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Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera
BACKGROUND: Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Commercially available IgG-ELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacte...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968261/ https://www.ncbi.nlm.nih.gov/pubmed/33726780 http://dx.doi.org/10.1186/s12917-021-02828-7 |
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author | Tian, Yaqin Xu, Zuobo Wen, Yukang Yang, Mei Ning, Yaru Wang, Zhaodi Ding, Honglei |
author_facet | Tian, Yaqin Xu, Zuobo Wen, Yukang Yang, Mei Ning, Yaru Wang, Zhaodi Ding, Honglei |
author_sort | Tian, Yaqin |
collection | PubMed |
description | BACKGROUND: Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Commercially available IgG-ELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection. Establishment of an ELISA to detect anti-M. hyopneumoniae IgG in convalescent sera will facilitate the evaluation of the M. hyopneumoniae status of pig farms. RESULTS: In this study, we expressed and purified recombinant Mhp366-N protein, which contains an epitope recognized by M. hyopneumoniae convalescent sera but not hyperimmune sera, for use as a coating antigen. For the M. hyopneumoniae convalescent serum IgG-ELISA, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time and colorimetric reaction time were 0.25 µg/mL, 2.5 % skim milk, 1 h, 1:500, 0.5 h, 1:10,000, 1 h and 15 min, respectively. Validation of the M. hyopneumoniae convalescent serum IgG-ELISA showed a cut-off value of 0.323, the intra-assay CV ranged from 3.27 to 7.26 %, the inter-assay CV ranged from 3.46 to 5.93 %, and the assay was able to differentiate convalescent sera from antibodies to 7 other porcine respiratory pathogens. The convalescent serum IgG-ELISA detected no anti-M. hyopneumoniae IgG in hyperimmune serum samples while a commercial IgG-ELISA identified 95/145 of these sera as positive. The accuracy of the M. hyopneumoniae convalescent serum IgG-ELISA was comparable to the sIgA-ELISA but better than the commercial IgG-ELISA. CONCLUSIONS: The convalescent serum IgG-ELISA is a reproducible, sensitive, and specific indirect ELISA to detect anti-M. hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera. This ELISA could be used to carry out large scale surveillance of M. hyopneumoniae infection in pig farms regardless of vaccination status. |
format | Online Article Text |
id | pubmed-7968261 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-79682612021-03-22 Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera Tian, Yaqin Xu, Zuobo Wen, Yukang Yang, Mei Ning, Yaru Wang, Zhaodi Ding, Honglei BMC Vet Res Methodology Article BACKGROUND: Immunization of pigs with an inactivated Mycoplasma hyopneumoniae vaccine (bacterin) generates hyperimmune serum that contains high concentrations of anti-M. hyopneumoniae IgG. Commercially available IgG-ELISA kits cannot distinguish between anti-M. hyopneumoniae IgG in inactivated bacterin-induced hyperimmune sera and convalescent sera resulting from natural M. hyopneumoniae infection. Establishment of an ELISA to detect anti-M. hyopneumoniae IgG in convalescent sera will facilitate the evaluation of the M. hyopneumoniae status of pig farms. RESULTS: In this study, we expressed and purified recombinant Mhp366-N protein, which contains an epitope recognized by M. hyopneumoniae convalescent sera but not hyperimmune sera, for use as a coating antigen. For the M. hyopneumoniae convalescent serum IgG-ELISA, the optimal antigen concentration, blocking buffer, blocking time, dilution of serum, incubation time with serum, secondary antibody dilution, secondary antibody incubation time and colorimetric reaction time were 0.25 µg/mL, 2.5 % skim milk, 1 h, 1:500, 0.5 h, 1:10,000, 1 h and 15 min, respectively. Validation of the M. hyopneumoniae convalescent serum IgG-ELISA showed a cut-off value of 0.323, the intra-assay CV ranged from 3.27 to 7.26 %, the inter-assay CV ranged from 3.46 to 5.93 %, and the assay was able to differentiate convalescent sera from antibodies to 7 other porcine respiratory pathogens. The convalescent serum IgG-ELISA detected no anti-M. hyopneumoniae IgG in hyperimmune serum samples while a commercial IgG-ELISA identified 95/145 of these sera as positive. The accuracy of the M. hyopneumoniae convalescent serum IgG-ELISA was comparable to the sIgA-ELISA but better than the commercial IgG-ELISA. CONCLUSIONS: The convalescent serum IgG-ELISA is a reproducible, sensitive, and specific indirect ELISA to detect anti-M. hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera. This ELISA could be used to carry out large scale surveillance of M. hyopneumoniae infection in pig farms regardless of vaccination status. BioMed Central 2021-03-16 /pmc/articles/PMC7968261/ /pubmed/33726780 http://dx.doi.org/10.1186/s12917-021-02828-7 Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data. |
spellingShingle | Methodology Article Tian, Yaqin Xu, Zuobo Wen, Yukang Yang, Mei Ning, Yaru Wang, Zhaodi Ding, Honglei Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera |
title | Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera |
title_full | Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera |
title_fullStr | Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera |
title_full_unstemmed | Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera |
title_short | Development of an indirect ELISA for detection of anti-Mycoplasma hyopneumoniae IgG in naturally infected pathogen-induced convalescent sera |
title_sort | development of an indirect elisa for detection of anti-mycoplasma hyopneumoniae igg in naturally infected pathogen-induced convalescent sera |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968261/ https://www.ncbi.nlm.nih.gov/pubmed/33726780 http://dx.doi.org/10.1186/s12917-021-02828-7 |
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