Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase

Autores
Alonso, Maria del Rosario; Leiva, Laura Cristina Ana; Gay, Claudia Carolina; Fusco, Luciano Sebastian
Año de publicación
2019
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Phosphodiesterases (PDE) belong to a super-family of enzymes that have multiple roles in the metabolism of nucleotides. These enzyme classes have played such a critical role as tools in the field of molecular biology, but their function is little known today. Here, we evaluated the humoral response induced by a PDE from Crotalus durissus terrificus (CDT) venom used as antigen. The PDE of the Crotalus durissus terrificus venom (CDT-PDE) was purified by gel filtration chromatography (Sephadex G-75) and ion exchange chromatography (HiTrap Q-FF). The CDT-PDE activity was tested by chromogenic reaction with sodium salt of bis (p-nitrophenyl) phosphate and the purity was monitored by SDS-PAGE. BALB /c mice (n= 5) were immunized subcutaneously with 0.3, 0.6 and1.2 µg of enzyme (CDT-PDE) on days 1, 15 and 30, respectively. Freund's complete adjuvant was used in the first immunization and incomplete adjuvant in the booster. On day 50, mice were sacrificed and plasma antibody titers were evaluated by ELISA and cross-reactions with Bothrops alternatus and B. diporus venoms were analyzed by dot blotting. Mice plasma showed a specific response to CDT-PDE and antibodies gave titers around 1/1600. About of dot blotting test, C.d.t venom and it’s PDE showed strong spots were noted in each case, but partial and negative reactions were noted on B. diporus and B. alternatus, respectively. The neutralization capacity of PDE- antibodies was 40 and 20 % when different amounts of serum were analyzed (36.66 µl; 18.33 µl). These results demonstrated that CDT-PDE produces a humoral response. PDE-CDT antibodies recognize the crotalic PDE and, to a lesser extent, the PDE present in the venom of B. diporus. These antibodies can be used as tools in the study of the effect of PDE on platelet aggregation, as well as in the purification of PDE by affinity chromatography to improve the performance in the isolation from CDT venom.
Fil: Alonso, Maria del Rosario. 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
Fil: 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; Argentina
Fil: Gay, Claudia 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; Argentina
Fil: 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; Argentina
Reunión Anual de Sociedades de Biociencia
Mar del Plata
Argentina
Sociedad Argentina de Investigación Clínica
Asociación Argentina de Farmacología Experimental
Sociedad Argentina de Biología
Sociedad Argentina de Protozoología
Asociación Argentina de Nanomedicinas
Asociación Argentina de Ciencia y Tecnología de Animales de Laboratorio
Materia
ANTI-PHOSPHODIESTERASE
CROSS-REACTION
BOTHROPS
SNAKE VENOMS
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/155025
Acceder
id CONICETDig_b4668bff96700ec6fefd198b02c6e8f4
oai_identifier_str oai:ri.conicet.gov.ar:11336/155025
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesteraseAlonso, Maria del RosarioLeiva, Laura Cristina AnaGay, Claudia CarolinaFusco, Luciano SebastianANTI-PHOSPHODIESTERASECROSS-REACTIONBOTHROPSSNAKE VENOMShttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Phosphodiesterases (PDE) belong to a super-family of enzymes that have multiple roles in the metabolism of nucleotides. These enzyme classes have played such a critical role as tools in the field of molecular biology, but their function is little known today. Here, we evaluated the humoral response induced by a PDE from Crotalus durissus terrificus (CDT) venom used as antigen. The PDE of the Crotalus durissus terrificus venom (CDT-PDE) was purified by gel filtration chromatography (Sephadex G-75) and ion exchange chromatography (HiTrap Q-FF). The CDT-PDE activity was tested by chromogenic reaction with sodium salt of bis (p-nitrophenyl) phosphate and the purity was monitored by SDS-PAGE. BALB /c mice (n= 5) were immunized subcutaneously with 0.3, 0.6 and1.2 µg of enzyme (CDT-PDE) on days 1, 15 and 30, respectively. Freund's complete adjuvant was used in the first immunization and incomplete adjuvant in the booster. On day 50, mice were sacrificed and plasma antibody titers were evaluated by ELISA and cross-reactions with Bothrops alternatus and B. diporus venoms were analyzed by dot blotting. Mice plasma showed a specific response to CDT-PDE and antibodies gave titers around 1/1600. About of dot blotting test, C.d.t venom and it’s PDE showed strong spots were noted in each case, but partial and negative reactions were noted on B. diporus and B. alternatus, respectively. The neutralization capacity of PDE- antibodies was 40 and 20 % when different amounts of serum were analyzed (36.66 µl; 18.33 µl). These results demonstrated that CDT-PDE produces a humoral response. PDE-CDT antibodies recognize the crotalic PDE and, to a lesser extent, the PDE present in the venom of B. diporus. These antibodies can be used as tools in the study of the effect of PDE on platelet aggregation, as well as in the purification of PDE by affinity chromatography to improve the performance in the isolation from CDT venom.Fil: Alonso, Maria del Rosario. 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: Gay, Claudia 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: 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; ArgentinaReunión Anual de Sociedades de BiocienciaMar del PlataArgentinaSociedad Argentina de Investigación ClínicaAsociación Argentina de Farmacología ExperimentalSociedad Argentina de BiologíaSociedad Argentina de ProtozoologíaAsociación Argentina de NanomedicinasAsociación Argentina de Ciencia y Tecnología de Animales de LaboratorioFundación Revista Medicina2019info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/155025Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase; Reunión Anual de Sociedades de Biociencia; Mar del Plata; Argentina; 2019; 11-110025-76801669-9106CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://biologia.org.ar/downloads/Borrador_REV-MEDICINA-REUNION-2019-20200403.pdfInternacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:09:55Zoai:ri.conicet.gov.ar:11336/155025instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 10:09:56.139CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase
title Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase
spellingShingle Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase
Alonso, Maria del Rosario
ANTI-PHOSPHODIESTERASE
CROSS-REACTION
BOTHROPS
SNAKE VENOMS
title_short Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase
title_full Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase
title_fullStr Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase
title_full_unstemmed Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase
title_sort Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase
dc.creator.none.fl_str_mv Alonso, Maria del Rosario
Leiva, Laura Cristina Ana
Gay, Claudia Carolina
Fusco, Luciano Sebastian
author Alonso, Maria del Rosario
author_facet Alonso, Maria del Rosario
Leiva, Laura Cristina Ana
Gay, Claudia Carolina
Fusco, Luciano Sebastian
author_role author
author2 Leiva, Laura Cristina Ana
Gay, Claudia Carolina
Fusco, Luciano Sebastian
author2_role author
author
author
dc.subject.none.fl_str_mv ANTI-PHOSPHODIESTERASE
CROSS-REACTION
BOTHROPS
SNAKE VENOMS
topic ANTI-PHOSPHODIESTERASE
CROSS-REACTION
BOTHROPS
SNAKE VENOMS
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Phosphodiesterases (PDE) belong to a super-family of enzymes that have multiple roles in the metabolism of nucleotides. These enzyme classes have played such a critical role as tools in the field of molecular biology, but their function is little known today. Here, we evaluated the humoral response induced by a PDE from Crotalus durissus terrificus (CDT) venom used as antigen. The PDE of the Crotalus durissus terrificus venom (CDT-PDE) was purified by gel filtration chromatography (Sephadex G-75) and ion exchange chromatography (HiTrap Q-FF). The CDT-PDE activity was tested by chromogenic reaction with sodium salt of bis (p-nitrophenyl) phosphate and the purity was monitored by SDS-PAGE. BALB /c mice (n= 5) were immunized subcutaneously with 0.3, 0.6 and1.2 µg of enzyme (CDT-PDE) on days 1, 15 and 30, respectively. Freund's complete adjuvant was used in the first immunization and incomplete adjuvant in the booster. On day 50, mice were sacrificed and plasma antibody titers were evaluated by ELISA and cross-reactions with Bothrops alternatus and B. diporus venoms were analyzed by dot blotting. Mice plasma showed a specific response to CDT-PDE and antibodies gave titers around 1/1600. About of dot blotting test, C.d.t venom and it’s PDE showed strong spots were noted in each case, but partial and negative reactions were noted on B. diporus and B. alternatus, respectively. The neutralization capacity of PDE- antibodies was 40 and 20 % when different amounts of serum were analyzed (36.66 µl; 18.33 µl). These results demonstrated that CDT-PDE produces a humoral response. PDE-CDT antibodies recognize the crotalic PDE and, to a lesser extent, the PDE present in the venom of B. diporus. These antibodies can be used as tools in the study of the effect of PDE on platelet aggregation, as well as in the purification of PDE by affinity chromatography to improve the performance in the isolation from CDT venom.
Fil: Alonso, Maria del Rosario. 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
Fil: 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; Argentina
Fil: Gay, Claudia 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; Argentina
Fil: 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; Argentina
Reunión Anual de Sociedades de Biociencia
Mar del Plata
Argentina
Sociedad Argentina de Investigación Clínica
Asociación Argentina de Farmacología Experimental
Sociedad Argentina de Biología
Sociedad Argentina de Protozoología
Asociación Argentina de Nanomedicinas
Asociación Argentina de Ciencia y Tecnología de Animales de Laboratorio
description Phosphodiesterases (PDE) belong to a super-family of enzymes that have multiple roles in the metabolism of nucleotides. These enzyme classes have played such a critical role as tools in the field of molecular biology, but their function is little known today. Here, we evaluated the humoral response induced by a PDE from Crotalus durissus terrificus (CDT) venom used as antigen. The PDE of the Crotalus durissus terrificus venom (CDT-PDE) was purified by gel filtration chromatography (Sephadex G-75) and ion exchange chromatography (HiTrap Q-FF). The CDT-PDE activity was tested by chromogenic reaction with sodium salt of bis (p-nitrophenyl) phosphate and the purity was monitored by SDS-PAGE. BALB /c mice (n= 5) were immunized subcutaneously with 0.3, 0.6 and1.2 µg of enzyme (CDT-PDE) on days 1, 15 and 30, respectively. Freund's complete adjuvant was used in the first immunization and incomplete adjuvant in the booster. On day 50, mice were sacrificed and plasma antibody titers were evaluated by ELISA and cross-reactions with Bothrops alternatus and B. diporus venoms were analyzed by dot blotting. Mice plasma showed a specific response to CDT-PDE and antibodies gave titers around 1/1600. About of dot blotting test, C.d.t venom and it’s PDE showed strong spots were noted in each case, but partial and negative reactions were noted on B. diporus and B. alternatus, respectively. The neutralization capacity of PDE- antibodies was 40 and 20 % when different amounts of serum were analyzed (36.66 µl; 18.33 µl). These results demonstrated that CDT-PDE produces a humoral response. PDE-CDT antibodies recognize the crotalic PDE and, to a lesser extent, the PDE present in the venom of B. diporus. These antibodies can be used as tools in the study of the effect of PDE on platelet aggregation, as well as in the purification of PDE by affinity chromatography to improve the performance in the isolation from CDT venom.
publishDate 2019
dc.date.none.fl_str_mv 2019
dc.type.none.fl_str_mv info:eu-repo/semantics/publishedVersion
info:eu-repo/semantics/conferenceObject
Reunión
Journal
http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/155025
Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase; Reunión Anual de Sociedades de Biociencia; Mar del Plata; Argentina; 2019; 11-11
0025-7680
1669-9106
CONICET Digital
CONICET
url http://hdl.handle.net/11336/155025
identifier_str_mv Assessment of neutralizing capacity of antibodies raised in mice against a Crotalus durissus terrificus phosphodiesterase; Reunión Anual de Sociedades de Biociencia; Mar del Plata; Argentina; 2019; 11-11
0025-7680
1669-9106
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://biologia.org.ar/downloads/Borrador_REV-MEDICINA-REUNION-2019-20200403.pdf
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
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dc.coverage.none.fl_str_mv Internacional
dc.publisher.none.fl_str_mv Fundación Revista Medicina
publisher.none.fl_str_mv Fundación Revista Medicina
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
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repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
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