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Doctoral Thesis
DOI
10.11606/T.87.2015.tde-04122015-141425
Document
Author
Full name
Eduardo Aliprandini
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2015
Supervisor
Committee
Moro, Ana Maria (President)
Boscardin, Silvia Beatriz
Lepique, Ana Paula
Rosa, Daniela Santoro
Stephano, Marco Antonio
Title in Portuguese
Obtenção de anticorpos monoclonais humanos antitetânicos.
Keywords in Portuguese
Anticorpos monoclonais humanos
Linfócitos B de memória
Plasmablastos
Separação single cell
Tétano
Toxina tetânica
Abstract in Portuguese
Anticorpos monoclonais (AcMos) para uso terapêutico correspondem a uma área importante na indústria de biofármacos, em especial os AcMos humanos, que apresentam menor probabilidade de elicitar imunogenicidade. O objetivo deste trabalho consistiu em obter AcMos humanos antitetânicos através da separação de linfócitos B produtores de anticorpos específicos utilizando o antígeno ou de plasmablastos. As células foram coletadas de doadores após vacinação e separadas por equipamento de cell sorter. As regiões variáveis dos anticorpos foram amplificadas e clonadas em vetores de expressão, que foram usados para transfectar transitoriamente células HEK293-F. O uso da toxina tetânica conjugada independentemente com dois marcadores, biotina e Alexa Fluor® 647, possibilitou a separação específica de linfócitos B produtores de AcMos antitetânicos, que foram avaliados por ELISA, western blotting e pela inibição da ligação da toxina ao gangliosídio GT1b. O ensaio in vivo mostrou proteção total dos animais contra a toxina tetânica quando três AcMos foram usados em conjunto.
Title in English
Anti-tetanus human monoclonal antibodies.
Keywords in English
Human monoclonal antibodies
Memory B cells
Plasmablasts
Single cell separation
Tetanus
Tetanus toxin
Abstract in English
Monoclonal antibodies (mAbs) for therapeutic use correspond to a major area of the biopharmaceutical industry, especially human mAbs that are less prone to elicit immunogenicity. The objective of this work was to obtain anti-tetanus human mAbs through separation of memory B lymphocytes producing specific antibodies stained with the antigen or plasmablasts. Cells were collected from peripheral blood of donors after vaccination and separated through cell sorting. The variable regions of the antibodies were amplified and cloned in expression vectors for transient transfection of HEK293-F cells. The staining with the tetanus toxin labeled independently with two markers, biotin and Alexa Fluor® 647 allowed the separation of specific B lymphocytes producing anti-tetanus mAbs. The antibodies expressed were evaluated by ELISA, western blotting and the inhibition of the binding of the tetanus toxin to the ganglioside GT1b. The in vivo neutralization assay showed that a pool of three different mAbs were able to protect mice against the tetanus toxin.
 
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Release Date
2017-12-03
Publishing Date
2015-12-07
 
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