生物样品中蓖麻毒素的双夹心酶联免疫定量检测方法及应用

A quantitative sandwich enzyme-linked immunosorbent assay for ricin in biological samples and its application

目的建立定量检测生物样品中蓖麻毒素的双夹心ELISA方法并研究毒素在大鼠体内的分布。方法利用抗蓖麻毒素的单克隆抗体(MAb)3D74和辣根过氧化物酶(HRP)标记的小鼠抗蓖麻毒素单克隆抗体4C13,建立了定量检测血清和组织样品中蓖麻毒素的双夹心ELISA方法,并对方法学进行了必要的验证。结果建立的双抗夹心ELISA方法检测组织中蓖麻毒素的浓度范围为1.25～320 ng/ml,最低定量限为2.5 ng/ml,日内和日间精密度分别小于3.5%和9%。将该方法应用于蓖麻毒素组织分布的定量测定,结果显示,大鼠静脉染毒后毒素可以在心、肝、脾、肺、肾、肠和脂肪等组织分布,以脾脏的分布为最高,其次为肝脏,染毒后0.5和2 h脾与血清的比值分别为3.9和7.2。在脑和肌肉中未检出毒素。结论建立的双夹心ELISA方法简便、灵敏,可以用于定量检测血清和组织等生物样品中的毒素含量,为蓖麻毒素的毒物代谢动力学研究及毒素中毒的快速检测提供了技术支撑。

Objective To develop and validate a sandwich ELISA method for the quantitative analysis of riein in serum and tissue samples. Methods The sandwich ELISA method was established by using mouse anti-riein monoelonal antibody 3D74 and HRP-labeled mouse anti-riein monoelonal antibody 4C13. The necessary validation of the method was performed in biological samples. Results The quantitative deteetion range of the assay was from 1.25 to 320 ng/ml, with the lower limit of quantification of 2.5 ng/ml. The RSD of inter-day variability and intra-day variability was less than 3.5% and 9% ,respectiveLy. This assay was used to quantitatively analyze the riein tissue distribution. The results indieated that, the toxin could be detected in a number of tissues after iv exposure, including heart, liver, spleen, lungs, kidneys, intestine and fat. Among these tissues, the highest toxin level was found in spleen and the next was liver. The concentration ratio between spleen and serum at 0.5 and 2 h after exposure was 3.9 and 7.2, respectively. No detectable riein was found in brain and muscle. Conclusion The established sandwich ELISA method is proved to be simple and sensitive. It has been successfully applied to quantitatively analyze riein in serum and various tissues. The method could also been used for toxi- eokinetie study, as well as for rapid riein deteetion in biological samples.