通过免疫聚合酶链反应测定法检测蓖麻毒素和其他核糖体失活蛋白。

Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay.

作者信息

Lubelli Chiara, Chatgilialoglu Alexandros, Bolognesi Andrea, Strocchi Paola, Colombatti Marco, Stirpe Fiorenzo

机构信息

Department of Experimental Pathology, University of Bologna, via S. Giacomo 14, I-40126 Bologna, Italy.

出版信息

Anal Biochem. 2006 Aug 1;355(1):102-9. doi: 10.1016/j.ab.2006.05.003. Epub 2006 May 19.

DOI:10.1016/j.ab.2006.05.003

PMID:16762307

Abstract

Ribosome-inactivating proteins (RIPs) are plant proteins with enzymatic activity, classified as type 1 (single chain) or type 2 (two chains). They are identified as rRNA N-glycosidases (EC 3.2.2.22) and cause an irreversible inhibition of protein synthesis. Among type 2 RIPs, there are potent toxins (ricin is the best known) that are considered as potential biological weapons. The development of a fast and sensitive method for the detection of biological agents is an important tool to prevent or deal with the consequences of intoxication. In this article, we describe a very sensitive immuno-polymerase chain reaction (IPCR) assay for the detection of RIPs-a type 1 RIP (dianthin) and a type 2 RIP (ricin)-that combines the specificity of immunological analysis with the exponential amplification of PCR. The limit of detection (LOD) of the technique was compared with the LODs of the conventional immunological methods enzyme-linked immunosorbent assay (ELISA) and fluorescent immunosorbent assay (FIA). The LOD of IPCR was more than 1 million times lower than that of ELISA, allowing the detection of 10 fg/ml of dianthin and ricin. The possibility to detect ricin in human serum was also investigated, and a similar sensitivity was observed (10 fg/ml). IPCR appears to be the most sensitive method for the detection of ricin and other RIPs.

摘要

核糖体失活蛋白（RIPs）是具有酶活性的植物蛋白，分为1型（单链）或2型（双链）。它们被鉴定为rRNA N-糖苷酶（EC 3.2.2.22），可导致蛋白质合成的不可逆抑制。在2型RIPs中，有一些强效毒素（蓖麻毒素最为人所知）被视为潜在的生物武器。开发一种快速灵敏的生物制剂检测方法是预防或应对中毒后果的重要工具。在本文中，我们描述了一种非常灵敏的免疫聚合酶链反应（IPCR）检测方法，用于检测RIPs——一种1型RIP（石蒜毒素）和一种2型RIP（蓖麻毒素），该方法将免疫分析的特异性与PCR的指数扩增相结合。将该技术的检测限（LOD）与传统免疫方法酶联免疫吸附测定（ELISA）和荧光免疫吸附测定（FIA）的检测限进行了比较。IPCR的检测限比ELISA低100多万倍，能够检测到10 fg/ml的石蒜毒素和蓖麻毒素。还研究了在人血清中检测蓖麻毒素的可能性，观察到了类似的灵敏度（10 fg/ml）。IPCR似乎是检测蓖麻毒素和其他RIPs最灵敏的方法。