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Producción de anticuerpos monoclonales que reconozcan proteínas de membrana en células de memoria en aotus spp

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dc.contributor.advisorPatarroyo, Manuel A.
dc.creatorCubides Amézquita, Jenner Rodrigo
dc.creator.degreeMagíster en Ciencias con Énfasis en Genética Humanaspa
dc.creator.degreetypeFull timespa
dc.date.accessioned2019-08-21T20:44:18Z
dc.date.available2019-08-21T20:44:18Z
dc.date.created2019-06-05
dc.date.issued2019
dc.descriptionA pesar de que varios candidatos a la vacuna contra la malaria se encuentran actualmente ensayos de fase clínica 2 y 3, sus mecanismos de protección inmunológica no se comprenden completamente y la duración de la protección de la vacuna se desconoce en gran medida. El modelo del mono Aotus spp. ha sido recomendado por la Organización Mundial de la Salud para el estudio de los candidatos a la vacuna contra la malaria debido a las fuertes similitudes con la patología y los antecedentes genéticos observados en humanos. Sin embargo, la falta de anticuerpos específicos para los marcadores de superficie molecular de las células inmunes en Aotus, ha retrasado los avances en la investigación de la malaria. Entre los anticuerpos monoclonales comerciales (mAbs) para los marcadores moleculares de células T de memoria CD19 +, CD27 + B y CD4 +, CD45RO +, solo los del clon SK3 reconocieron las células T Aotus CD4. En este trabajo, los enfoques bioinformáticos fueron usados para diseñar péptidos antigénicos que corresponden a las regiones extracelulares de las proteínas de membrana CD19, CD27, CD4 y CD45RO, para producir mAbs. 1746 clones de hibridoma resultantes reconocieron los marcadores de superficie molecular de las células inmunes por citometría de flujo y el 30% de ellos se unen al péptido sintético por ELISA. Los mAbs, CD194G12A3G3, CD275F11C11, CD45H3D10 y CD45RO3A8G1 se unieron al 17,7%, 40,1%, 27,4 y 51% de los PBMC de Aotus con alta afinidad (100 ng / 106 células) pero solo mostraron afinidad media a las células humanas (300 ng / 106 células) en análisis FACS. En los ensayos de doble marcaje de las células B y T, mostró que los mAbs CD194G12A3G3 y CD275F11C11 reconocieron el 15,9%, y CD45H3D10 y CD45RO3A8G1 se unieron al 20,6% de las PBMC de Aotus, lo que sugiere que los mAbs reconocieron los marcadores de proteínas de membrana de las células B y T. Estos mAbs son útiles para la identificación y el seguimiento de las células de memoria en el modelo de Aotus para dilucidar qué células inmunes humanas pueden mediar la protección contra la malaria.spa
dc.description.abstractEven though several malaria vaccine candidates are currently in clinical phase 2, and 3 trials, their mechanisms of immune protection are not fully understood, and durations of vaccine protection are largely unknown. The Aotus monkey model has been recommended by the World Health Organization for the study of malaria vaccine candidates because of strong similarities to pathology and genetic background observed in humans. However, the lack of antibodies specific for molecular surface markers of immune cells in Aotus have delayed advances in malaria research. Among commercial monoclonal antibodies (mAbs) for human CD19+, CD27+ B cell and CD4+, CD45RO+ memory T cell molecular markers, only those of the clone SK3 recognized Aotus CD4 T cells. Here, bioinformatics approaches were used to design antigenic peptides that correspond to the extracellular regions of the membrane proteins CD19, CD27, CD4, and CD45RO, to produce mAbs. 1746 resulting hybridoma clones recognized molecular surface markers of immune cells by flow cytometry and 30% of them bond to the synthetic peptide by ELISA. The mAbs, CD194G12A3G3, CD275F11C11, CD45H3D10, and CD45RO3A8G1 bound to 17.7%, 40.1%, 27.4 and 51% of the Aotus’ PBMCs with high affinity (100 ng/106 cells) but displayed only medium affinity to human cells (300 ng/106 cells) in FACS analyses. Double staining of B and T cells showed that the mAbs CD194G12A3G3 and CD275F11C11 recognized 15.9%, and CD45H3D10 and CD45RO3A8G1 bound to 20.6% of Aotus’ PBMCs, suggesting that the mAbs recognized membrane protein markers of memory B and T cells. These mAbs are useful for the identification and tracking of memory cells in the Aotus model to elucidate which human immune cells may mediate protection against malariaspa
dc.description.embargo2021-08-22 01:01:01: Script de automatizacion de embargos. info:eu-repo/date/embargoEnd/2021-08-21
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dc.identifier.doihttps://doi.org/10.48713/10336_20148
dc.identifier.urihttp://repository.urosario.edu.co/handle/10336/20148
dc.language.isospaspa
dc.publisherUniversidad del Rosariospa
dc.publisher.departmentEscuela de Medicina y Ciencias de la Saludspa
dc.publisher.programMaestría en Ciencias con Énfasis en Genética Humanaspa
dc.rightsAtribución-NoComercial-SinDerivadas 2.5 Colombiaspa
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dc.source.instnameinstname:Universidad del Rosariospa
dc.source.reponamereponame:Repositorio Institucional EdocURspa
dc.subjectInmunologíaspa
dc.subjectAnticuerpo Monoclonalspa
dc.subjectCitometría de flujospa
dc.subject.ddcFisiología humanaspa
dc.subject.lembInmunologíaspa
dc.subject.lembAnticuerposspa
dc.subject.lembCitometría de flujospa
dc.titleProducción de anticuerpos monoclonales que reconozcan proteínas de membrana en células de memoria en aotus sppspa
dc.title.TranslatedTitleMonoclonal Antibodies for the Tracking of Aotus spp. Memory T and B Cellseng
dc.typemasterThesiseng
dc.type.documentTesisspa
dc.type.hasVersioninfo:eu-repo/semantics/acceptedVersion
dc.type.spaTesis de maestríaspa
local.department.reportEscuela de Medicina y Ciencias de la Saludspa
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Maestría en Genética Humana