• JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
 
  Bookmark and Share
 
 
Master's Dissertation
DOI
Document
Author
Full name
Noeli Maria Espindola
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 1999
Supervisor
Committee
Vaz, Adelaide Jose (President)
Cunha, Regina Ayr Florio da
Ueda, Mirthes
Title in Portuguese
T. solium e T. crassiceps: Caracterização dos antígenos e obtenção de anticorpos monoclonais
Keywords in Portuguese
Anticorpo monoclonal
Cisticercose
Imunologia
Neurologia
Parasitologia
Reação cruzada
Taenia crassiceps
Taenia solium
Abstract in Portuguese
O objetivo deste trabalho foi a obtenção de anticorpos monoclonais a partir da imunização de camundongos com antígenos de Taenia crassiceps (Tcra), que possibilitassem identificar antígenos referentes à Taenia solium (Tso) responsável pela infecção humana. Na caracterização por SDS-PAGE, o antígeno de Tso mostrou um padrão heterogêneo de composição de peptídeos, enquanto o de líquido vesicular de Tcra (LV-Tcra) se apresentou mais homogêneo com elevada concentração de peptídeos de peso molecular inferior a 20kD. Para o estudo da cinética de produção de anticorpos, foram utilizados camundongos BALB/c inoculados com antígeno LV-Tcra e total de Taenia solium (T-Tso), agrupados em 6 grupos de camundongos e 1 grupo controle, a saber: GI: salina; GII: 20µg de LV-Tcra; GIII e GV: 50µg de LV-Tcra; GIV: ~50µg de gel<30kD-Tcra; GVI: 50µg de eluato <30kD-Tcra e GVII: 50µg de T-Tso. Nos grupos de camundongos imunizados com Tcra foram detectados bons níveis de anticorpos IgM, IgG e IgA pelo ELISA-Tcra a partir do 15°dia após imunização, exceto o GIV que não apresentou anticorpos IgM. No blot-Tcra os anticorpos anti-Tcra mostraram reatividade para os peptídeos <20kD. As três classes de imunoglobulinas anti-Tcra dos GII e GIII apresentaram reatividade anti-Tso no teste de ELISA. As amostras do GIV não demonstraram anticorpos IgM e IgG e o GVI não apresentou IgG com reatividade anti-Tso. No blot-Tso, GII, GIII e GVI apresentaram anticorpos IgM que identificaram o peptídeo 30kD e o GIV apresentou anticorpos IgM que identificaram os peptídeos 60, 43, 30 e 12kD. Os anticorpos IgG identificaram o 12kD de Tso em todos os grupos inclusive nos GIV e GVI, cujas amostras haviam apresentado ELISA-Tso negativo. Os anticorpos IgA presentes nos animais dos GIII, GIV e GVI identificaram os peptídeos 65, 50, 30 e 12kD de Tso. Os animais do GVII imunizados com Tso apresentaram anticorpos IgM por ELISA-Tso a partir do 5° dia, IgG a partir do 15º dia e anticorpos IgA a partir do 39° dia após imunização. Os anticorpos IgM identificaram especificamente os peptídeos 60, 43, 30 e 12kD; IgG os peptídeos 95, 75, 67, 60, 45, 40-35, 25, 18 e 12kD e IgA os peptídeos 45, 40-35, 28-25 e 18Kd de Tso. Esses anticorpos reagiram cruzadamente com antígeno Tcra em menor intensidade quando comparado com a reatividade específica. Os peptídeos 72, 35, 30, 24 e 14kD de Tcra foram identificados por anticorpos IgM, 95, 70, 50, 30, 18 e 14kD por IgG e 70, 37, 35, 30, 18 e 14kD de Tcra por IgA. A fusão com células esplênicas de camundongos imunizados com 50µg de LV-Tcra e células de mieloma P3X63Ag8.653 foi realizada no 40° dia após a imunização. A triagem dos clones híbridos por ELISA detectou 36 clones secretores de lgM e 117 clones secretores de IgG, que identificaram o antígeno de Tcra e 40 clones secretores de IgM e 10 clones secretores de IgG que identificaram Tso. Após o teste de triagem, foram selecionados alguns clones para serem transferidos para placa de 24 cavidades e destes, 16 clones secretaram anticorpos com especificidade para LV-Tcra com reatividade com T-Tso. Dois clones 7 e 33 reconheceram os peptídeos 18 e 14-12kD no blot-Tcra.
Title in English
T. solium and T. crassiceps: Characterization of the antigens and production of monoclonal antibodies
Keywords in English
Crossreactivity
Cysticercosis
Imunologia
Monoclonal antibodies
Neurologia
Parasitologia
Taenia crassiceps
Taenia solium
Abstract in English
The objective of this study was to perform the production of monoclonal antibodies (MAb) using BALB/c mice immunized with T. crassiceps (Tcra) antigen for identification of Taenia solium (Tso) antigens in human infection. Both antigens vesicular fluid of Tcra (VF-Tcra) and total of Tso (T-Tso) were submitted to analysis by means of SDS-PAGE: Tso antigen presented several peptides from 8 to 158kD, while VF-Tcra antigen was rich in <20kD peptides. Female BALB/c mice were divided into seven groups: GI-control; GII immunized with 20µg of VF-Tcra; GIII and GV with 50µg of VF-Tcra; GIV with ~50µg de gel<30kD-Tcra; GVI with 50 µg antigen eluate <30kD-Tcra, and GVII with 50µg of T-Tso, in order to study antibody production kinetic. IgM, IgG and IgA antibodies were detected by means of ELISA-Tcra, starting on 15th day in serum samples from all groups of mice immunized with Tcra, except from GIV which presented no detectable IgM. On western blot-Tcra (blot-Tcra), these antibodies presented reactivity with <20kD Tcra peptides. Analysis of cross-reactivity showed that groups anti-Tcra antibodies presented anti-Tso cross-reactivity, with exception of GVI which presented no IgG antibodies; and GIV presented no IgM and IgG antibodies when analyzed on ELISA-Tso. On blot-Tso, anti-Tcra IgM antibodies in samples from GII and GV weakly recognized peptide of 30kD, and peptides 60, 43, 30 and 12kD were reactive in samples from GIV. Samples from all groups of mice, IgG anti-Tcra antibodies identified 12kD peptide of Tso. IgA antibodies in sera from GIII, GIV and GV identified 65, 50, 30 and 12kD peptides of Tso. Samples from GVII group immunized with T-Tso identified IgM antibodies on ELISA-Tso starting on 5th day, and IgG antibodies starting on 15th day with increasing levels after booster. This group presented high IgA levels in assayed samples. On blot-Tso, IgM antibodies identified peptides of 60, 43, 30, and 12kD, IgG antibodies reacted with 95, 75, 60, 67, 45, 40-35, 25, 18, and 12kD peptides, and IgA antibodies identified peptides of 45 , 40-35, 28-25 and 18kD. These antibodies presented cross-reactivity with Tcra antigen: peptides of 72, 35, 30, 24 and 14kD were identified by IgM antibodies, 95, 70, 50, 30, 18 and 14kD by IgG antibodies, and 70, 37, 35, 30, 18 and 14kD of Tcra by IgA antibodies. For spleen cells fusion BALB/c mice immunized with VF-Tcra antigen and mieloma cells P3X63-Ag8.653 were used. After cell fusion 36 IgM-clones and 117 IgG-clones were detected. These clones specifically reacted with Tcra antigen. It was also detected 40 IgM-clones and 10 IgG-clones which recognized Tso antigen. By immunoblotting, two clones identified 8-14 and 18kD peptides of VF-Tcra.
 
WARNING - Viewing this document is conditioned on your acceptance of the following terms of use:
This document is only for private use for research and teaching activities. Reproduction for commercial use is forbidden. This rights cover the whole data about this document as well as its contents. Any uses or copies of this document in whole or in part must include the author's name.
Publishing Date
2019-11-28
 
WARNING: Learn what derived works are clicking here.
All rights of the thesis/dissertation are from the authors
CeTI-SC/STI
Digital Library of Theses and Dissertations of USP. Copyright © 2001-2020. All rights reserved.