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
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- 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

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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.

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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.

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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.

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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

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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

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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

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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

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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

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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

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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

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