Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor
Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor
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DOI：
10.1016/j.talanta.2005.08.054
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发表时间：
2005-12-15
期刊：
影响因子：
6.1
通讯作者：
Imato, T
中科院分区：
文献类型：
--
作者：
Matsumoto, K;Torimaru, A;Imato, T
制备了抗三硝基苯基（TNP）衍生物的多克隆抗体，并将其应用于表面等离子体共振（SPR）生物传感器检测三硝基甲苯（TNT）。将TNP-KLH偶联物注射家兔体内，用G蛋白纯化血清，获得多克隆抗TNP-KLH抗体。方面的抗TNP抗体对各种硝基芳香族化合物，如交叉反应性和亲和力，进行了表征。还评价了抗TNP抗体与TNT之间的亲和力的温度依赖性。TNT的定量是基于间接竞争免疫测定的原理，其中在溶液中存在游离TNT的情况下，TNP-β-丙氨酸-卵清蛋白（TNP-β-ala-OVA）与抗TNP抗体之间的免疫反应被抑制。通过胺偶联将TNP-β-ala-OVA固定到Au表面上的葡聚糖基质上。发现将游离TNT的混合物添加到抗TNP抗体中由于TNT的抑制作用而降低了入射角偏移。该免疫测定法在3 × 10 ~（-11）~ 3 × 10 ~（-7）μ g/ml浓度范围内对TNT的检测具有很好的灵敏度。为了提高传感器的灵敏度，使用了抗兔IgG抗体。流动游离TNT和抗TNP抗体的混合物后，注射抗兔IgG抗体，并测量入射角偏移。观察到信号放大，检测限提高到1 × 10 ~（-11）g/ml。(c)2005 Elsevier B. V.保留所有权利。
A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-beta-alanine-ovalbumin (TNP-beta-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-beta-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3 x 10(-11) to 3 x 10(-7) g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1 x 10(-11) g/ml. (c) 2005 Elsevier B.V. All rights reserved.