其他(ELISA比色免疫检测) 2010

Triacetone triperoxide (TATP): hapten design and development of antibodies.

Langmuir : the ACS journal of surfaces and colloids Walter MA, Pfeifer D, Kraus W, Emmerling F, Schneider RJ, Panne U, Weller MG
阅读原文 PDF DOI PubMed

组成图示

Triacetone triperoxide (TATP): hapten... 传感器构成示意图

点击图片查看大图 · 依据论文自动绘制

传感器类型

其他(ELISA比色免疫检测)

检测对象

TATP(三乙酰基三过氧化氢,triacetone triperoxide);样品基质:ELISA标准溶液(甲醇/水体系,原文未明确实际样品基质)

检测原理

该检测采用竞争ELISA免疫比色原理。固相TATP hapten-BSA捕获免疫小鼠血清中的抗TATP抗体;TATP hapten-HRP作为酶标记竞争物与待测TATP竞争结合抗体。当样品中TATP浓度升高时,更多抗体被TATP占据,结合到固相上的HRP减少,HRP催化TMB氧化生成的有色产物减少,吸光度降低。信号通过比色法读出,并用四参数逻辑函数拟合浓度-吸光度关系。该方法依赖抗体对TATP三过氧环结构的特异性识别,酶催化TMB提供显色放大,但未使用HCR、RCA或CRISPR等核酸放大策略。

检测灵敏度

LOD: 65 μg L-1;另一血清 LOD: 870 μg L-1;C值/拐点: 2.5 mg L-1 和 5.6 mg L-1;抗体亲和力: 5.7 μmol L-1

效应效果

该文重点报道抗TATP抗体的开发,而非完整传感器器件性能。竞争ELISA显示,两只免疫小鼠血清对TATP的检出限分别为65 μg/L和870 μg/L,曲线拐点C值为2.5 mg/L和5.6 mg/L,说明抗体可识别微克级TATP。作者计算抗体亲和力为5.7 μmol/L,认为偏低,可能源于TATP在生理温度下构象柔性。文中未报告RSD、长期稳定性、实际样品加标回收率,也未与HPLC、质谱或qPCR等方法进行定量对比。作者强调,高选择性抗体有助于构建低假阳性的TATP生物传感器表面和现场快速检测,是开发TATP传感系统的基础。

传感器的构成

  • 固相载体:ELISA微孔板(原文未指明材质,用于固定免疫原)
  • 包被免疫原:TATP hapten-BSA(半抗原偶联牛血清白蛋白,作为固相抗原)
  • 识别元件:抗TATP多克隆抗体(免疫小鼠血清,特异性识别TATP/半抗原)
  • 竞争标记物:TATP hapten-HRP(半抗原偶联辣根过氧化物酶,作为酶标记竞争物)
  • 底物/显色剂:TMB(3,3',5,5'-四甲基联苯胺,被HRP氧化显色)
  • 读出信号:比色吸光度(竞争ELISA中吸光度随TATP浓度变化)

中文摘要

三乙酰基三过氧化氢(TATP)是一种简易爆炸物,因合成成本低且难以直接检测而构成安全威胁。高选择性抗体可为检测提供必要特异性。本研究以丙酮、过氧化氢和7-氧辛酸为原料设计并合成了与TATP结构高度相似的羧基化半抗原,并通过NMR确认其结构。作者测定了TATP·甲醇(1:1)溶剂合物及TATP衍生物的单晶X射线结构,发现两者均具有D3对称性并采用扭曲船-椅构象。将半抗原偶联到牛血清白蛋白(BSA)后免疫BALB/c小鼠,成功诱导针对TATP的免疫反应,并在小鼠血清中检测到选择性抗体。基于该抗体的酶联免疫吸附试验(ELISA)对TATP的检出限为65 μg/L,表明该抗体可用于开发TATP生物传感器系统。

英文摘要

Triacetone triperoxide (TATP), an improvised explosive, is a potential security threat because of its cost-efficient synthesis and the difficulty in detecting it. A highly selective antibody could provide the necessary specificity to the detection process. To obtain antibodies, a hapten made from acetone, hydrogen peroxide, and 7-oxooctanoic acid has been designed, synthesized, and confirmed by NMR that displays the utmost similarity to the analyte. The single-crystal X-ray structures of the solvated species TATP·methanol (1:1) and the TATP derivate were determined. In both compounds, the molecules exhibit D(3) symmetry and adopt a twisted boat-chair conformation. The hapten was coupled to bovine serum albumin, and mice were immunized. An immune response against TATP was elicited, and selective antibodies were detected in the mouse serum, which should be very useful for the development of a TATP biosensor system. An ELISA with a limit of detection for TATP of 65 μg L(-1) is shown.