Producción de anticuerpos monoclonales que reconozcan proteínas de membrana en células de memoria en aotus spp
A 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 s...
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| Formato: | Tesis de maestría (Master Thesis) |
| Lenguaje: | Español (Spanish) |
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Universidad del Rosario
2019
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| Acceso en línea: | http://repository.urosario.edu.co/handle/10336/20148 |
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ir-10336-20148 |
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EdocUR - Universidad del Rosario |
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Español (Spanish) |
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Inmunología Anticuerpo Monoclonal Citometría de flujo Fisiología humana Inmunología Anticuerpos Citometría de flujo |
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Inmunología Anticuerpo Monoclonal Citometría de flujo Fisiología humana Inmunología Anticuerpos Citometría de flujo Cubides Amézquita, Jenner Rodrigo Producción de anticuerpos monoclonales que reconozcan proteínas de membrana en células de memoria en aotus spp |
| description |
A 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. |
| author2 |
Patarroyo, Manuel A. |
| author_facet |
Patarroyo, Manuel A. Cubides Amézquita, Jenner Rodrigo |
| format |
Tesis de maestría (Master Thesis) |
| author |
Cubides Amézquita, Jenner Rodrigo |
| author_sort |
Cubides Amézquita, Jenner Rodrigo |
| title |
Producción de anticuerpos monoclonales que reconozcan proteínas de membrana en células de memoria en aotus spp |
| title_short |
Producción de anticuerpos monoclonales que reconozcan proteínas de membrana en células de memoria en aotus spp |
| title_full |
Producción de anticuerpos monoclonales que reconozcan proteínas de membrana en células de memoria en aotus spp |
| title_fullStr |
Producción de anticuerpos monoclonales que reconozcan proteínas de membrana en células de memoria en aotus spp |
| title_full_unstemmed |
Producción de anticuerpos monoclonales que reconozcan proteínas de membrana en células de memoria en aotus spp |
| title_sort |
producción de anticuerpos monoclonales que reconozcan proteínas de membrana en células de memoria en aotus spp |
| publisher |
Universidad del Rosario |
| publishDate |
2019 |
| url |
http://repository.urosario.edu.co/handle/10336/20148 |
| _version_ |
1712098445761708032 |
| spelling |
ir-10336-201482021-08-22T06:01:02Z Producción de anticuerpos monoclonales que reconozcan proteínas de membrana en células de memoria en aotus spp Monoclonal Antibodies for the Tracking of Aotus spp. Memory T and B Cells Cubides Amézquita, Jenner Rodrigo Patarroyo, Manuel A. Díaz Arévalo, Diana Inmunología Anticuerpo Monoclonal Citometría de flujo Fisiología humana Inmunología Anticuerpos Citometría de flujo A 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. Even 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. 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12,111491 |