荧光免疫层析技术定量检测蓖麻毒素

A Fluorescent Microsphere Immunochromatography Method for Quantitative Detection of Ricin Toxin

为了建立免疫层析技术定量检测蓖麻毒素试剂,首先依据蓖麻毒素对半乳糖的特异性结合能力,采用亲和层析技术对蓖麻种子进行亲和层析纯化;其次采用凝胶过滤层析技术,获得更纯的天然蓖麻毒素纯品;最后以天然的蓖麻毒素脱毒为免疫原,分别免疫获得鼠抗相思中毒素单克隆抗体和兔抗蓖麻毒素多克隆抗体,利用获得的鼠抗蓖麻毒素单克隆抗体作为标记抗体,标记羧基荧光微球(激发波长365nm,发射波长610nm)在NC膜上包被兔抗蓖麻毒素多克隆体作为检测线、包被羊抗鼠IgG抗体作为质控线,建立蓖麻毒素双抗体夹心定量检测试剂,并对该试剂进行性能评价。结果显示,纯化后的蛋白经SDS-PAGE处理后,分为分子量为65kDa左右的单条带、分子量为31kDa左右的双条带和33kDa左右的双条带,分别为Ricin毒素A链和B链。用TotalLab2.0软件分析纯化率为96%;检测试剂配合荧光免疫检测仪在0~50ng/mL的检测范围时,拟合曲线R2>0.990,线性范围为1~10ng/mL,R2>0.990,最低检出限为1ng/mL,且批间、批内重复性均小于15%。此法特异性良好,常温稳定性14个月,样本检测能力良好,可在食品和环境中应用蓖麻毒素进行定量检测。

In order to develop an immunochromatographic reagent for the detection of ricin toxin, firstly, based on the specific binding ability of ricin to galactose, affinity chromatography is used to purify ricin seeds;secondly, gel filtration chromatography is used to obtain more pure natural ricin products;finally, natural ricin is used. Monoclonal antibodies against Acacia toxin in mice and polyclonal antibodies against ricin in rabbits are obtained by immunization with toxin detoxified as immunogen. Combining the labeling technology, the specific mouse monoclonal antibodies are conjuncted with fluorescence nanoparticles(excitation spectrum peak is 365 nm;emission spectrum peak is 610 nm), ricin rabbit polyclonal antibodies are sprayed on the test zone to capture object protein in the samples, and goat anti-mouse IgG is sprayed on the control zone for quality control to develop lateral flow immunochromatographic strips. The results show that protein estimation is done by Lowry’s method and PAGE both under native conditions and in presence of SDS. When subjected to native PAGE, the purified protein shows a single band about molecular weight of 65 kDa, otherwise two bands about 31 kDa and 33 kDa subjected to SDS-PAGE, which are equal as ricin toxin A chain and B chain. The purification rate is analyzed as 96% by TotalLab2.0 software. In the quantitative test, the fluorescence intensity is measured by a portable strip reader and a standard curve is obtained from 0 to 50 ng/mL,(R2 >0.990), and the linear rangeand is from 1 to 10 ng/mL(R2 >0.990). The cut-off value(sensitivity) of immunofluorescence detection stripes is in detecting. Ricin is 1 ng/mL. Besides, both the variation coefficients are less than 15%. The method has good specificity, stability at room temperature for 14 months and good detection ability. It can be rapid screening tools for rapid detection of the Ricin toxin in food and environmental samples.