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使用酶联免疫吸附测定（ELISA）、酶联免疫斑点测定（ELISPOT）和毒素中和试验来测量艰难梭菌毒素对抗体反应影响的方案。

Protocol to measure the impact of Clostridioides difficile toxins on antibody responses using ELISA, ELISPOT, and toxin-neutralization assays.

作者信息

Norman Kaylee M, Lang Gillian A, Ballard Jimmy D, Lang Mark L

机构信息

Department of Microbiology and Immunology, University of Oklahoma Health Sciences, Oklahoma City, OK 73104, USA.

Department of Microbiology and Immunology, University of Oklahoma Health Sciences, Oklahoma City, OK 73104, USA.

出版信息

STAR Protoc. 2025 Jun 20;6(2):103754. doi: 10.1016/j.xpro.2025.103754. Epub 2025 Apr 8.

DOI:10.1016/j.xpro.2025.103754
PMID:40202837
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12008137/
Abstract

To determine the effects of bacterial toxins on the humoral immune response, accurate measurement of antibody quantity, specificity, and function is critical. Here, we present a murine injection protocol for bacterial toxins and vaccine antigens and collection of plasma via retro-orbital eye bleeds. We describe steps for measuring antigen-specific antibody titers, antibody-secreting plasma cell abundance, and in vitro function of those antibodies. This protocol may be adapted to measure the impact of several bacterial toxins on humoral immunity. For complete details on the use and execution of this protocol, please refer to Norman et al..

摘要

为了确定细菌毒素对体液免疫反应的影响，准确测量抗体的数量、特异性和功能至关重要。在此，我们介绍一种用于注射细菌毒素和疫苗抗原的小鼠实验方案，以及通过眶后眼球取血收集血浆的方法。我们描述了测量抗原特异性抗体滴度、分泌抗体的浆细胞丰度以及这些抗体体外功能的步骤。该方案可用于测量多种细菌毒素对体液免疫的影响。有关该方案使用和实施的完整详细信息，请参考诺曼等人的研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/3ca03d5867bc/gr7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/6fa77d1f7070/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/61efd808c3b4/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/9c076d15fec7/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/185d9a2539a5/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/dbad343adc8a/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/c73d78d055f1/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/ddb131659da1/gr6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/3ca03d5867bc/gr7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/6fa77d1f7070/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/61efd808c3b4/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/9c076d15fec7/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/185d9a2539a5/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/dbad343adc8a/gr4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/c73d78d055f1/gr5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/ddb131659da1/gr6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ed36/12008137/3ca03d5867bc/gr7.jpg

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本文引用的文献

1
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Cell Rep. 2024 May 28;43(5):114245. doi: 10.1016/j.celrep.2024.114245. Epub 2024 May 17.
2
Important Considerations for ELISpot Validation.酶联免疫斑点分析（ELISpot）验证的重要考虑因素。
Methods Mol Biol. 2024;2768:1-13. doi: 10.1007/978-1-0716-3690-9_1.
3
Deletion of a 19-Amino-Acid Region in Clostridioides difficile TcdB2 Results in Spontaneous Autoprocessing and Reduced Cell Binding and Provides a Nontoxic Immunogen for Vaccination.
艰难梭菌 TcdB2 中 19 个氨基酸区域的缺失导致自发自我加工以及细胞结合减少，并为疫苗接种提供了无毒的免疫原。
Infect Immun. 2019 Jul 23;87(8). doi: 10.1128/IAI.00210-19. Print 2019 Aug.
4
Mastering the Computational Challenges of Elispot Plate Evaluation.掌握酶联免疫斑点（ELISPOT）板评估的计算挑战
Methods Mol Biol. 2018;1808:9-30. doi: 10.1007/978-1-4939-8567-8_2.
5
Standardization and validation issues of the ELISPOT assay.酶联免疫斑点试验（ELISPOT）的标准化与验证问题。
Methods Mol Biol. 2005;302:51-86. doi: 10.1385/1-59259-903-6:051.