时间分辨荧光免疫法检测日本鳗鲡的产酸克雷伯氏菌

引用本文: 卢洁, 郭松林, 冯建军, 王艺磊, 朱洪泉, 林鹏. 时间分辨荧光免疫法检测日本鳗鲡的产酸克雷伯氏菌[J]. 分析化学, 2012, 40(11): 1709-1714. doi: 10.3724/SP.J.1096.2012.20139 shu

Citation: LU Jie, GUO Song-Lin, FENG Jian-Jun, WANG Yi-Lei, ZHU Hong-Quan, LIN Peng. Time-Resolved Fluoroimmunoassay for Detecting Klebsiella Oxytoca in Anguilla Janponica[J]. Chinese Journal of Analytical Chemistry, 2012, 40(11): 1709-1714. doi: 10.3724/SP.J.1096.2012.20139 shu

时间分辨荧光免疫法检测日本鳗鲡的产酸克雷伯氏菌

通讯作者: 林鹏

基金项目:
本文系国家自然科学基金(Nos. 30972281, 31001136) (Nos. 30972281, 31001136)

福建省高等学校新世纪优秀人才支持计划(闽教科[2007]20号) (闽教科[2007]20号)

集美大学创新团队基金项目(No. 2010A001)资助 (No. 2010A001)

摘要: 从皮肤点状出血的日本鳗鲡中分离出产酸克雷伯氏菌菌株B12,经福尔马林灭活后,注射新西兰兔,制备免疫抗血清,G蛋白亲和层析纯化得到抗体(IgG),以固定于微孔板的IgG作为捕获抗体,Eu³⁺螯合物标记的IgG作为检测抗体,建立了产酸克雷伯氏菌的夹心型时间分辨荧光免疫检测法(TRFIA),并研究了抗体浓度、免疫时间及离解时间对检测的影响。本方法检出限为5.0×10³ cfu/mL; 线性范围5.0×10³-1.0×10⁷ cfu/mL,相关系数达0.99以上。交叉反应表明其具有较高的特异性,板内和板间相对标准偏差分别为5.64%和2.27%。将本方法标准化后,检测了29份人工感染的日本鳗鲡样品,包括鳃、肾、肠、肝和肌肉组织,结果满意。

关键词:

English

Time-Resolved Fluoroimmunoassay for Detecting Klebsiella Oxytoca in Anguilla Janponica

1.
Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Key Laboratory of Science and Technology for Aquaculture and Food Safety, Fisheries College, Jimei University, Xiamen 361021, China

Corresponding author: 林鹏

Received Date: 25 February 2012
Fund Project:
本文系国家自然科学基金(Nos. 30972281, 31001136)

福建省高等学校新世纪优秀人才支持计划(闽教科[2007]20号)

集美大学创新团队基金项目(No. 2010A001)资助

Abstract: Klebsiella Oxytoca B12, isolated from diseased Anguilla janponica, was used for rabbit anti-serum preparation by injecting the formalin-inactivated bacteria. The rabbit immunoglobulin (IgG) was purified by SPA affinity column. A microplate sandwich immunoassay for the detection of K. Oxytoca was developed utilizing time-resolved fluoroimmunoassay (TRFIA). Antibodies immobilized on microtiter plates were used to capture the bacteria. The bound antigen-antibody complexes were then detected using the same antibodies labeled with Eu³⁺ chelates. The effects of antibody concentration, immunoreaction time and dissociation time on assay were studied during assay development. The limit of detection was 5.0×10³ cfu/mL and the linear range was 5.0×10³-1.0×10⁷ cfu/mL with the correlation coefficients more than 0.99. Cross reaction showed that it had high specificity. The intra-assay and inter-assay standard deviation were 5.6% and 2.3%, respectively. The method was optimized to detect the tissues including gill, kidney, intestine, liver and muscle from infected K. Oxytoca with satisfactory results.

Key words:

1. [1]
  1 LIANG Xu-Fang. Fisheries Science, 2005, 24(4): 24-261 LIANG Xu-Fang. Fisheries Science, 2005, 24(4): 24-26
2. [2]
  梁旭方. 水产科学, 2005, 24(4): 24-26梁旭方. 水产科学, 2005, 24(4): 24-26
3. [3]
  2 DONG Jie, YU Lan, ZHAO Yu, FAN Hai-Jun, ZHAO Yue-Ping. Chinese Journal of Infectious Diseases, 2002, 20(5): 315-3162 DONG Jie, YU Lan, ZHAO Yu, FAN Hai-Jun, ZHAO Yue-Ping. Chinese Journal of Infectious Diseases, 2002, 20(5): 315-316
4. [4]
  董捷, 于兰, 赵郁, 樊海军, 赵月萍. 中华传染病杂志, 2002, 20(5): 315-316董捷, 于兰, 赵郁, 樊海军, 赵月萍. 中华传染病杂志, 2002, 20(5): 315-316
5. [5]
  3 FAN Jing-Feng, LI Guang, WANG Bin, MA Cheng, LIANG Yu-Bo. Journal of Fisheries of China, 2006, 30(1): 113-1173 FAN Jing-Feng, LI Guang, WANG Bin, MA Cheng, LIANG Yu-Bo. Journal of Fisheries of China, 2006, 30(1): 113-117
6. [6]
  樊景凤, 李光, 王斌, 马成, 梁玉波. 水产学报, 2006, 30(1): 113-117樊景凤, 李光, 王斌, 马成, 梁玉波. 水产学报, 2006, 30(1): 113-117
7. [7]
  4 LI Chong-Shi, LI Qiang, LIU Hai-Yan, YE Shi-Gen, LI Hua. Journal of Guangdong Ocean University, 2010, 30(4): 17-214 LI Chong-Shi, LI Qiang, LIU Hai-Yan, YE Shi-Gen, LI Hua. Journal of Guangdong Ocean University, 2010, 30(4): 17-21
8. [8]
  李重实, 李强, 刘海燕, 叶仕根, 李华. 广东海洋大学学报, 2010, 30(4): 17-21李重实, 李强, 刘海燕, 叶仕根, 李华. 广东海洋大学学报, 2010, 30(4): 17-21
9. [9]
  5 BAI Xue-Mei, LI Tai-Yuan, YANG Xing, LI Yan-Ru, SUN Dan-Dan, DONG Dan. Fisheries Science, 2010, 29(10): 613-6155 BAI Xue-Mei, LI Tai-Yuan, YANG Xing, LI Yan-Ru, SUN Dan-Dan, DONG Dan. Fisheries Science, 2010, 29(10): 613-615
10. [10]
  白雪梅, 李太元, 杨兴, 李艳茹, 孙丹丹, 董丹. 水产科学, 2010, 29(10): 613-615白雪梅, 李太元, 杨兴, 李艳茹, 孙丹丹, 董丹. 水产科学, 2010, 29(10): 613-615
11. [11]
  6 WANG Jun, YAN Qing-Pi, SU Yong-Quan, ZHANG Wen. Marine Sciences, 2002, 26(007): 1-46 WANG Jun, YAN Qing-Pi, SU Yong-Quan, ZHANG Wen. Marine Sciences, 2002, 26(007): 1-4
12. [12]
  王军, 鄢庆枇, 苏永全, 张纹. 海洋科学, 2002, 26(007): 1-4王军, 鄢庆枇, 苏永全, 张纹. 海洋科学, 2002, 26(007): 1-4
13. [13]
  7 Jiang H F, Wang G L, Zhang W Z, Liu X Y, Ye Z Q, Jin D Y, Yuan J L, Liu Z G. J. Fluoresc., 2010, 20(1):321-3287 Jiang H F, Wang G L, Zhang W Z, Liu X Y, Ye Z Q, Jin D Y, Yuan J L, Liu Z G. J. Fluoresc., 2010, 20(1):321-328
14. [14]
  8 Kupstat A, Kumke M U, Hildebrandt N. Analyst, 2011, 136(5): 1029-10358 Kupstat A, Kumke M U, Hildebrandt N. Analyst, 2011, 136(5): 1029-1035
15. [15]
  9 LIU Yuan, LIU Rong-Rong, LIU Xian-Jin, FANG Dun-Huang, SONG Cun-Man, Huovinen Tuomas, Vehniäinen Markus, Lövgren Timo. Chinese J. Anal. Chem., 2012, 40(7): 1114-11179 LIU Yuan, LIU Rong-Rong, LIU Xian-Jin, FANG Dun-Huang, SONG Cun-Man, Huovinen Tuomas, Vehniäinen Markus, Lövgren Timo. Chinese J. Anal. Chem., 2012, 40(7): 1114-1117
16. [16]
  刘媛, 刘蓉蓉, 刘贤金, 方敦煌, 宋春满, Huovinen Tuomas, Vehniäinen Markus, Lövgren Timo. 分析化学, 2012, 40(7): 1114-1117刘媛, 刘蓉蓉, 刘贤金, 方敦煌, 宋春满, Huovinen Tuomas, Vehniäinen Markus, Lövgren Timo. 分析化学, 2012, 40(7): 1114-1117
17. [17]
  10 Chafer-Pericas C, Maquieira A, Puchades R, Miralles J, Moreno A, Pastor-Navarro N, Espinos F. Anal. Chim. Acta, 2010, 662(2): 177-18510 Chafer-Pericas C, Maquieira A, Puchades R, Miralles J, Moreno A, Pastor-Navarro N, Espinos F. Anal. Chim. Acta, 2010, 662(2): 177-185
18. [18]
  11 Zhang Z, Liu J F, Shao B, Jiang G. Environ. Sci. Technol., 2009, 44(3): 1030-103511 Zhang Z, Liu J F, Shao B, Jiang G. Environ. Sci. Technol., 2009, 44(3): 1030-1035
19. [19]
  12 WANG Hao-Dan, ZHOU Shen. Labeling Techniques in Biomedical Domain. Beijing: Peoples Health Press, 1995: 16512 WANG Hao-Dan, ZHOU Shen. Labeling Techniques in Biomedical Domain. Beijing: Peoples Health Press, 1995: 165
20. [20]
  王浩丹, 周申. 生物医学标记示踪技术. 北京: 人民卫生出版社, 1995: 165王浩丹, 周申. 生物医学标记示踪技术. 北京: 人民卫生出版社, 1995: 165
21. [21]
  13 ZHU Li-Ping, CHEN Xue-Qing. Experimental Methods Used in Immunology. Beijing: People’s Military Medical Press, 2000: 18-2213 ZHU Li-Ping, CHEN Xue-Qing. Experimental Methods Used in Immunology. Beijing: People’s Military Medical Press, 2000: 18-22
22. [22]
  朱立平,陈学清. 免疫学常用实验方法. 北京:人民军医出版社, 2000: 18-22朱立平,陈学清. 免疫学常用实验方法. 北京:人民军医出版社, 2000: 18-22
23. [23]
  14 Niu C G, Liu J, Qin P Z,Zeng G M, Ruan M, He H. Anal. Biochem., 2011, 409(2): 244-24814 Niu C G, Liu J, Qin P Z,Zeng G M, Ruan M, He H. Anal. Biochem., 2011, 409(2): 244-248
24. [24]
  15 Caruhel P, Mazier C, Kunde J, Morgenthaler N G, Darbouret B. Clin. Biochem., 2009, 42(7-8): 725-72815 Caruhel P, Mazier C, Kunde J, Morgenthaler N G, Darbouret B. Clin. Biochem., 2009, 42(7-8): 725-728
25. [25]
  16 Sellrie F, Beck M, Hildebrandt N, Micheel B. Anal. Methods, 2010, 2(9): 1298-130116 Sellrie F, Beck M, Hildebrandt N, Micheel B. Anal. Methods, 2010, 2(9): 1298-1301
26. [26]
  17 HU Da-Yan, QIAN Dong, LIU Wen, ZHANG You-Ping, PAN Qing-Qing, CHEN Chang-fu. Journal of Huazhong Agricultural University, 2008, 27(1): 80-8517 HU Da-Yan, QIAN Dong, LIU Wen, ZHANG You-Ping, PAN Qing-Qing, CHEN Chang-fu. Journal of Huazhong Agricultural University, 2008, 27(1): 80-85
27. [27]
  胡大雁, 钱冬, 刘问, 张友平, 潘清清, 陈昌福. 华中农业大学学报, 2008, 27(1): 80-85胡大雁, 钱冬, 刘问, 张友平, 潘清清, 陈昌福. 华中农业大学学报, 2008, 27(1): 80-85
28. [28]
  18 WANG Chong-Ming, YANG Bing, SONG Xiao-Ling, Huan Jie. Marine Fisheries Research, 1999, 20(1): 30-3418 WANG Chong-Ming, YANG Bing, SONG Xiao-Ling, Huan Jie. Marine Fisheries Research, 1999, 20(1): 30-34
29. [29]
  王崇明, 杨冰, 宋晓玲, 黄倢. 海洋水产研究, 1999, 20(1): 30-34王崇明, 杨冰, 宋晓玲, 黄倢. 海洋水产研究, 1999, 20(1): 30-34

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

时间分辨荧光免疫法检测日本鳗鲡的产酸克雷伯氏菌

通讯作者: 林鹏

English

Time-Resolved Fluoroimmunoassay for Detecting Klebsiella Oxytoca in Anguilla Janponica

Corresponding author: 林鹏

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

时间分辨荧光免疫法检测日本鳗鲡的产酸克雷伯氏菌

通讯作者: 林鹏

English

Time-Resolved Fluoroimmunoassay for Detecting Klebsiella Oxytoca in Anguilla Janponica

Corresponding author: 林鹏

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

CCS Chemistry：颜徐州、鲍哲南：你的皮肤可能是一片SPMs