Development of an ELISPOT assay for numerating IFN-γ-secreting T cells in chicken using novel monoclonal antibodies

IF 4.2 1区农林科学 Q1 AGRICULTURE, DAIRY & ANIMAL SCIENCE

Poultry Science Pub Date : 2025-07-10 DOI:10.1016/j.psj.2025.105524

Zhou Zhou , Ji Wu , Huining Zhang , Xinjie Yuan , Ye Meng , Lina Chen , Yi Yang , Xiaoli Hao , Shaobin Shang

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Abstract

Cellular immune responses play critical roles in the control of pathogenic infection. The measurement of antigen-specific IFN-γ-secreting T cells via an enzyme-linked immunospot assay (ELISPOT) is a valuable method for the evaluation of cellular immune responses. However, in chicken, few of monoclonal antibodies (mAbs) against chicken IFN-γ (chIFN-γ) are suitable for this application. In this study, three anti-chIFN-γ mAbs (2B10, 3F10, 5A7) were generated by immunization with pcDNA-chIFN-γ plasmid and recombinant Hela cell line stably expressing chIFN-γ (Hela-chIFN-γ). Indirect immunofluorescence assay showed that these mAbs specifically recognized eukaryotically-expressed chIFN-γ in DF-1 cells and natural chIFN-γ secreted by mitogen-activated chicken splenocytes. Furthermore, using 3F10 as a capture antibody and biotinylated 5A7 as a detection antibody, an ELISPOT assay was established for numerating IFN-γ-secreting T cells of chicken. This chIFN-γ ELISPOT assay had higher reactivity than a commercially available kit and showed no cross-reactivity with activated lymphocytes from geese and ducks. This assay was further applied to detect the frequency of MDV antigen-specific IFN-γ-secreting T cells in the spleen of CVI988-immunized chickens. Collectively, we developed and validated an ELISPOT assay for detecting IFN-γ-secreting T cells in chickens using novel anti-chIFN-γ mAbs. Our study provides an important immunological tool for in-depth analysis of cellular immune response in chicken after infection or vaccination.

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用新型单克隆抗体测定鸡体内分泌IFN-γ的T细胞的ELISPOT方法的建立

细胞免疫应答在病原性感染的控制中起关键作用。通过酶联免疫斑点法（ELISPOT）测量抗原特异性IFN-γ分泌T细胞是评估细胞免疫反应的一种有价值的方法。然而，在鸡中，很少有针对鸡IFN-γ (chIFN-γ)的单克隆抗体（mab）适合于这种应用。本研究用稳定表达chIFN-γ (Hela-chIFN-γ)的pcDNA-chIFN-γ质粒和重组Hela细胞系免疫制备了3种抗chIFN-γ单克隆抗体（2B10、3F10、5A7）。间接免疫荧光实验表明，这些单克隆抗体特异性识别真核表达的DF-1细胞和由丝裂原激活的鸡脾细胞分泌的天然chIFN-γ。以3F10为捕获抗体，生物素化5A7为检测抗体，建立了鸡分泌IFN-γ T细胞的ELISPOT计数法。该chIFN-γ ELISPOT检测比市售试剂盒具有更高的反应性，并且与鹅和鸭的活化淋巴细胞无交叉反应。本实验进一步应用于检测cvi988免疫鸡脾脏中MDV抗原特异性IFN-γ分泌T细胞的频率。总之，我们开发并验证了使用新型抗chifn -γ单克隆抗体检测鸡体内分泌IFN-γ的T细胞的ELISPOT方法。本研究为深入分析鸡感染或接种后的细胞免疫反应提供了重要的免疫学工具。

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来源期刊

Poultry Science 农林科学-奶制品与动物科学

CiteScore

7.60

自引率

15.90%

发文量

审稿时长

94 days

期刊介绍： First self-published in 1921, Poultry Science is an internationally renowned monthly journal, known as the authoritative source for a broad range of poultry information and high-caliber research. The journal plays a pivotal role in the dissemination of preeminent poultry-related knowledge across all disciplines. As of January 2020, Poultry Science will become an Open Access journal with no subscription charges, meaning authors who publish here can make their research immediately, permanently, and freely accessible worldwide while retaining copyright to their work. Papers submitted for publication after October 1, 2019 will be published as Open Access papers. An international journal, Poultry Science publishes original papers, research notes, symposium papers, and reviews of basic science as applied to poultry. This authoritative source of poultry information is consistently ranked by ISI Impact Factor as one of the top 10 agriculture, dairy and animal science journals to deliver high-caliber research. Currently it is the highest-ranked (by Impact Factor and Eigenfactor) journal dedicated to publishing poultry research. Subject areas include breeding, genetics, education, production, management, environment, health, behavior, welfare, immunology, molecular biology, metabolism, nutrition, physiology, reproduction, processing, and products.