CN106841611A - 一种基于ibv串联抗原s‑m‑n的免疫磁性微球‑elisa检测ibv抗体的方法 - Google Patents
一种基于ibv串联抗原s‑m‑n的免疫磁性微球‑elisa检测ibv抗体的方法 Download PDFInfo
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- CN106841611A CN106841611A CN201710080119.8A CN201710080119A CN106841611A CN 106841611 A CN106841611 A CN 106841611A CN 201710080119 A CN201710080119 A CN 201710080119A CN 106841611 A CN106841611 A CN 106841611A
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- Prior art keywords
- ibv
- magnetic microsphere
- antibody
- elisa
- antigen
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- 241000711450 Infectious bronchitis virus Species 0.000 title claims abstract description 47
- 102000004965 antibodies Human genes 0.000 title claims abstract description 25
- 108090001123 antibodies Proteins 0.000 title claims abstract description 25
- 238000002965 ELISA Methods 0.000 title claims abstract description 22
- 102000032510 antigens Human genes 0.000 title claims abstract description 21
- 108700005831 antigens Proteins 0.000 title claims abstract description 21
- 239000000427 antigens Substances 0.000 title claims abstract description 19
- 239000004005 microspheres Substances 0.000 title claims abstract description 19
- 241000287828 Gallus gallus Species 0.000 claims abstract description 21
- 210000002966 Serum Anatomy 0.000 claims abstract description 17
- 238000005576 amination reactions Methods 0.000 claims abstract description 6
- 201000009910 diseases by infectious agent Diseases 0.000 claims abstract description 6
- 230000036039 immunity Effects 0.000 claims abstract description 6
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound 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O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 101710010216 Capsid Proteins Proteins 0.000 claims abstract 2
- 241000700605 Viruses Species 0.000 claims description 6
- 241000711404 Avian avulavirus 1 Species 0.000 claims description 3
- 241000712461 unidentified influenza virus Species 0.000 claims description 3
- 241001260012 Bursa Species 0.000 claims description 2
- 239000000203 mixtures Substances 0.000 claims description 2
- 239000002245 particles Substances 0.000 claims description 2
- 239000007790 solid phases Substances 0.000 claims description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N silicon Chemical compound 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[Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims 1
- 229910052710 silicon Inorganic materials 0.000 claims 1
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- GWEVSGVZZGPLCZ-UHFFFAOYSA-N titan oxide Chemical compound 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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Abstract
本发明涉及一种检测禽传染性支气管炎病毒抗体的ELISA检测方法，该方法以IBV S–M–N片段串联蛋白为包被抗原、以氨基化二氧化硅磁性微球为固相支持物。包被抗原包含了IBV 3种主要结构蛋白（ S、M、N）的抗原表位，是一种多表位重组蛋白，通过在大肠杆菌中表达获得，避免了IBV鸡胚增殖、提纯的繁琐过程。该方法检测鸡血清中的IBV抗体，具有较好的特异性、重复性，可用于鸡群免疫状态的评估，也可以用于鸡群IBV感染风险的评估。
Description
一种基于IBV串联抗原S-M-N的免疫磁性微球-ELISA检测IBV 抗体的方法
技术领域
[0001] 本发明涉及生物检测技术领域，所检测的是鸡血清中IBV主要抗原蛋白的抗体，可 用于鸡群免疫状态的评估，也可以用于鸡群IBV感染风险的评估。
背景技术
[0002] 禽传染性支气管炎（Infectious Bronchitis, IB)是危害养禽业的重大传染病之 一，是由冠状病毒科冠状病毒属的禽传染性支气管炎病毒（Infectious Bronchitis Virus，IBV)引起的一种急性、高度接触性传染病，可引起小鸡呼吸系统和肾脏损伤、产蛋 鸡群产蛋量下降和蛋品质降低。疫苗免疫是防制本病的主要措施，进行血清抗体水平监测 可以为鸡群免疫效果的评价、免疫程序的合理制定等提供依据，对于本病的防控具有重要 意义。因IBV没有血凝性，且用C型魏氏梭菌等处理病毒后建立的HI检测方法不稳定，因此没 有广泛用于IBV抗体检测。ELISA是一种敏感、快速、操作简便的抗体检测方法，通常采用全 病毒作为包被抗原。全病毒ELISA方法在理论上能够检测针对该病毒所有抗原蛋白的抗体， 能够较全面地反映血清中IBV抗原蛋白的抗体水平。然而，全病毒ELISA方法需要在鸡胚中 增殖获得IBV，过程繁琐，病毒的纯化操作困难，最外层的纤突蛋白容易掉落，且存在病毒灭 活不彻底的生物安全隐患。目前有进口的IDEXX ELISA试剂盒用于IBV抗体检测，但该试剂 盒价格高，在生产上未得到广泛应用。全病毒制备困难、成本高的一个解决方法是用重组多 表位蛋白作为替代。多个抗原或抗原表位的联合是具有潜力的检测抗原，且通过外源表达 的方式使得制备简单，近年来已有多项以多表位串联蛋白用作ELISA包被抗原的研究。常规 ELISA方法通常以酶标板作为固相支持物。磁性微球作为一种新型固相支持物，相比于平坦 的固相载体，具有比表面积大、偶联容量高的特点，以磁性微球为固相支持物的免疫学检测 技术具有敏感性高、检测时间短的优点。
发明内容
[0003] 本发明涉及一种检测禽传染性支气管炎病毒抗体的检测方法，是以IBV S-M-N片 段串联蛋白为包被抗原、磁性微球为固相支持物建立的免疫磁性微球-ELISA检测IBV抗体 的方法，检测的是鸡血清中的IBV抗体，可用于鸡群免疫状态的评估，也可以用于鸡群IBV感 染风险的评估。
附图说明
[0004] 图1是本发明实施例1提供的IBV S-M-N片段串联基因的组成图； 图2是本发明实施例2提供的氨基化二氧化硅磁性微球的TEM表征图； 图3是本发明实施例2提供的ELISA操作流程图。
具体实施方式
[0005] 以下实施例仅用于进一步说明本发明，但不应理解为对本发明的限制。其中，IBV M41毒株购自中国兽医药品监察所，由本实验室保存。大肠杆菌BL21(DE3)感受态细胞购自 大连宝生物工程公司。pET32a(+)由生科院白林含老师实验室惠赠。IBV阳性血清购自中国 兽医药品监察所。标准阴性血清采自SPF鸡。待检鸡血清样品采集自免疫鸡群。新城疫病毒、 禽流感病毒(H9亚型）和传染性法氏囊病病毒的阳性血清购自哈尔滨维科生物技术开发公 司。
[0006] I. IBV S-M-N片段串联抗原的制备 根据文献报道的IBV S、M、N蛋白的抗原表位鉴定情况，结合2种在线预测软件对IBV B 细胞表位的预测结果，在综合分析抗原表位的分布情况后选出了 8个抗原片段。它们的氨基 酸位置分别是：Sl 蛋白 166-247、501-515; S2 蛋白 8-30 ;M蛋白 181-210;N蛋白 6-88、118-133、 218-264、304-385。将8个抗原片段以柔性氨基酸间隔、按顺序进行串联，构建S-M-N片段串 联基因（基因组成示意图见图1)，并连接至pET32a⑴后构建了重组表达载体，随后在大肠 杆菌BL21 (DE3)中进行诱导表达并纯化，大小约61.5kDa，并伴随一个截短蛋白产物，都能和 IBV阳性血清反应。
[0007] 2. IBV ELISA抗体检测方法的建立 2.1免疫磁性微球的制备 利用反相微乳液法制备了氨基化二氧化硅磁性微球，粒径约50_70nm，分散性较好、均 一性良好(图2)。将IBV S-M-N片段串联抗原在室温条件下偶联至磁性微球表面制成免疫磁 性微球溶液，偶联缓冲液为0. OlM磷酸盐缓冲液(pH 6.5)，偶联时间为3h。
[0008] 2.2 ELISA条件确定 按照常规ELISA的操作步骤建立基于免疫磁性微球的ELISA检测IBV抗体的方法，并对 各项反应条件进行了优化，确定免疫磁性微球溶液用量为80yL，血清稀释倍数为1:500，HRP 标记的驴抗鸡抗体稀释倍数为1:5000，血清作用时间和二抗孵育时间均为30!^11。阴阳性血 清的临界值确定为0.302。
[0009] 2.3 ELISA操作步骤 ELISA具体操作步骤(图3):⑴将80yL免疫磁性微球溶液和IOOyL经抗体稀释液稀释的 待检血清混合，37 °C振荡孵育30min;⑵磁性分离2min，弃上清;PBST洗涤3次;⑶加入100μ L经抗体稀释液稀释的HRP标记的驴抗鸡IgY(IgG)，37°C振荡孵育30min; ⑷磁性分离2min，弃上清;PBST洗涤3次；(5)加入IOOyL TMB底物溶液，避光显色5-IOmin; (6)加入50yL终止液(2M H2S(M)终止反应;(7)磁性分离2min，吸取上清IOOyL转移到 酶标板中，在酶标仪上测定各孔的OD450值。
[0010] 2.4特异性试验 分别对新城疫病毒、禽流感病毒(H9)和鸡传染性法氏囊病毒的阳性血清进行检测，结 果均为阴性，具有较好的特异性(表1)。
[0011] 表1特异性试验结果
2.5重复性试验 分别选取5份血清进行批内重复性试验和批间重复性试验，每份进行4个重复。结果表 明批内变异系数范围为3.08%-7.17%(表2)，批间变异系数范围为5.23%-10.09% (表3)，具有 较好的重复性。
[0012] 表2批内重复性试验结果
表3批间重复性试验结果
Claims (3)
1. 一种基于IBV S-M-N片段串联抗原的免疫磁性微球-ELISA检测IBV抗体的方法，其特征在于:构建一种包含了IBV S、M、N片段的串联抗原，并偶联至氨基化二氧化硅磁性微球表面制成免疫磁性微球，建立了以IBV S-M-N片段串联蛋白为包被抗原、磁性微球为固相支持物的ELI SA检测IBV抗体的方法。
2. 根据权利要求1所述的一种基于IBV S-M-N片段串联抗原的免疫磁性微球-ELISA检测IBV抗体的方法，其特征在于:所述抗原由IBV 3种主要结构蛋白（S、M、N)的抗原片段串联组成;所述磁性微球为氨基化二氧化硅磁性微球;所检测的是鸡血清中的IBV抗体，可用于鸡群免疫状态的评估，也可以用于鸡群IBV感染风险的评估。
3. 根据权利要求1所述，IBV S-M-N片段串联蛋白的大小约61.5kDa;所述氨基化二氧化硅磁性微球粒径约50-70nm;所述免疫磁性微球-ELISA检测IBV抗体的方法特异性较好，对新城疫病毒、禽流感病毒(H9)和鸡传染性法氏囊病毒的阳性血清的检测结果均为阴性;批内、批间变异系数分别为3.08%-7.17%、5.23%-10.09%，重复性较好。
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| CN104569413A (zh) * | 2015-01-12 | 2015-04-29 | 中国科学院苏州生物医学工程技术研究所 | 一种血小板抗体检测的免疫纳米磁珠酶联免疫检测方法 |
| CN105699653A (zh) * | 2014-11-25 | 2016-06-22 | 北京市肝病研究所 | 一种超敏感超顺磁性纳米免疫微球及其检测gp73抗原的方法 |
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| CN101533011A (zh) * | 2009-04-10 | 2009-09-16 | 北京倍爱康生物技术有限公司 | 一种日本血吸虫虫卵抗体磁微粒分离酶联免疫检测方法 |
| CN103926398A (zh) * | 2014-04-30 | 2014-07-16 | 洛阳惠尔纳米科技有限公司 | 一种免疫磁珠的制备方法 |
| CN105699653A (zh) * | 2014-11-25 | 2016-06-22 | 北京市肝病研究所 | 一种超敏感超顺磁性纳米免疫微球及其检测gp73抗原的方法 |
| CN104569413A (zh) * | 2015-01-12 | 2015-04-29 | 中国科学院苏州生物医学工程技术研究所 | 一种血小板抗体检测的免疫纳米磁珠酶联免疫检测方法 |
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