胞内劳森菌MltA蛋白单克隆抗体的制备及阻断ELISA抗体检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.11.025

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (11): 5660-5669.doi: 10.11843/j.issn.0366-6964.2025.11.025

胞内劳森菌MltA蛋白单克隆抗体的制备及阻断ELISA抗体检测方法的建立

孙心如¹^,²(), 武文清¹^,², 罗亚娟¹^,², 谢锐¹^,², 彭长江¹^,², 华琳¹^,², 吴斌¹^,²^,³^,*(), 彭忠¹^,²^,³^,*()

1. 华中农业大学动物医学院农业微生物学国家重点实验室, 武汉 430070
2. 生猪健康养殖协同创新中心, 武汉 430070
3. 湖北洪山实验室, 武汉 430070

收稿日期:2025-02-10 出版日期:2025-11-23 发布日期:2025-11-27
通讯作者: 吴斌,彭忠 E-mail:sunxinru@webmail.hzau.edu.cn;wub@mail.hzau.edu.cn;pengzhong@mail.hzau.edu.cn
作者简介:孙心如(2000-)，女，山东临沂人，硕士生，主要从事猪疫病诊断及防控研究，E-mail: sunxinru@webmail.hzau.edu.cn
基金资助:
国家生猪技术创新中心先导科技项目(NCTIP-XD/B11)

Preparation of Monoclonal Antibodies against MltA Protein of Lawsonia intracellularis and Establishment of Blocking ELISA Antibody Detection Method

SUN Xinru¹^,²(), WU Wenqing¹^,², LUO Yajuan¹^,², XIE Rui¹^,², PENG Changjiang¹^,², HUA Lin¹^,², WU Bin¹^,²^,³^,*(), PENG Zhong¹^,²^,³^,*()

1. State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
2. Collaborative Innovation Center for Healthy Pig Breeding, Wuhan 430070, China
3. Hubei Hongshan Laboratory, Wuhan 430070, China

Received:2025-02-10 Online:2025-11-23 Published:2025-11-27
Contact: WU Bin, PENG Zhong E-mail:sunxinru@webmail.hzau.edu.cn;wub@mail.hzau.edu.cn;pengzhong@mail.hzau.edu.cn

摘要/Abstract

摘要：

本研究旨在建立一种检测胞内劳森菌(Lawsonia intracellularis)抗体的阻断ELISA方法。采用原核表达的胞内劳森菌重组MltA蛋白免疫小鼠，制备获得单克隆抗体，通过Western blot和间接免疫荧光(IFA)鉴定了单克隆抗体的特异性。以重组蛋白MltA为包被抗原，待检血清为一抗，建立了检测胞内劳森菌抗体的阻断ELISA方法。确定了阻断ELISA方法最佳反应条件，并对方法的灵敏度、特异性、重复性及符合率进行分析。结果显示: 所建立方法的灵敏度和特异性分析显示在胞内劳森菌阳性血清稀释度为1∶4时仍可稳定检出阳性，且与猪流行性腹泻病毒、伪狂犬病病毒及大肠杆菌阳性血清无交叉反应；重复性试验结果显示，批内重复试验的变异系数为1.4%~9.3%，批间重复试验的变异系数为1.0%~9.6%；与IFA结果对比，本研究所建立方法的敏感性为95.91%，特异性为80.65%，总符合率为85.92%。本研究对胞内劳森菌外膜蛋白MltA进行原核表达和纯化，制备了针对MltA的单克隆抗体，建立了一种检测胞内劳森菌抗体的阻断ELISA方法，可用于猪增生性肠病的临床诊断。

关键词: 胞内劳森菌, 单克隆抗体, 阻断ELISA, 重组MltA蛋白

Abstract:

This study aims to develop a blocking ELISA method for detecting antibodies against Lawsonia intracellularis. The recombinant MltA protein of L. intracellularis, expressed in a prokaryotic system, was used to immunize mice. Monoclonal antibodies were successfully prepared, and their specificity was confirmed through Western blot and indirect immunofluorescence assay (IFA). A blocking ELISA method for detecting L. intracellularis antibodies was developed, utilizing the recombinant MltA protein as the coating antigen and the test serum as the primary antibody. The optimal reaction conditions for the blocking ELISA method were established, and the method's sensitivity, specificity, repeatability, and coincidence rate were thoroughly evaluated. The analysis of the sensitivity and specificity of the established method demonstrated that positive results could be consistently detected at a 1∶4 dilution of L. intracellularis-positive serum. No cross-reactivity was observed with porcine epidemic diarrhea virus, pseudorabies virus, or Escherichia coli positive sera. The repeatability test results revealed that the coefficient of variation (CV) for intra-assay repeatability ranged from 1.4% to 9.3%, while the CV for inter-assay repeatability ranged from 1.0% to 9.6%. Compared with the IFA result, the method developed in this study achieved a sensitivity of 95.91%, a specificity of 80.65%, and an overall agreement rate of 85.92%.This study conducted the prokaryotic expression and purification of the L. intracellularis outer membrane protein MltA. Monoclonal antibodies targeting MltA were successfully prepared. A blocking ELISA method for detecting L. intracellularis antibodies was developed, which can be effectively applied to the clinical diagnosis of porcine proliferative enteropathy.

Key words: Lawsonia intracellularis, monoclonal antibody, blocking ELISA, recombinant MltA protein

中图分类号:

S852.61

孙心如, 武文清, 罗亚娟, 谢锐, 彭长江, 华琳, 吴斌, 彭忠. 胞内劳森菌MltA蛋白单克隆抗体的制备及阻断ELISA抗体检测方法的建立[J]. 畜牧兽医学报, 2025, 56(11): 5660-5669.

SUN Xinru, WU Wenqing, LUO Yajuan, XIE Rui, PENG Changjiang, HUA Lin, WU Bin, PENG Zhong. Preparation of Monoclonal Antibodies against MltA Protein of Lawsonia intracellularis and Establishment of Blocking ELISA Antibody Detection Method[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5660-5669.

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胞内劳森菌MltA蛋白单克隆抗体的制备及阻断ELISA抗体检测方法的建立

Preparation of Monoclonal Antibodies against MltA Protein of Lawsonia intracellularis and Establishment of Blocking ELISA Antibody Detection Method

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方法 Method		IFA
方法 Method		+	-	合计Total
阻断ELISA Blocking ELISA	+	47	18	65
	-	2	75	77
	合计	49	93	142