Preparation of a Polyclonal Antibody and a Bioassay for Nitroaromatic Compounds by an Enzyme-Linked Immunosorbent Assay Technique and a Surface Plasmon Resonance Biosensor
Preparation of a Polyclonal Antibody and a Bioassay for Nitroaromatic Compounds by an Enzyme-Linked Immunosorbent Assay Technique and a Surface Plasmon Resonance Biosensor
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多克隆抗体的制备以及酶联免疫吸附测定技术和表面等离子共振生物传感器对硝基芳香族化合物的生物测定
作者：
Takatoshi Sakai;Akira Torimaru;Katsura Shinahara;Kiyoshi Matsumoto;N. Miura;T. Imato;K. Toko
利用多克隆抗TNP抗体和N-（TNP）-卵清蛋白（TNP-OVA）偶联物，建立了基于表面等离子体共振（SPR）的2，4，6-三硝基苯基衍生物（TNP衍生物）的高灵敏、高选择性检测方法。将TNP-牛血清白蛋白（TNP-BSA）偶联物注射小鼠，血清经蛋白G纯化后获得多克隆抗TNP抗体。采用物理吸附法将TNP-OVA固定在SPR传感器芯片的Au膜上。TNP-0 VA固定化传感器的入射角偏移随着抗TNP抗体浓度的增加而急剧增加，直到20 μg/ml，并且仅稍微高于该浓度增加。由于TNP衍生物的抑制作用，在抗TNP抗体溶液中加入TNP衍生物可以减小入射角的偏移。三硝基甲苯（TNT）的最低检测限为1×10 - 7 g/ml，TNP-6-氨基己酸（TNP-aha）的最低检测限为3×10 - 9 g/ml。进行抗TNP抗体的亲和常数的评价。SPR数据的分析进行了朗缪尔等温线和免疫反应的平衡状态的假设。抗TNP抗体与固定化TNP-OVA的结合常数（K1）为6.4× 106 M-1。当抗体浓度为20和10 μg/ml时，抗体与TNP-aha（K2）的结合常数分别为2.7和8.5× 106 M-1。
Sensitive and selective detection of 2, 4, 6-trinitrophenyl derivatives (TNP derivatives) based on surface plasmon resonance (SPR) was performed using polyclonal anti-TNP antibody and N-(TNP)-ovalbumin (TNP-OVA) conjugate. TNP-bovine serum albumin (TNP-BSA) conjugate was injected into the mice, and polyclonal anti-TNP antibody was gained after purification of the sera using protein G. TNP-OVA was immobilized on the Au film of the SPR sensor chip by physical adsorption. The incidence angle shift of the TNP-OVA immobilized sensor increased steeply with increasing concentration of anti-TNP antibody up to 20 μg/ml and increased only slightly above this concentration. The additions of TNP derivatives into the anti-TNP antibody solution were found to decrease the incidence angle shift because of the inhibition effects of TNP derivatives. The lowest detection limit for trinitrotoluene (TNT) by SPR was 1×10 -7 g/ml, whereas for TNP-6-aminohexanoic acid (TNP-aha) it was 3×10 -9 g/ml. Evaluations of affinity constants of anti-TNP antibody were performed. Analyses of SPR data were carried out by assumptions of the Langmuir isotherm and equilibrium state of immunoreaction. The value of association constant between anti-TNP antibody and immobilized TNP-OVA (K 1 ) was 6.4×10 6 M -1 . The values of association constants between antibody and TNP-aha (K 2 ) were 2.7 and 8.5×10 6 M -1 when 20 and 10 μg/ml antibody were used, respectively.