| Ricin extracted from castor seeds is classified into type II ribosome inactivating protein. It is a heterodimer glycoprotein of approximately 66KD, consisting of A and B subunits which are covalently connected by a single disulfide bond. The characteristics of ricin, including the high stability, extreme toxicity, easy to be produced and used, make it potential threatened biochemical warfare. For half a century, ricin played a central role in several terrorist incidents in different countries. It has been a significant global threat to public safety because of its feature favored by the majority of extremist groups and terrorists.Recently, many countries, including United States, Germany, French, and Russia put a huge financial and human resource for development of the prevention and therapy in response to the ricin poisoning. It has been established multiple approaches for ricin detection, including radioimmunoassay, enzyme-linked immunosorbent assay, immune PCR, biological sensor, capillary electrophoresis and mass spectrometry. Although the approaches of ricin detection have become more and more variety, and the accuracy has been remarkably improved through the high-tech development, most of them are extremely limited via the numerous factors, such as expensive devices, long-time testing period and/or strait requirements for operation, resulting of the vacancies of appropriate methods of ricin detection in response to incidents and terrorism attacks.Aiming to establish a rapid and convenient method for ricin detection, a simple and precise approach was developed to detect the residual ricin in biological samples via establishing the ricin-poisoning animal mode. The profile of in vivo metabolic kinetics and distribution of ricin was investigated by this method in animal modes established by different routes of ricin exposure. The reliability was as well as evaluated via pathological analysis on different tissues of ricin-poisoned animals.Experimental methods (1) Rapid ricin detection of colloidal gold test strip was used to detect the residual ricin in intoxicated animal, including serum and tissue homogenates. (2) Sandwich ELISA was established via two ricin-specific monoclonal antibodies for the toxic detection. (3) Slices of tissue samples were prepared for HE staining and the determination of ricin distribution supporting the result of metabolic kinetics quantified by sandwich ELISA assay. Results (1) In the detection ricin in serum samples using the colloidal gold test strip, it is demonstrated that this method is only suitable to detect the bio-samples containing high concentration ricin because the interruption from the serum component decreased the detection sensitivity of this method. (2) Sandwich ELISA assay established via the combination of two mouse-anti-ricin monoclonal antibodies, 3D74 and horseradish peroxidase-labeled 4C13, was used for ricin detection. The detection limit is as low as 2.5 ng/ml of toxin in PBS. When it was used to measure the standard curve of ricin diluted by tissues homogenate, the absorbance values were lower than the counterpart diluted by PBS, indicating that some component could interrupt the antigen-antibody specific binding. The influence depended on the type tissue homogenate used. To eliminate the interference from homogenate, the standard curves of ricin diluted by normal tissue homogenates were used to detect the toxin in animal tissues. The results showed that this method had good reproducibility and stability for ricin detection. (3) ELISA method was used to detect the ricin in tissue samples of poisoned animal in different exposure, including intravenous, intramuscular, and intragastric. For the intravenous poisoning mode, the high concentration of ricin was detected in the spleen and liver with rich blood supply, but it was undetectable in brain and muscle. For intramuscular exposure, ricin was accumulated at the injection position, showing the tardiness distribution compared with intravenous and intragastric exposure animal. The concentration of ricin gradient decreased in muscle, whereas displaying a peak value in serum and spleen. Additionally, ricin could be detectable in liver, kidney, intestine, as well as heart, intensity, and thymus. For intragastric exposure animal, ricin was mainly detected in the intestine and its concentration gradually decreased over time. Additional positive signal was demonstrated in kidney, but no detectable signals in serum and other tissues. (4) Residual ricin in animal tissues could be detected by immunostaining. In the intravenous exposure animal mode ricin could be detected in heart, liver, spleen, kidney and lung with the positive correlation between poisoning dose and signal intensity. For the intramuscular injection, the positive signals were displayed in the heart, liver, spleen, lung, kidney, intestine and muscle, showing different tendencies over poisoning time. The stained intensity showed gradual increase in lung, kidney and spleen, but decrease in heart, liver, intestine and muscle, no positive results in thymus and testis. The result of immunohistochemical staining was accordant with the data from Sandwich ELISA assay. The results mentioned above indicated that colloid gold immunochromatographic strip is more suitable to detect the environmental samples. When it is used in the analysis of medical and biological samples, some bio-components could interfere the binding of ricin with its antibody and decrease the sensitivity of detection. The Sandwich ELISA established by the ricin specific monoclonal antibodies displayed a good reproducibility and stability which make it satisfying the requirement of ricin rapid detection. This research additionally supported a good understanding of the distribution and metabolism of ricin in animal poisoned in different exposure pathway, dose, and to find a reasonable opportunity to provide a scientific basis for treatment. |
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