基于IgY的ELISA定量检测甲鱼系统性败血症球状病毒
PDF下载 (374)夏小磊,李登峰 *,彭 姣,周永强,郭安南,刘联国,陈 炯.基于IgY的ELISA定量检测甲鱼系统性败血症球状病毒[J].宁波大学学报(理工版),2016,29(4):13-19.DOI:
XIA Xiao-lei,LI Deng-feng *,PENG Jiao,ZHOU Yong-qiang,GUO An-nan,LIU Lian-guo,CHEN Jiong.Development of ELISA for Quantitative Detection of Soft-shelled Turtle Systemic Sepsis Spherical Virus (STSSSV)[J].Journal of Ningbo University(Natural Science & Engineering Edition),2016,29(4):13-19.DOI:
| Title: | Development of ELISA for Quantitative Detection of Soft-shelled Turtle Systemic Sepsis Spherical Virus (STSSSV) |
| 作者: | 夏小磊, 李登峰 *, 彭 姣, 周永强, 郭安南, 刘联国, 陈 炯 |
| Author(s): | XIA Xiao-lei, LI Deng-feng *, PENG Jiao, ZHOU Yong-qiang, GUO An-nan, LIU Lian-guo, CHEN Jiong |
| 关键词: | 甲鱼系统性败血症球状病毒; 卵黄抗体; 酶联免疫吸附实验; 检测 |
| Keywords: | soft-shelled turtle systemic sepsis spherical virus; IgY; ELISA; detection |
| 分类号: | S917.1 |
| 文献标识码: | A |
| 摘要: | 为了对甲鱼系统性败血症球状病毒(STSSSV)进行快速检测, 以抗STSSSV卵黄抗体(egg yolk immunoglobulin, IgY)为一抗, 以羊抗鸡IgG(HRP-IgG)为二抗, 建立STSSSV检测的间接酶联免疫吸附(ELISA)与血凝抑制(HI)实验, 对两者进行了比较. 运用ELISA对感染病毒甲鱼的血清、肝脏、粪便、及饲养环境水样进行了STSSSV检测. 用ELISA分析了病毒稀释倍数及其2的对数值与OD492nm值间的线性关系, 建立STSSSV的ELISA定量检测方法. 结果显示: ELISA较HI更灵敏, 其对STSSSV的检测灵敏度为3.98ng?mL-1, 能够在携带该病毒尚未显症的隐性感染的甲鱼血清和甲鱼粪便与养殖水中检测出STSSSV. ELISA对其他水产病毒无交叉反应, 特异性强. ELISA定量检测限度范围为0.031~4μg?mL-1. 本研究建立的间接ELISA方法为STSSSV的定性、定量提供了准确有效的检测手段. |
| Abstract: | Based on chicken immunoglobulin of yolk (IgY), we have developed an indirect ELISA for detection of soft-shelled turtle systemic sepsis spherical virus (STSSSV). Chicken IgY against STSSSV is used as the primary antibody, while an HRP conjugated goat anti-chicken IgG is used as the secondary antibody. By comparing ELISA with hemagglutination inhibition test (HI), the linear relationship between OD492nm values and the dilution of STSSSV, as well as log 2 of the dilutions is established and analyzed, base on which a quantitative ELISA is developed. The presented method has been applied to test various infected samples including sera, liver, feces and culturing water. The results show that the minimum detection limit of the developed ELISA method is 3.98 ng?mL-1, and there has been no cross-reaction to other aquatic virus. The detection range of quantitative ELISA is limited to 0.031~4 μg?mL-1. Sera contains the highest viral load. Even in latent period, STSSSV in sera can be detected, which is helpful for early diagnosis. |
| 参考文献 /References: | [1] 李登峰, 周永强, 刘联国, 等. 抗甲鱼系统性败血症球状病毒的卵黄抗体及其制备方法和应用: 中国, CN 103073640[P]. 2015-07-15. [2] 李登峰, 周永强, 刘联国, 等. 甲鱼系统性败血症球状病毒的鸡胚培养方法: 中国, CN102628031[P]. 2013- 10-02. [3] 李登峰, 周永强, 刘联国, 等. 一种甲鱼系统性败血症球状病毒灭活疫苗及其制备方法: 中国, CN102626 514[P]. 2013-10-02. [4] HONG L J, LI F Z, ZHEN Z F, et al. Detection of grass carp reovirus (GCRV) with monoclonal antibodies[J]. Archives of Virology, 2014, 159(4):649-655. [5] NISHIKI I, MINAMI T, ITAMI T, et al. Cloning and expression of a surface immunogenic protein in Strepto- coccus dysgalactiae isolated from fish and its application in enzyme-linked immunosorbent assays to diagnose S. dysgalactiae infections in fish[J]. Journal of Fish Diseases, 2014, 37(12):1031-1039. [6] 程顺峰. 牙鲆淋巴囊肿病毒双抗体夹心ELISA检测方法的建立[J]. 水产科学, 2009, 28(7):374-377. [7] 陈连颐. 卵黄抗体的特性、应用及开发前景[J]. 中国禽业导刊, 2007, 24(21):36-37. [8] CARLANDER D, STALBERG J, LARSSON A. Chicken antibodies: a clinical chemistry perspective[J]. Upsala Journal of Medical Sciences, 1999, 104(3):179-189. [9] DILONARDO A D, MARCANTE M L, POGGIALI F, et al. Egg yolk antibodies against the E7 oncogenic protein of human papillomavirus type 16[J]. Archives of Virology, 2001, 146(1):117-125. [10] CIPOLLA A, CORDEVIOLA J, TERZOLO H, et al. Campylobacter fetus diagnosis: Direct immunofluores- cence comparing chicken IgY and rabbit IgG conjugates [J]. Alternatives to Animal Experimentation, 2001, 18(3): 165-170. [11] WU C, YANG R, ZHOU J, et al. Production and charac- terisation of a novel chicken IgY antibody raised against C-terminal peptide from human thymidine kinase[J]. Journal of Immunological Methods, 2003, 277(1):157-169. [12] SCHADE R, CALZADO E G, SARMIENTO R, et al. Chicken egg yolk antibodies(IgY-technology): A review of progress in production and use in research and human and veterinary medicine[J]. Alternatives to Laboratory Animals, 2005, 33(2):129-154. [13] 尤昕, 时娜, 赵秀文, 等. 心肌特异表达基因p93的原核表达及鸡卵黄抗体的制备[J]. 中华检验医学杂志, 2005, 28(10):1060-1063. [14] DAI Y C, WANG Y Y, ZHANG X F, et al. Evaluation of anti-norovirus IgY from egg yolk of chickens immunized with norovirus P particles[J]. Journal of Virological Methods, 2012, 186(1):126-131. [15] PARMA Y R, CHACANA P A, LUCCHESI P M, et al. Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies[J]. Frontiers in Cellular and Infection Microbiology, 2012, 84(2):1-8. [16] 张晓华, ROBERTSON P, AUSTIN B, 等. 检测海洋弧菌的酶联免疫吸附实验研究[J]. 青岛海洋大学学报, 1997, 27(3):326-331. [17] 柴顺秀, 马花, 韩英, 等. 牛病毒性腹泻病毒重组E2蛋白PPA-ELISA方法的建立及临床应用[J]. 动物医学进展, 2013,34(6):106-110. [18] 吴晓春, 邓灯, 程顺峰, 等. 鱼肠道弧菌间接ELISA快速检测法的建立[J]. 水产科学, 2013, 32(5):284-288. [19] CHEN C C, TAI Y C, SHEN S C, et al. Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase[J]. Food Chemistry, 2006, 95 (2):213-220. [20] EMPEY C C, HOKAMA Y, YABUSAKI K, et al. Development of an enzyme-linked immunosorbent assay for the detection of ciguatoxin in fish tissue using chicken immunoglobulin Y[J]. Journal of Clinical Laboratory Analysis, 2008, 22(4):239-245. [21] BENTES G A, LANZARINI N M, LIMA L R, et al. Using immunoglobulin Y as an alternative antibody for the detection of hepatitis a virus in frozen liver sections[J]. Memórias Do Instituto Oswaldo Cruz, 2015, 110(4):577-585. [22] NAFEA N M, SABBAH M A, ALSUHAIL R, et al. Development of hen antihepatitis B antigen IgY-based conjugate for ELISA assay[J]. Advanced Biomedical Research, 2015, 11:2197-2207. [23] DAI Y C, ZHANG X F, TAN M, et al. A dual chicken IgY against rotavirus and norovirus[J]. Antiviral Research, 2013, 97(3):293-300. [24] 施少华, 傅光华, 万春和, 等. 检测鸭坦布苏病毒卵黄抗体间接ELISA方法的建立[J]. 养禽与禽病防治, 2012(2):2-4. [25] 张艳, 吴旭干, 杨帆, 等. 三疣梭子蟹卵黄磷蛋白纯化及其ELISA测定方法[J]. 水产学报, 2011, 35(8): 1146-1157. [26] 李培育, 刁晓明, 盛晓洒, 等. 瓦氏黄颡鱼卵黄脂磷蛋白(Lv)的纯化、性质签定及其抗血清的研制[J]. 水产学报, 2010, 34(1):116-125. [27] 章雪明, 胡仁莉, 俞小红. 间接ELISA和HI方法检测禽流感[J]. 养禽与禽病防治, 2010(6):8-10. [28] 贾杏林, 陈焕春, 王祥, 等. 猪乙型脑炎间接ELISA与血凝抑制试验方法的比较[J]. 中国畜牧兽医, 2011, 38(2):157-159. |
| 备注/Memo: | 收稿日期: 2015?03?15. 宁波大学学报（理工版）网址: http://journallg.nbu.edu.cn/ 基金项目: 浙江省公益性技术应用研究计划项目（2011C32004）; 宁波市农业攻关项目（2011C10004）; 宁波大学王宽诚幸福基金. 第一作者: 夏小磊（1988?）, 男, 安徽合肥人, 在读硕士研究生, 主要研究方向: 水产动物病害防治. E-mail: xiaxiaolei2010@126.com *通信作者: 李登峰（1975?）, 女, 湖南双峰人, 副教授, 主要研究方向: 水产动物病害防治. E-mail: lidengfeng@nbu.edu.cn 宁波大学学报（理工版）网址：http://journallg.nbu.edu.cn/ |
