Development and comparison of two immunoassays for the detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance
Development and comparison of two immunoassays for the detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance
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DOI：
10.1016/j.snb.2005.04.013
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发表时间：
2006-03-30
影响因子：
8.4
通讯作者：
Miura, N
中科院分区：
文献类型：
--
作者：
Shankaran, DR;Matsumoto, K;Miura, N
本文介绍了两种基于竞争抑制的2，4，6-三硝基甲苯（TNT）免疫检测方法的建立和比较。在免疫测定中使用两种多克隆抗体;一种由2，4，6-三硝基苯酚-牛血清白蛋白缀合物（抗TNP-BSA Ab，山羊IgG）制备，另一种由2，4，6-三硝基苯基-匙孔戚血吸虫缀合物（抗TNPh-KLH Ab，兔IgG）制备。一个固相免疫分析，包括物理固定的TNP-BSA结合物被允许与这些抗体的相互作用和共振角的变化，由于生物分子的相互作用进行了监测，通过表面等离子体共振（SPR）。TNT的定量基于抗体与TNP-BSA缀合物结合的共振角位移的变化，其在溶液中存在TNT的情况下发生。两种抗体均显示出对TNT的高度亲和性和对相关硝基芳族衍生物化合物如2，4-二硝基甲苯（DNT）、1，3-二硝基苯（DNB）、2-氨基-4，6-二硝基甲苯（2A-4，6-DNT）、4-氨基-2，6-二硝基甲苯（4A-2，6-DNT）和TNP的不同程度的交叉反应性。本实验室制备的抗TNPh-KLH抗体的检测限为0.006 ng/ml（6 ppt），与市售抗TNP-BSA抗体检测TNT的检测限（0.095 ng/ml或95 ppt）相当，甚至更好。用甘氨酸-HCl缓冲液（pH 2.0）制备的胃蛋白酶溶液用于再生传感器表面。对于抗TNP-BSA Ab和抗TNPh-KLH Ab免疫系统，单个免疫循环的持续时间分别类似于19分钟和类似于13分钟。该方法简单、灵敏度高，可用于TNT的定量分析。(c)2005 Elsevier B. V.保留所有权利。
This paper describes the development and comparison of two immunoassays for the detection of 2,4,6-trinitrotoluene (TNT) based on competitive inhibition. Two polyclonal antibodies were used in the immunoassay; one was prepared from 2,4,6-trinitrophenol-bovine serum albumin conjugate (anti-TNP-BSA Ab, goat IgG) and another from 2,4,6-trinitrophenyl-keyhole limpet hemocyanine conjugate (anti-TNPh-KLH Ab, rabbit IgG). A solid-phase immunoassay that consisted of physically immobilized TNP-BSA conjugate was allowed to interact with these antibodies and the resonance angle changes due to the biomolecular interactions were monitored by surface plasmon resonance (SPR). The quantification of TNT was based on the change in the resonance angle shift for binding of antibodies with a TNP-BSA conjugate, which occurred in the presence of TNT in solution. Both antibodies showed a high degree of affinity for TNT and varying degrees of cross-reactivity to the related nitroaromatic derivative compounds, such as 2,4-dinitrotoluene (DNT), 1,3-dinitrobenzene (DNB), 2-amino-4,6-dintitrotoluene (2A-4,6-DNT), 4-amino-2,6-dinitrotoluene (4A-2,6-DNT) and TNP. The anti-TNPh-KLH Ab prepared in our laboratory showed a detection limit of 0.006 ng/ml (6 ppt), which was comparable to or even better than the detection limit (0.095 ng/ml or 95 ppt) of a commercially available anti-TNP-BSA Ab for detection of TNT. A pepsin solution prepared with a glycine-HCl buffer (pH 2.0) was used for regeneration of the sensor surface. The duration for a single immunocycle was similar to 19 and similar to 13 min for anti-TNP-BSA Ab and anti-TNPh-KLH Ab immunosystems, respectively. The proposed immunoassay is simple and possesses good analytical characteristics for quantification of TNT. (c) 2005 Elsevier B.V. All rights reserved.