Research On The Qualitative And Quantitative Detection Of Ricin In The Various Complicated Matrices

Ricin is an extraordinary toxin protein extracted from the beans of the castor oil plant Ricinus communis. It is a typeⅡribosome-inactivating protein(RIPⅡ)with the molecular weight ~66 kDa. Ricin is composed of two disulfide-linked polypeptide chains with different functions. The enzymatically active A chain can inactivate ribosome RNA (rRNA) and therefore inhibit protein synthesis, eventually causes cell death. The B chain has two galactose-binding sites and can bind to the surface of cell where it comes into contacting with carbohydrate-binding sites and thereby facilitates endocytosic uptake of the protein. As a consequence of its high toxicity, availability, ease of preparation, and the lack of medical countermeasures, ricin has been considered to be a likely biological toxin for military or terrorist uses. Towards to this potential agent for bioterrorism, establishing a fast and sensitive method has been an important tool for preventing and dealing with the consequences of intoxication.By far the most common methods of detection for ricin are immunological methods based on antibodies. Due to the rapid and sensitivity characterization of these methods could achieve analysis of target molecules, this assays have been adapted and optimized for the qualitative and quantitative detection of ricin in contaminated or poisoned samples. However, in practice these assays are not always unambiguous because of false positives from cross reactivity of antibodies with other molecules which posses similar structures with target molecules, such as ricin and Ricinus communis Agglutinin 120 (RCA120). RCA120 is composed of two A chains and two B chains with sequence homology to ricin A and B chains greater than 85%, but RCA120 has only a fraction of ricin's toxic potency. Because of the high similarity of RCA120 to ricin, the two proteins are very difficult to distinguish exactly. Many immunological methods currently in use for ricin detection cannot distinguish the two proteins. Thus it would be advantageous to use two (or more) detective technologies to confirm the presence of ricin. Since the mid-1990s, there have been more and more modern analysis instruments developed in protein assay field. Advances in biological mass spectrometry now allow sensitive detection, accurate mass measurement, and structural analysis of a wide range of biomolecules. Thus, mass spectrometry techniques involving either electrospray ionization(ESI) or matrix-assisted laser desorption/ionization (MALDI) are now being increasingly developed for the detection and absolute quantification of proteins in biological environments.The purpose of this thesis is to establish qualitative and quantitative methods in enzyme-linked immunosorbent assay (ELISA) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for biotoxins protein ricin in various matrices. This paper consists of 3 parts. In the first chapter, we overviewed the physical and chemical properties of ricin. Also, we summarized the signs and symptoms of ricin exposure and the development of detection methods for ricin in recent years. The objectives, contents and new insights of this dissertation were briefly outlined at the end of this chapter.In the second chapter, we established three determination methods in enzyme-linked immunosorbent assay, namely optical absorption, fluorescent and chemiluminescent immunoassay for quantitation of biotoxins protein ricin in various matrices. Under individually optimized conditions, method validation including standard curve, linear range, sensitivity, accuracy and specificity were investigated in these three methods. The results showed that chemiluminescent immunoassay could provide wider linear range (from 0.02 to 5.5μg/L with correlation coefficient 0.999), higher sensitivity (limit of detection was 0.005μg/L), and could be adopted as a simple, rapid and robust approach. Otherwise, three methods were applied to measure the spiked ricin in water, beverage and human serum samples. The recoveries and the accuracies were qualified for examination standard.In the third chapter, we established a method of liquid chromatography-tandem mass spectrometry for qualitative and quantitative detection of ricin. First of all, among the 14 ricin peptides observed by LC-Q-TOF/MS, three of them were chosen for detection based on several parameters such as detection sensitivity and specificity toward ricin forms. Whereafter,we established a rapid, accuracy and high specificity quantification method of ricn with multiple reaction monitoring mode. Furthermore, in order to achieve the more sensitive quantitative determination of ricin in complex samples and clinical samples, in this thesis we used an approach based on immunocapture by magnetic beads with anti ricin antibodies to concentrate the samples and to remove unrelated proteins potentially coexisting in the media to be tested. With the use of this pretreatment coupled to MS determination, ricin can be quantified as low as 1.25 ng/mL in human serum. At last, this method has been successful used in quantitative determination of serum samples collected from poisoned rats. The result showed that we could also determine about 10 ng/mL of the residual ricin in rat serum at the time point of eight hours.