猪流行性腹泻病毒特异性SIgA抗体间接ELISA检测方法的建立及应用

朱海侠

doi:10.19303/j.issn.1008-0384.2023.09.002

猪流行性腹泻病毒特异性SIgA抗体间接ELISA检测方法的建立及应用

doi: 10.19303/j.issn.1008-0384.2023.09.002

朱海侠

兆丰华生物科技（福州）有限公司，福建福州　350014

基金项目: 兆丰华生物科技（福州）有限公司内部项目（2019年）

详细信息

作者简介:
朱海侠（1986 —），女，硕士，主要从事分子生物学相关研究，E-mail：happyhaixia1101@126.com

中图分类号: S858.28
计量
- 文章访问数: 453
- HTML全文浏览量: 211
- PDF下载量: 14
- 被引次数: 0
出版历程
- 收稿日期: 2022-07-27
- 修回日期: 2023-03-18
- 网络出版日期: 2023-10-25
- 刊出日期: 2023-09-28

An Indirect ELISA Assay for Detecting Specific SIgA Antibody of Porcine Epidemic Diarrhea Virus

ZHU Haixia

JOFUNHWA Biotechnology (Fuzhou) Co., Ltd., Fuzhou, Fujian　350014, China

摘要

摘要: 目的快速了解猪群感染猪流行性腹泻病毒或免疫猪流行性腹泻病毒相关疫苗后猪群特异性SIgA抗体水平。方法对猪流行性腹泻病毒（Porcine epidemic diarrhea virus，PEDV）ZJ08株进行纯化，采用纯化的病毒作为包被抗原，以辣根过氧化物酶标记的SC（Secretory component，SC）多克隆抗体为标记抗体，通过优化检测条件，建立乳汁中猪流行性腹泻病毒特异性SIgA抗体间接ELISA检测方法。结果成功建立了猪流行性腹泻病毒SIgA抗体检测方法。PEDV抗原的最佳包倍浓度为0.6 μg·孔⁻¹，样本最佳稀释比例为1∶5，作用时间1 h；SC标记抗体最适稀释比例为1∶100，作用时间1.5 h。建立的ELISA方法能够特异性检测样本中抗PEDV SIgA抗体，与PoRV（Porcine rotavirus，PoRV）、TGEV（Transmissible gastroenteritis virus，TGEV）的阳性乳汁均无交叉反应；批内、批间重复试验的变异系数小于10%，具有良好的特异性和重复性。通过临床采集的乳汁样品测定发现，样本中PEDV IgA抗体阳性率达到98.1%，而采用针对特异性SIgA的抗体方法检测，其阳性率仅为69.2%，样本中IgA和SIgA抗体符合率仅为70.6%，提示仅仅依据PEDV IgA抗体水平并不能很好地反映猪群免疫该疫苗后特异性黏膜免疫的真实水平。结论建立的PEDV特异性SIgA抗体检测方法可为分析、评估猪群免疫PEDV疫苗后乳汁中特异SIgA抗体产生及猪群的保护情况提供可靠依据。
- 猪流行性腹泻病毒 /
- 间接ELISA /
- SIgA抗体
Abstract: Objective A method for detecting SIgA antibodies of porcine epidemic diarrhea virus (PEDV) was established. Methods Using a purified PEDV ZJ08 strain as coating antigen and the antibody for secretory component(SC) labeled by horseradish peroxidase (HRP) as secondary antibody, an indirect ELISA assay with optimized conditions was developed to detect the specific SIgA antibody of PEDV. Result The optimized assay applied a working antigen concentration of 0.6 μg per well, a 1∶5 sample dilution, a coating at 37 ℃ for 1 h, and an 100× dilution of the labeled antibody for 1.5 h incubation at 37 ℃. Other than the target detection, the assay did not react with porcine rotavirus or transmissible gastroenteritis virus. The inter- or intra-batch variations of the assay on test results were less than 10%. On the milk from vaccinated pigs, a 98.1% positive detection on IgA, which was significantly higher than 69.2% on SIgA and merely 70.6% on combined IgA and SIgA, was achieved indicating the deficiency of a single IgA test for an accurate determination on the mucosal immunity of the animal. Conclusion The established assay appeared to appropriately reveal the specific SIgA antibody produced in pigs for protection of the livestock from PEDV by the vaccination.
- Porcine epidemic diarrhea virus /
- indirect ELISA /
- SIgA antibody

HTML全文

图 1 不同浓度蔗糖层PEDV RT-PCR检测结果

M：1000 DNA Marker；1～3：40%～60%层带未稀释、100倍稀释、1000倍稀释；4～6为30%层带未稀释、100倍稀释、1000倍稀释；7～9为20%层带未稀释、100倍稀释、1000倍稀释。

Figure 1. RT-PCR detections of PEDV on sucrose layers

M：DNA ladder DL1000; 1–3: on not diluted 40%–60% sucrose layer and on those diluted 100× and 1 000×, respectively; 4–6: on not diluted 30% sucrose layer and on those diluted 100× and 1 000×, respectively; 7–9 on not diluted 20% sucrose layer and on those diluted 100× and 1 000×, respectively.

下载: 全尺寸图片幻灯片

图 2 Western Blot分析PET32a-SC重组蛋白的表达

M：蛋白 Marker；1：SC 蛋白通过 His 标签和羊抗鼠二抗的反应检测。

Figure 2. PET32a-SC protein expression by western blot

M: Protein marker; 1: SC protein tested by His tag and goat anti-mouse secondary antibody.

下载: 全尺寸图片幻灯片

图 3 PET32a-SC蛋白SDS-PAGE分析

M：蛋白 marker；1、2：纯化后 PET32a-SC。

Figure 3. SDS-PAGE on expressed PET32a-SC protein

M: Protein marker; 2-3: purified protein of PET32a-SC.

下载: 全尺寸图片幻灯片

表 1 抗原最佳包被浓度及乳汁最佳稀释度的确定（OD_{450 nm}）

Table 1. Optimal antigen concentration and milk dilution determined according to OD_{450 nm}

样本稀释度 Sample dilution	抗原稀释度 Antigen dilution/(μg·孔⁻¹)	P	N	P/N
1∶5	1.2	0.821	0.130	6.32
	0.6	0.663	0.083	7.99
	0.3	0.627	0.096	6.53
	0.15	0.584	0.075	7.79
1∶10	1.2	0.725	0.131	5.53
	0.6	0.572	0.096	5.96
	0.3	0.494	0.093	5.31
	0.15	0.455	0.077	5.91
1∶20	1.2	0.69	0.132	5.23
	0.6	0.563	0.105	5.36
	0.3	0.472	0.115	4.10
	0.15	0.436	0.099	4.40
1∶30	1.2	0.519	0.128	4.05
	0.6	0.412	0.107	3.85
	0.3	0.324	0.117	2.77
	0.15	0.271	0.111	2.44

下载: 导出CSV

表 2 最佳包被条件的确定

Table 2. Optimum conditions for coating

指标 Index	包被条件 Coating condition
指标 Index	4 ℃包被过夜 4 ℃ wrap overnight	37 ℃ 1 h	37 ℃ 2 h
P	0.657	0.572	0.620
N	0.130	0.082	0.081
P/N	5.05	6.98	7.65

下载: 导出CSV

表 3 最佳封闭条件的确定

Table 3. Optimum conditions for blocking

指标 Index	封闭浓度 Closure condition			封闭条件（37 ℃） Closure condition
指标 Index	1%	2%	5%	1 h	2 h	3 h
P	1.180	1.121	1.034	1.110	1.070	1.077
N	0.241	0.213	0.220	0.282	0.226	0.255
P/N	4.89	5.26	4.68	3.94	4.73	4.22

下载: 导出CSV

表 4 最佳乳汁样本工作条件的确定

Table 4. Optimum working conditions for milk specimens

指标 Index	孵育时间 Incubation time
指标 Index	37 ℃ 45 min	37 ℃ 60 min	37 ℃ 90 min
P	0.638	0.639	0.654
N	0.096	0.085	0.087
P/N	6.65	7.52	7.52

下载: 导出CSV

表 5 最佳标记抗体工作条件的确定

Table 5. Optimum working conditions for HRP-labeled secondary antibody

指标 Index	稀释比例 dilution				作用时间 Reaction conditions
指标 Index	1∶50	1∶100	1∶200	1∶300	37 ℃ 45 min	37 ℃ 60 min	37 ℃ 90 min
P	0.819	0.891	0.709	0.706	0.746	0.796	0.867
N	0.168	0.162	0.172	0.159	0.146	0.150	0.160
P/N	4.88	5.5	4.12	4.44	5.11	5.31	5.42

下载: 导出CSV

表 6 底物最佳反应时间的确定

Table 6. Optimum reaction time for TMB

指标 Index	孵育时间 Incubation time
指标 Index	15 min	20 min	25 min	30 min
P	0.869	0.934	1.019	1.089
N	0.136	0.136	0.215	0.187
P/N	6.39	6.87	4.93	5.82

下载: 导出CSV

表 7 乳汁样本的重复性检验结果

Table 7. Repeatability of whey samples

编号 Number	组内重复 Intra group repetition		组间重复 Intergroup duplication
编号 Number	$\overline { X}\pm { {S D} }$	变异系数CV/%	$\overline { X}\pm { {S D} }$	变异系数CV/%
1	0.167±0.009	0.3	0.168±0.040	1
2	0.225±0.005	0.2	0.223±0.052	2
3	0.249±0.006	0.2	0.252±0.009	1
4	0.352±0.017	0.6	0.342±0.032	2
5	0.304±0.006	0.2	0.298±0.028	1
6	0.340±0.009	0.3	0.335±0.025	2
7	0.159±0.040	1.3	0.162±0.059	3
8	0.641±0.002	0.1	0.623±0.066	6
9	0.174±0.021	0.8	0.165±0.032	5
10	0.161±0.013	0.5	0.157±0.035	1

下载: 导出CSV

参考文献(22)

[1]	杨朋霞, 闫若潜, 王华俊, 等. 基于猪流行性腹泻病毒N蛋白间接ELISA抗体检测方法的建立及初步应用 [J]. 中国预防兽医学报, 2019, 41(10):1021−1026. YANG P X, YAN R Q, WANG H J, et al. Establishment and preliminary application of an indirect ELISA for detecting antibody based on N protein of porcine epidemic diarrhea variant virulent strain [J]. Chinese Journal of Preventive Veterinary Medicine, 2019, 41(10): 1021−1026.(in Chinese)
[2]	陈建飞. 猪流行性腹泻病毒全基因组序列分析及感染性cDNA克隆的构建[D]. 北京: 中国农业科学院, 2012: 3. CHEN J F. Analysis of complete genome sequences and construction of an infectious cDNA clone of porcine epidemic diaririea virus[D]. Beijing: Chinese Academy of Agricultural Sciences, 2012: 3. (in Chinese)
[3]	LI Y C, WU Q X, HUANG L L, et al. An alternative pathway of enteric PEDV dissemination from nasal cavity to intestinal mucosa in swine [J]. Nature Communications, 2018, 9: 3811. doi: 10.1038/s41467-018-06056-w
[4]	LI W T, LI H, LIU Y B, et al. New variants of porcine epidemic diarrhea virus, China, 2011 [J]. Emerging Infectious Diseases, 2012, 18(8): 1350−1353. doi: 10.3201/eid1803.120002
[5]	CHEN Q, LI G W, STASKO J, et al. Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States [J]. Journal of Clinical Microbiology, 2014, 52(1): 234−243. doi: 10.1128/JCM.02820-13
[6]	SONG Q Y, STONE S, DREBES D, et al. Characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions [J]. Virus Research, 2016, 226: 85−92. doi: 10.1016/j.virusres.2016.06.002
[7]	JUNG K, SAIF L J, WANG Q H. Porcine epidemic diarrhea virus (PEDV): An update on etiology, transmission, pathogenesis, and prevention and control [J]. Virus Research, 2020, 286: 198045. doi: 10.1016/j.virusres.2020.198045
[8]	PARK J E, KANG K J, RYU J H, et al. Porcine epidemic diarrhea vaccine evaluation using a newly isolated strain from Korea [J]. Veterinary Microbiology, 2018, 221: 19−26. doi: 10.1016/j.vetmic.2018.05.012
[9]	POONSUK K, GIMÉNEZ-LIROLA L G, ZHANG J Q, et al. Does circulating antibody play a role in the protection of piglets against porcine epidemic diarrhea virus? [J]. PLoS One, 2016, 11(4): e0153041. doi: 10.1371/journal.pone.0153041
[10]	陈冰清, 葛艳, 司伏生, 等. 猪流行性腹泻病毒诱导的猪肠道-乳腺-sIgA轴以及免疫机制探讨 [J]. 微生物学通报, 2021, 48(11):4240−4249. doi: 10.13344/j.microbiol.china.210041 CHEN B Q, GE Y, SI F S, et al. Gut-mammary-sIgA axis and immunologic mechanisms induced by porcine epidemic diarrhea virus [J]. Microbiology China, 2021, 48(11): 4240−4249.(in Chinese) doi: 10.13344/j.microbiol.china.210041
[11]	孙立旦, 陈立功, 周双海, 等. 猪流行性腹泻病毒血清IgA抗体间接ELISA方法的建立 [J]. 中国兽医学报, 2019, 39(3):381−386. doi: 10.16303/j.cnki.1005-4545.2019.03.01 SUN L D, CHEN L G, ZHOU S H, et al. Indirect ELISA for detecting specific IgA antibody in serum to porcine epidemic diarrhea virus [J]. Chinese Journal of Veterinary Science, 2019, 39(3): 381−386.(in Chinese) doi: 10.16303/j.cnki.1005-4545.2019.03.01
[12]	谷长维, 谷长乐, 胡博. 猪流行性腹泻病毒血清中特异性IgG抗体间接ELISA检测方法的建立 [J]. 中国动物传染病学报, 2022, 30(3):106−112. doi: 10.3969/j.issn.1674-6422.2022.3.zgsyjscb202203015 GU C W, GU C L, HU B. Development of indirect ELISA assay for detection of porcine epidemic diarrhea virus antibodies in sera of naturally infected pigs [J]. Chinese Journal of Animal Infectious Diseases, 2022, 30(3): 106−112.(in Chinese) doi: 10.3969/j.issn.1674-6422.2022.3.zgsyjscb202203015
[13]	DIEL D G, LAWSON S, OKDA F, et al. Porcine epidemic diarrhea virus: An overview of current virological and serological diagnostic methods [J]. Virus Research, 2016, 226: 60−70. doi: 10.1016/j.virusres.2016.05.013
[14]	高冉, 刘建波, 陈建飞, 等. 抗猪SIgA SC片段单克隆抗体的制备及鉴定 [J]. 中国预防兽医学报, 2018, 40(5):455−458. GAO R, LIU J B, CHEN J F, et al. Preparation and identification of monoclonal antibodies against porcine SIgA SC fragment [J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40(5): 455−458.(in Chinese)
[15]	丛广义, 刘建波, 时洪艳, 等. 猪流行性腹泻病毒特异性SIgA抗体间接ELISA检测方法的建立及应用 [J]. 中国预防兽医学报, 2019, 41(8):812−817. CONG G Y, LIU J B, SHI H Y, et al. Establishment and application of indirect ELISA for detection of porcine epidemic diarrhea virus specific SIgA [J]. Chinese Journal of Preventive Veterinary Medicine, 2019, 41(8): 812−817.(in Chinese)
[16]	李春喜, 姜丽娜, 邵云. 生物统计学[M]. 5版. 北京: 科学出版社, 2016.
[17]	SUN R Q, CAI R J, CHEN Y Q, et al. Outbreak of porcine epidemic diarrhea in suckling piglets, China [J]. Emerging Infectious Diseases, 2012, 18(1): 161−163. doi: 10.3201/eid1801.111259
[18]	王永恒, 周孟云, 李昱辰, 等. 猪流行性腹泻病毒黏液SIgA抗体ELISA检测方法的建立 [J]. 中国兽医科学, 2020, 50(1):10−19. doi: 10.16656/j.issn.1673-4696.2020.0004 WANG Y H, ZHOU M Y, LI Y C, et al. Establishment of ELISA for detecting porcine epidemic diarrhea virus specific SIgA in mucus [J]. Chinese Veterinary Science, 2020, 50(1): 10−19.(in Chinese) doi: 10.16656/j.issn.1673-4696.2020.0004
[19]	朱利塞, 李菲, 王娟, 等. 母猪初乳中猪流行性腹泻病毒SIgA抗体ELISA检测方法的建立及应用 [J]. 中国动物传染病学报, 2023, 31(1):55−60. doi: 10.19958/j.cnki.cn31-2031/s.20211103.006 ZHU L S, LI F, WANG J, et al. Development of an ELISA method for detection of SIgA antibodies against porcine epidemic diarrhea virus in sow colostrum [J]. Chinese Journal of Animal Infectious Diseases, 2023, 31(1): 55−60.(in Chinese) doi: 10.19958/j.cnki.cn31-2031/s.20211103.006
[20]	丁振江, 库旭钢, 闫贵伟, 等. 猪流行性腹泻IgA抗体间接ELISA检测方法的建立 [J]. 畜牧与兽医, 2014, 46(11):1−5. DING Z J, KU X G, YAN G W, et al. Development of an indirect ELISA method for detection of IgA antibody against porcine epidemic diarrhea virus [J]. Animal Husbandry & Veterinary Medicine, 2014, 46(11): 1−5.(in Chinese)
[21]	王治平, 张云静, 孙玉洁, 等. 基于单克隆抗体捕获的猪血清和乳汁中PEDVIgA抗体间接ELISA方法的建立和评估 [J]. 中国畜牧兽医, 2020, 47(7):2248−2255. WANG Z P, ZHANG Y J, SUN Y J, et al. Development and evaluation of an indirect ELISA for detection of porcine epidemic diarrhea virus IgA in pig serum and milk captured by monoclonal antibody [J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(7): 2248−2255.(in Chinese)
[22]	柏家果, 杜琛, 王若木, 等. 猪流行性腹泻病毒特异性SIgA 间接ELISA检测方法的建立及初步应用 [J]. 中国兽医学报, 2023, 43(3):455−461. BAI J G, DU C, WANG R M, et al. Establishment and preliminary application ofindirect ELISA method for detection of specific SIgA against porcine epidemic diarrhea virus [J]. Chinese Journal of Veterinary Science, 2023, 43(3): 455−461.(in Chinese)