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

Abstract

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

CLC Number:

S852.61

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.

Figures/Tables 5

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References 35

1	FRISK C S , WAGNER J E . Experimental hamster enteritis: an electron microscopic study[J]. Am J Vet Res, 1977, 38 (11): 1861- 1868. doi: 10.2460/ajvr.1977.38.11.1861
2	YEH J Y . Prevalence and associated risk factors for Lawsonia intracellularis infection infarmed rabbits: A serological and molecular cross-sectional study in South Korea[J]. Front Vet Sci, 2023, 10, 1058113. doi: 10.3389/fvets.2023.1058113
3	NIWA H , HIGUCHI T , FUJII S , et al. Prevalence of equine proliferative enteropathy in Hidaka district, Hokkaido, over five seasons[J]. J Equine Sci, 2022, 33 (4): 71- 74. doi: 10.1294/jes.33.71
4	罗万和, 程会娟, 李振霞, 等. 胞内劳森菌检测方法研究进展[J]. 中兽医医药杂志, 2019, 38 (2): 93- 96.
	LUO W H , CHENG H J , LI Z X , et al. Research progress on detection methods for Lawsonia Intracellularis[J]. Journal of Traditional Chinese Veterinary Medicine and Pharmacology, 2019, 38 (2): 93- 96.
5	郑新添, 杨小燕. 猪增生性肠炎诊断技术研究进展[J]. 中国畜牧兽医, 2011, 38 (4): 203- 206.
	ZHENG X T , YANG X Y . Research progress in diagnostic techniques for porcine proliferative enteritis[J]. China Animal Husbandry & Veterinary Medicine, 2011, 38 (4): 203- 206.
6	KROLL J J , ROOF M B , HOFFMAN L J , et al. Proliferative enteropathy: a global enteric disease of pigs caused by Lawsonia intracellularis[J]. Animal Health Research Reviews, 2005, 173- 197.
7	刘佩红, 王建, 沈莉萍, 等. 上海地区猪增生性肠炎的血清学调查[J]. 动物医学进展, 2007, 168 (9): 108- 109.
	LIU P H , WANG J , SHEN L P , et al. A Serological investigation of porcine proliferative enteritis in the shanghai region[J]. Progress in Veterinary Medicine, 2007, 168 (9): 108- 109.
8	CHOUET S , PRIETO C , MIELI L , et al. Patterns of exposure to Lawsonia intracellularis infection on European pig farms.[J]. Vet Rec, 2003, 152 (1): 14. doi: 10.1136/vr.152.1.14
9	WU Z X , LING Y , TIAN D Y , et al. Seroprevalence of Lawsonia intracellularis antibodies in intensive pig farms in China[J]. BMC Vet Res, 2014, 10 (1): 100. doi: 10.1186/1746-6148-10-100
10	XIAO N , LI J , LI M , et al. Isolation and in vitro cultivation of Lawsonia intracellularis from China[J]. Vet Microbiol, 2022, 270, 109457. doi: 10.1016/j.vetmic.2022.109457
11	唐娜, 沈志强, 管宇, 等. 国外猪增生性肠炎诊断方法研究进展[J]. 养猪, 2009 (3): 54- 56.
	TANG N , SHEN Z Q , GUAN Y , et al. Research progress on diagnostic methods for porcine proliferative enteritis in foreign countries[J]. Swine Production, 2009 (3): 54- 56.
12	DERALD J H . Lawsonia intracellularis: economic losses associated with ileitis[J]. Int Pig Topics, 2019 (7): 34.
13	MARIE-ANNE P , CONNIE J G , DENISE T , et al. Subclinical ileitis: Diagnostic and performance parameters in a multi-dose mucosal homogenate challenge model[J]. J Swine Health Product, 2012, 20 (3): 137- 141. doi: 10.54846/jshap/697
14	庄金秋, 梅建国, 姚春阳, 等. 猪增生性肠炎实验室诊断方法研究进展[J]. 养猪, 2022 (6): 121- 128.
	ZHUANG J Q , MEI J G , YAO C Y , et al. Research progress on laboratory diagnostic methods for porcine proliferative enteritis[J]. Swine Production, 2022 (6): 121- 128.
15	李敏雪, 李剑男, 周红, 等. 基于SodC单克隆抗体的胞内劳森菌IPMA抗原检测方法的建立及应用[J]. 中国农业科学, 2021, 54 (20): 4478- 4486.
	LI M X , LI J N , ZHOU H , et al. Establishment and application of an IPMA antigen detection method for Lawsonia intracellularis using a SodC monoclonal antibody[J]. Scientia Agricultura Sinica, 2021, 54 (20): 4478- 4486.
16	COLLINS A , GONSALVES J , FELL S , et al. Comparison of a commercial ELISA with an indirect fluorescent antibody test to detect antibodies to Lawsonia intracellularis in experimentally challenged pigs[J]. Aust Vet J, 2012, 90 (3): 97- 99. doi: 10.1111/j.1751-0813.2011.00865.x
17	WATARAI M , YAMATO Y , HORIUCHI N , KIM S , et al. Enzyme-linked immunosorbent assay to detect Lawsonia intracellularis in rabbits with proliferative enteropathy[J]. J Vet Med Sci, 2004, 66 (6): 735- 737. doi: 10.1292/jvms.66.735
18	裴艳艳, 张梦琳, 许浒, 等. 猪胞内劳森菌抗体间接ELISA检测方法的建立及应用[J]. 中国预防兽医学报, 2024, 46 (1): 55-60, 69.
	PEI Y Y , ZHANG M L , XU H , et al. Development and application of an indirect ELISA for detecting antibodies against Lawsonia intracellularis in pigs[J]. Chinese Journal of Preventive Veterinary Medicine, 2024, 46 (1): 55-60, 69.
19	廖荣莉, 郑金, 熊梦霞, 等. 基于胞内劳森菌重组flgE蛋白间接ELISA检测方法的建立及应用[J]. 中国预防兽医学报, 2021, 43 (2): 150- 157.
	LIAO R L , ZHENG J , XIONG M X , et al. Development and application of an indirect ELISA assay based on the recombinant flgE protein of Lawsonia intracellularis[J]. Chinese Journal of Preventive Veterinary Medicine, 2021, 43 (2): 150- 157.
20	吴艳阳, 杨东东, 高冬生, 等. 胞内劳森菌LsaA蛋白的原核表达及间接ELISA抗体检测方法的建立[J]. 畜牧兽医学报, 2018, 49 (4): 786- 793. doi: 10.11843/j.issn.0366-6964.2018.04.016
	WU Y Y , YANG D D , GAO D S , et al. Prokaryotic expression of the LsaA protein from Lawsonia intracellularis and establishment of an indirect ELISA-based antibody detection method[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49 (4): 786- 793. doi: 10.11843/j.issn.0366-6964.2018.04.016
21	王丹丹, 周俊明, 倪艳秀, 等. 胞内劳森氏菌间接ELISA抗体检测方法的建立及应用[J]. 中国预防兽医学报, 2022, 44 (4): 396- 401.
	WANG D D , ZHOU J M , NI Y X , et al. Establishment and application of an indirect ELISA antibody detection method for Lawsonia intracellularis[J]. Chinese Journal of Preventive Veterinary Medicine, 2022, 44 (4): 396- 401.
22	佟泽. 泰安及周边地区猪增生性肠炎感染情况调查和胞内劳森菌培养方法建立[D]. 泰安: 山东农业大学, 2020.
	TONG Z. Investigation of porcine proliferative enteritis infection in Tai'an and surrounding areas and development of a culture method for Lawsonia intracellularis[D]. Tai'an: Shandong Agricultural University, 2020. (in Chinese)
23	周银玲. 胞内劳森菌表面蛋白的表达及ELISA方法建立[D]. 武汉: 华中农业大学, 2024.
	ZHOU Y L. Expression of surface proteins of Lawsonia intracellularis and establishment of an ELISA method[D]. Wuhan: Huazhong Agricultural University, 2024. (in Chinese)
24	李复坤. 胞内劳森菌LI0710和LI0649基因的原核表达及ELISA检测方法的建立[D]. 佛山: 佛山科学技术学院, 2022.
	LI F K. Prokaryotic expression of LI0710 and LI0649 genes from Lawsonia intracellularis and development of an ELISA detection method[D]. Foshan: Foshan University, 2022. (in Chinese)
25	MAGDALENA J , PER W , ANN N , et al. Evaluation of a blocking ELISA for the detection of antibodies against Lawsonia intracellularis in pig sera[J]. Acta Vet Scandinavica, 2011, 53 (1): 23- 23. doi: 10.1186/1751-0147-53-23
26	MCORIST S , SMITH S H , GREEN L E . Estimate of direct financial losses due to porcine proliferative enteropathy[J]. Vet Rec, 1997, 140 (22): 579. doi: 10.1136/vr.140.22.579
27	GUEDES R M , GEBHART C J , WINKELMAN N L , et al. Comparison of different methods for diagnosis of porcine proliferative enteropathy[J]. Can J Vet Res, 2002, 66 (2): 99.
28	张从钺, 周红, 蔺辉星, 等. 猪增生性肠病间接ELISA抗体检测试剂盒的研制与应用[J]. 中国农业科学, 2024, 57 (16): 3283- 3293.
	ZHANG C Y , ZHOU H , LIN H X , et al. Development and application of an indirect ELISA antibody detection kit for porcine proliferative enteropathy[J]. Scientia Agricultura Sinica, 2024, 57 (16): 3283- 3293.
29	LOMMATZSCH J , TEMPLIN M F , KRAFT A R , et al. Outer membrane localization of murein hydrolases: MltA, a third lipoprotein lytic transglycosylase in Escherichia coli[J]. J Bacteriol, 1997, 179 (17): 5465- 5470. doi: 10.1128/jb.179.17.5465-5470.1997
30	SCHEURWATER E , REID C W , CLARKE A J . Lytic transglycosylases: bacterial space-making autolysins.[J]. Int J Biochem Cell Biol, 2008, 40 (4): 586- 591. doi: 10.1016/j.biocel.2007.03.018
31	POWELL A J , LIU Z J , NICHOLAS R A , et al. Crystal structures of the lytic transglycosylase MltA from N. gonorrhoeae and E. coli: insights into interdomain movements and substrate binding[J]. J Mol Biol, 2006, 359 (1): 122- 136. doi: 10.1016/j.jmb.2006.03.023
32	JENNINGS G T , SAVINO S , MARCHETTI E , et al. GNA33 from Neisseria meningitidissero group B encodes a membrane-bound lytic transglycosylase (MltA)[J]. Eu J Biochem, 2002, 269, 3722- 2731. doi: 10.1046/j.1432-1033.2002.03064.x
33	李磊. 胞内劳森菌IPMA抗体检测方法的建立及猪源性胞内劳森菌在小鼠体内的复制[D]. 南京: 南京农业大学, 2014.
	LI L. Establishment of an IPMA antibody detection method for Lawsonia intracellularis and replication of porcine-derived Lawsonia intracellularis in mice[D]. Nanjing: Nanjing Agricultural University, 2014. (in Chinese)
34	GUEDES R M , GEBHART C J , WINKELMAN N L , et al. A comparative study of an indirect fluorescent antibody test and an immunoperoxidase monolayer assay for the diagnosis of porcine proliferative enteropathy[J]. J Vet Diagn Invest, 2002, 14 (5): 420- 423. doi: 10.1177/104063870201400512
35	CORZO CA , FRIENDSHIP R , DEWEY C , et al. Comparison of 2 serologic tests for the diagnosis of porcine proliferative enteropathy[J]. Can Vet J, 2005, 46 (5): 433- 435.

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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