Cargando…
Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp. spores and their developmental stages
BACKGROUND: Microsporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissu...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2017
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680761/ https://www.ncbi.nlm.nih.gov/pubmed/29121996 http://dx.doi.org/10.1186/s13071-017-2503-z |
_version_ | 1783277822034313216 |
---|---|
author | Izquierdo, Fernando Moura, Hercules Bornay-Llinares, Fernando Jorge Sriram, Rama Hurtado, Carolina Magnet, Ángela Fenoy, Soledad Visvesvara, Govinda del Aguila, Carmen |
author_facet | Izquierdo, Fernando Moura, Hercules Bornay-Llinares, Fernando Jorge Sriram, Rama Hurtado, Carolina Magnet, Ángela Fenoy, Soledad Visvesvara, Govinda del Aguila, Carmen |
author_sort | Izquierdo, Fernando |
collection | PubMed |
description | BACKGROUND: Microsporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult and immunological techniques such as immunofluorescence have proved to be a useful and reliable tool in the diagnosis of human microsporidiosis. For this reason, we have produced and characterized monoclonal antibodies (MAbs) specific for Encephalitozoon intestinalis (the second most frequent microsporidian infecting humans), and other Encephalitozoon species, that can be used in different diagnostic techniques. RESULTS: Seven MAbs were selected in accordance with their optical density (OD). Four (4C4, 2C2, 2E5 and 2H2) were isotype IgG2a; two (3A5 and 3C9) isotype IgG3, and one Mab, 1D7, IgM isotype. The selected monoclonal antibody-secreting hybridomas were characterized by indirect immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), Western blot, immunoelectron microscopy (Immunogold) and in vitro cultures. The study by IFAT showed different behavior depending on the MAbs studied. The MAbs 4C4, 2C2, 2E5 and 2H2 showed reactivity against epitopes in the wall of the spore (exospore and endospore) epitopes located in Encephalitozoon sp. spores, whereas the MAbs 3A5, 1D7 and 3C9 showed reactivity against internal epitopes (cytoplasmic contents or sporoplasm) of E. intestinalis spores. All MAbs recognized the developing parasites in the in vitro cultures of E. intestinalis. Additionally, 59 formalin-fixed stool samples that had been previously analyzed were screened, with 26 (44%) presenting microsporidian spores (18 samples with E. intestinalis and 8 samples with Enterocytozoon bieneusi). Detection of microsporidian spores by microscopy was performed using Calcofluor stain, Modified Trichrome, Quick-Hot Gram Chromotrope, as well as IFAT using MAbs 4C4, 2C2, 2E5 and 2H2. The 4 MAbs tested clearly recognized the larger spores corresponding to E. intestinalis, but showed no reactivity with Enterocytozoon bieneusi spores. The mass spectrometry and proteomic study revealed that the Mabs 4C4, 2C2, 2E5 and 2H2 recognized the Spore Wall Protein 1 (SWP1) as the antigenic target. CONCLUSIONS: The IFAT-positive MAbs exhibited excellent reactivity against spores and developmental stages, permitting their use in human and animal diagnosis. The epitopes recognized (exospore, endospore and cytoplasmic contents) by the different MAbs developed need further study, and may reveal potential targets for vaccine development, immunotherapy and chemotherapy. |
format | Online Article Text |
id | pubmed-5680761 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-56807612017-11-17 Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp. spores and their developmental stages Izquierdo, Fernando Moura, Hercules Bornay-Llinares, Fernando Jorge Sriram, Rama Hurtado, Carolina Magnet, Ángela Fenoy, Soledad Visvesvara, Govinda del Aguila, Carmen Parasit Vectors Research BACKGROUND: Microsporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult and immunological techniques such as immunofluorescence have proved to be a useful and reliable tool in the diagnosis of human microsporidiosis. For this reason, we have produced and characterized monoclonal antibodies (MAbs) specific for Encephalitozoon intestinalis (the second most frequent microsporidian infecting humans), and other Encephalitozoon species, that can be used in different diagnostic techniques. RESULTS: Seven MAbs were selected in accordance with their optical density (OD). Four (4C4, 2C2, 2E5 and 2H2) were isotype IgG2a; two (3A5 and 3C9) isotype IgG3, and one Mab, 1D7, IgM isotype. The selected monoclonal antibody-secreting hybridomas were characterized by indirect immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), Western blot, immunoelectron microscopy (Immunogold) and in vitro cultures. The study by IFAT showed different behavior depending on the MAbs studied. The MAbs 4C4, 2C2, 2E5 and 2H2 showed reactivity against epitopes in the wall of the spore (exospore and endospore) epitopes located in Encephalitozoon sp. spores, whereas the MAbs 3A5, 1D7 and 3C9 showed reactivity against internal epitopes (cytoplasmic contents or sporoplasm) of E. intestinalis spores. All MAbs recognized the developing parasites in the in vitro cultures of E. intestinalis. Additionally, 59 formalin-fixed stool samples that had been previously analyzed were screened, with 26 (44%) presenting microsporidian spores (18 samples with E. intestinalis and 8 samples with Enterocytozoon bieneusi). Detection of microsporidian spores by microscopy was performed using Calcofluor stain, Modified Trichrome, Quick-Hot Gram Chromotrope, as well as IFAT using MAbs 4C4, 2C2, 2E5 and 2H2. The 4 MAbs tested clearly recognized the larger spores corresponding to E. intestinalis, but showed no reactivity with Enterocytozoon bieneusi spores. The mass spectrometry and proteomic study revealed that the Mabs 4C4, 2C2, 2E5 and 2H2 recognized the Spore Wall Protein 1 (SWP1) as the antigenic target. CONCLUSIONS: The IFAT-positive MAbs exhibited excellent reactivity against spores and developmental stages, permitting their use in human and animal diagnosis. The epitopes recognized (exospore, endospore and cytoplasmic contents) by the different MAbs developed need further study, and may reveal potential targets for vaccine development, immunotherapy and chemotherapy. BioMed Central 2017-11-09 /pmc/articles/PMC5680761/ /pubmed/29121996 http://dx.doi.org/10.1186/s13071-017-2503-z Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Izquierdo, Fernando Moura, Hercules Bornay-Llinares, Fernando Jorge Sriram, Rama Hurtado, Carolina Magnet, Ángela Fenoy, Soledad Visvesvara, Govinda del Aguila, Carmen Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp. spores and their developmental stages |
title | Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp. spores and their developmental stages |
title_full | Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp. spores and their developmental stages |
title_fullStr | Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp. spores and their developmental stages |
title_full_unstemmed | Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp. spores and their developmental stages |
title_short | Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp. spores and their developmental stages |
title_sort | production and characterization of monoclonal antibodies against encephalitozoon intestinalis and encephalitozoon sp. spores and their developmental stages |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680761/ https://www.ncbi.nlm.nih.gov/pubmed/29121996 http://dx.doi.org/10.1186/s13071-017-2503-z |
work_keys_str_mv | AT izquierdofernando productionandcharacterizationofmonoclonalantibodiesagainstencephalitozoonintestinalisandencephalitozoonspsporesandtheirdevelopmentalstages AT mourahercules productionandcharacterizationofmonoclonalantibodiesagainstencephalitozoonintestinalisandencephalitozoonspsporesandtheirdevelopmentalstages AT bornayllinaresfernandojorge productionandcharacterizationofmonoclonalantibodiesagainstencephalitozoonintestinalisandencephalitozoonspsporesandtheirdevelopmentalstages AT sriramrama productionandcharacterizationofmonoclonalantibodiesagainstencephalitozoonintestinalisandencephalitozoonspsporesandtheirdevelopmentalstages AT hurtadocarolina productionandcharacterizationofmonoclonalantibodiesagainstencephalitozoonintestinalisandencephalitozoonspsporesandtheirdevelopmentalstages AT magnetangela productionandcharacterizationofmonoclonalantibodiesagainstencephalitozoonintestinalisandencephalitozoonspsporesandtheirdevelopmentalstages AT fenoysoledad productionandcharacterizationofmonoclonalantibodiesagainstencephalitozoonintestinalisandencephalitozoonspsporesandtheirdevelopmentalstages AT visvesvaragovinda productionandcharacterizationofmonoclonalantibodiesagainstencephalitozoonintestinalisandencephalitozoonspsporesandtheirdevelopmentalstages AT delaguilacarmen productionandcharacterizationofmonoclonalantibodiesagainstencephalitozoonintestinalisandencephalitozoonspsporesandtheirdevelopmentalstages |