mice with BaV was significantly affected (pneumonitis, p <0.05), in respect to those were BaV/Na2EDTA treated. Results demonstrated that the BaV/Na2EDTA immunogen has a lower organic impact with respect to BaV and, at the same time, providing a serum with high neutralizing capacity.Fil: López, Gisela Lumila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; ArgentinaFil: Hernandez, David Roque. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias. Instituto de Ictiología del Nordeste; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Leiva, Laura Cristina Ana. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Fusco, Luciano Sebastian. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Van de Velde, Andrea Carolina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaLXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología; IX Reunión Anual de ka Asociación Argentina de Nanomedicinas y VI Reunión Científica Regional de la Asociación Argentina de Ciencia y Tecnología de Animales de LaboratorioMar del PlataArgentinaSociedad Argentina de Investigación ClínicaAsociación Argentina de Farmacología ExperimentalSociedad Argentina de BiologíaAsociación Argentina de NanomedicinasAsociación Argentina de Ciencia y Tecnología de Animales de LaboratorioSociedad Argentina de Protozoologí

Bothrops alternatus venom pretrated with chelate agent for antiserum production

Snakebite envenoming is a major public health problem in tropical countries. The only specific therapies currently available for the treatment are antivenoms, which consist of polyclonal immunoglobulins purified from sera/plasma of horses or sheep immunized with doses sublethal of snake venom(s). Bothrops alternatus (BaV) venom induces a prominent tissue local damage, especially, hemorrhage, muscle damage and inflammation. These complex pathological phenomena are due to the concomitant action of metalloproteinases (SVMPs) in venoms and others edema-inducing components. In order to obtain a high titer of antibodies and reduced local damage in the animal, an alternative immunization protocol was proposed where the SVMPs action was blocked by using a chelate agent. For this proposal, the B. alternatus venom (10 mg/mL) was blocked by EDTA-Na2 (10 mM, BaV/ EDTA-Na2) and used as antigen. Previously to the inoculation, the excess of chelate was removed by passing the mixture on Sephadex G-25 column (venom without inhibitor was subjected to the same process) and the effective neutralization of SVMPs using azocasein as substrate was determined. Group of 5 Balb/c mice were immunized subcutaneously on days 0, 15 and 30 with BaV(15-30-45μg) or BaV/ EDTA-Na2 (45-90-135 μg) emulsified with Freund?s Adyuvant (complete first and incomplete-booster). Blood samples were collected by the animals tail tip on days 14, 29 and 41 of protocol immunization and it was destined to ELISA´s test. The results showed that the immunized animal with BaV/ EDTA-Na2had a higher titer (5.1x104) than those treated to BaV (1.3x104). Macroscopic analysis at the inoculation site of mice injected with Freund?s adjuvant showed local damage (with non-infectious abscesses) and hypertrophy of inguinal lymph nodes. Our results show that BaV/ EDTA-Na2 formulation, where the SVMPs are blocked, produced a higher humoral response compared with the produced by BaV. These preliminary results demonstrated the potential use of blocking the toxins with chelate to produce antivenom with less damage in the animals.Fil: López, Gisela Lumila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; ArgentinaFil: Van de Velde, Andrea Carolina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Leiva, Laura Cristina Ana. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Fusco, Luciano Sebastian. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaXX Annual Meeting of the Argentinean Biology Society and VII Meeting of the Uruguayan Society of BiosciencesBuenos AiresArgentinaSociedad Argentina de BiologíaSociedad Uruguaya de Biociencia

Use of venom blocked with chelating agent for anti-bothropic serum production

Snakes belonging to genus Bothrops are responsible for more than 85% of the bites occurring in South America. Bothrops alternatus is a pitviper widespread in this area and it is one of the most important species associated to snakebites not only in Argentina but also Brazil. Antivenoms are the only specific treatment for envenoming by snakebites. They are made by animals (e.g. horses or sheep) immunizations with sublethal doses of venom. Snake Venom Metalloproteinases (SVMPs) play an important role in envenomation, causing relevant local effects such as hemorrhage, edema and myotoxicity as well as systemic bleeding. They represent around ̴43.1% of the protein composition from B. alternatus venom and causing lesions during the immunization of animals. In this work, an alternative immunization protocol was developed in mice where SVMPs activity was previously blocked by Na2EDTA as chelating agent. For this proposal, the B. alternatus venom (BaV, 10 mg/mL) was treated with 50 mM Na2EDTA (1 h, 37°C) and excess chelator was removed by Sephadex G-25 chromatography. Proteolytic activity was assayed to control the block processing. Groups of 5 BALB/c mice were immunized s.v. on 0-15-30 days with BaV (15-30-45μg) and BaV/Na2EDTA (45-90-135 μg). Blood samples were collected on days 14-29-41 for antibody analysis. Sera from BaV/Na2EDTA protocol have a titer (5.1x104) higher than those treated with BaV (1.3x104). The neutralizing ability of both antivenoms was tested against proteolytic, coagulant and PLA2 activity, resulting that it was significantly higher (p <0.05) for sera from mice bowing to blocked venom. Histological analysis of mice lungs showed that pulmonary parenchyma from BaV immunized mice was significantly affected in relation to those BaV/Na2EDTA treated. In conclusion, BaV/Na2EDTA showed to be an appropriate immunogen, not only proved that serum has a high neutralizing capacity but also has a lower organic impact in animals to antivenom production.Fil: López, Gisela Lumila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Van de Velde, Andrea Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Hernandez, David Roque. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias. Instituto de Ictiología del Nordeste; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Leiva, Laura Cristina Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Fusco, Luciano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina7th International Toxinology MeetingOxfordInglaterraLibPubMedi

Comparative effectiveness of two adjuvants in antibody production against PLA2 from Crotaluss durissus terrificus venom

Antivenoms are produced by immunization of horses with snake venom and adjuvant, usually Freund´s adjuvant. Nevertheless, it is widely known that this procedure induces local and systemic adverse effects. In the present work we evaluate the avidity and neutralizing activity of IgG antibodies against PLA2 from C.d. terrificus venom using two different adjuvant formulations. For this proposal, Balb/c mice were subcutaneously immunized on days 0, 15 and 30 with 10-15μg PLA2/30μg CpG-ODN/Coa-ASC16 or 10-15μg PLA2/Freund´s Adjuvant (complete first and incomplete-booster). On day 50 mice serum was analyzed. Chaotropic agent (KSCN) was used to measure the antigen-antibody binding (relative avidity). The results showed that anti-PLA2 sera obtained by PLA2/CpG-ODN/Coa-ASC16 immunization presented lower avidity than PLA2/Freund´s Adjuvant because 0.75 M KSCN reduced the binding in 79% and 56% respectively, by ELISA assay. On the other hand, neutralization test by using Diheptanoyl Thio-PC (a synthetic chromogenic substrate PLA2) showed similar behavior between both antisera. In summary, CpG-ODN/Coa-ASC16 elicits a humoral response with a lesser avidity that Freund´s adjuvant but a similar neutralizing serum ability. In this way we add knowledge about an alternative innocuous adjuvant to be used on the process of antivenom production.Fil: López, Gisela Lumila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Yannet, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Moron, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Maletto, Belkys Angélica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Palma, Santiago Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; ArgentinaFil: Leiva, Laura Cristina Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Fusco, Luciano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina8 th International Toxinology MeetingOxfordInglaterraLibPubMedi