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Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats
BACKGROUND: Diseases caused by Toxoplasma gondii (T. gondii) have introduced serious threats to public health. There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prok...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10623812/ https://www.ncbi.nlm.nih.gov/pubmed/37924072 http://dx.doi.org/10.1186/s12917-023-03775-1 |
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author | Gao, Yafan Shen, Yu Fan, Jiyuan Ding, Haojie Zheng, Bin Yu, Haijie Huang, Siyang Kong, Qingming Lv, Hangjun Zhuo, Xunhui Lu, Shaohong |
author_facet | Gao, Yafan Shen, Yu Fan, Jiyuan Ding, Haojie Zheng, Bin Yu, Haijie Huang, Siyang Kong, Qingming Lv, Hangjun Zhuo, Xunhui Lu, Shaohong |
author_sort | Gao, Yafan |
collection | PubMed |
description | BACKGROUND: Diseases caused by Toxoplasma gondii (T. gondii) have introduced serious threats to public health. There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prokaryotic expression system and used as the detection antigen. The aim of this study was to evaluate and optimize a reliable indirect enzyme-linked immunosorbent assay (iELISA) method based on rAMA1 for the detection of antibodies against T. gondii in cats. RESULTS: The rAMA1-iELISA method was developed and optimized by the chessboard titration method. There were no cross-reactions between T. gondii-positive cat serum and positive serum for other pathogens, indicating that rAMA1-iELISA could only detect T. gondii in most cases. The lowest detection limit of rAMA1-iELISA was 1:3200 (dilution of positive serum), and the CV of repeated tests within batches and between batches were confirmed to be less than 10%. The results of 247 cat serum samples detected by rAMA1-iELISA (kappa value = 0.622, p < 0.001) were in substantial agreement with commercial ELISA. The ROC curve analysis revealed the higher overall check accuracy of rAMA1-iELISA (sensitivity = 91.7%, specificity = 93.6%, AUC = 0.956, 95% CI 0.905 to 1.000) than GRA7-based iELISA (sensitivity = 91.7%, specificity = 85.5%, AUC = 0.936, 95% CI 0.892 to 0.980). Moreover, the positive rate of rAMA1-iELISA (6.5%, 16/247) was higher than that of GRA7-based iELISA (3.6%, 9/247) and that of commercial ELISA kit (4.9%, 12/247). CONCLUSION: The iELISA method with good specificity, sensitivity, and reproducibility was established and can be used for large-scale detection of T. gondii infection in clinical cat samples. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12917-023-03775-1. |
format | Online Article Text |
id | pubmed-10623812 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-106238122023-11-04 Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats Gao, Yafan Shen, Yu Fan, Jiyuan Ding, Haojie Zheng, Bin Yu, Haijie Huang, Siyang Kong, Qingming Lv, Hangjun Zhuo, Xunhui Lu, Shaohong BMC Vet Res Research BACKGROUND: Diseases caused by Toxoplasma gondii (T. gondii) have introduced serious threats to public health. There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prokaryotic expression system and used as the detection antigen. The aim of this study was to evaluate and optimize a reliable indirect enzyme-linked immunosorbent assay (iELISA) method based on rAMA1 for the detection of antibodies against T. gondii in cats. RESULTS: The rAMA1-iELISA method was developed and optimized by the chessboard titration method. There were no cross-reactions between T. gondii-positive cat serum and positive serum for other pathogens, indicating that rAMA1-iELISA could only detect T. gondii in most cases. The lowest detection limit of rAMA1-iELISA was 1:3200 (dilution of positive serum), and the CV of repeated tests within batches and between batches were confirmed to be less than 10%. The results of 247 cat serum samples detected by rAMA1-iELISA (kappa value = 0.622, p < 0.001) were in substantial agreement with commercial ELISA. The ROC curve analysis revealed the higher overall check accuracy of rAMA1-iELISA (sensitivity = 91.7%, specificity = 93.6%, AUC = 0.956, 95% CI 0.905 to 1.000) than GRA7-based iELISA (sensitivity = 91.7%, specificity = 85.5%, AUC = 0.936, 95% CI 0.892 to 0.980). Moreover, the positive rate of rAMA1-iELISA (6.5%, 16/247) was higher than that of GRA7-based iELISA (3.6%, 9/247) and that of commercial ELISA kit (4.9%, 12/247). CONCLUSION: The iELISA method with good specificity, sensitivity, and reproducibility was established and can be used for large-scale detection of T. gondii infection in clinical cat samples. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12917-023-03775-1. BioMed Central 2023-11-03 /pmc/articles/PMC10623812/ /pubmed/37924072 http://dx.doi.org/10.1186/s12917-023-03775-1 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This 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/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://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 | Research Gao, Yafan Shen, Yu Fan, Jiyuan Ding, Haojie Zheng, Bin Yu, Haijie Huang, Siyang Kong, Qingming Lv, Hangjun Zhuo, Xunhui Lu, Shaohong Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats |
title | Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats |
title_full | Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats |
title_fullStr | Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats |
title_full_unstemmed | Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats |
title_short | Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats |
title_sort | establishment and application of an ielisa detection method for measuring apical membrane antigen 1 (ama1) antibodies of toxoplasma gondii in cats |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10623812/ https://www.ncbi.nlm.nih.gov/pubmed/37924072 http://dx.doi.org/10.1186/s12917-023-03775-1 |
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