Obtención de anticuerpos monoclonales de ratón contra proteasa de cisteína 5 recombinante de Entamoeba histolytica

• Autores: Juanita Trejos S., Jhon Castaño
• Localización: Revista MVZ Córdoba, ISSN 0122-0268, ISSN-e 1909-0544, Vol. 17, Nº. 2, 2012, págs. 3014-3023
• Idioma: español
• Títulos paralelos:
  • Production of monoclonal antibodies against cysteine protease 5 of Entamoeba histolytica
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• Resumen
  • español

    Objetivo. Obtener anticuerpos monoclonales de raton contra la proteasas de cisteina 5 (EhCP5) de Entamoeba histolytica. Materiales y metodos. Se inmunizaron ratones BALB/c por via intraperitoneal con adyuvante de Freund completo e incompleto con la proteina recombinante EhCP5 obtenida a partir del cultivo de E.coli DH5�¿ trasfectada con el vector recombinante pJC45 que expresa dicha proteina. Se selecciono el animal con mejor respuesta de anticuerpos. Al cual se le extrajo su bazo como fuente de linfocitos B, los cuales se fusionaron utilizando PEG con celulas de mieloma de raton SP2-0/Ag14. Se procedio a seleccion de los hibridomas y a la evaluacion de los sobrenadantes de las colonias que crecieron a los 7 dias mediante ELISA. Los hibridomas con valores mas altos de anticuerpos especificos contra la proteina EhCP5r se seleccionaron, y los clones obtenidos por diluciones limitantes fueron expandidos. Resultados. A partir de un clon secretor estable se purifico el anticuerpo monoclonal anti EhCP5r del isotipo IgG1 por cromatografia de afinidad con proteina G. Los clones fueron expandidos in vivo e in vitro. Con el anticuerpo purificado se disenaron tres sistemas de captura para evaluar la aplicabilidad del anticuerpo monoclonal anti EhCP5r como metodo inmunodiagnostico. Conclusiones. Se logro la produccion de un anticuerpo monoclonal especifico contra EhCP5r que permite diferenciar Entamoeba histolytica de Entamoeba dispar.

  • English

    Objective. Obtain mouse monoclonal antibodies against cysteine proteases 5 (EhCP5) of Entamoeba histolytica. Materials and methods. BALB/c mice were immunized intraperitoneally with complete and incomplete Freund adjuvant EhCP5 with the recombinant EhCP5 protein obtained from E.coli DH5�¿ culture transfected with the recombinant vector pJC45 that expresses said protein. The animal with the best antibody response was selected. Its spleen was extracted as a source of B-lymphocytes, which were merged using PEG miceSP2-0/Ag14 myeloma cells. The team proceeded to undergo the selection of the hybridomas and the evaluation of the supernatants of the colonies that grew after 7 days by ELISA. The hybridomas with higher values of specific antibodies against the protein EhCP5r were selected, and clones obtained by limiting dilution were expanded Results. With the use of a stable secreting clone the monoclonal antibody anti EhCP5r IgG1 isotype was purified by affinity chromatography with protein G. The clones were expanded in vivo and in vitro. Three capture systems were designed with the purified antibody to assess the applicability of the monoclonal antibody anti EhCP5r as an immunodiagnostic method. Conclusions. The production of a specific monoclonal antibody against EhCP5r was achieved to differentiate Entamoeba histolytica from Entamoeba dispar.