大鼠血清中人参皂甙Rb1的免疫检测

Immunodetection of ginsenoside Rb1 in rat serum

目的建立大鼠血清中人参皂甙Rb1(G-Rb1)的免疫检识方法。方法利用杂交瘤细胞技术制备抗G-Rb1单克隆抗体(mAb)。含G-Rb1的大鼠血清用甲醇沉淀蛋白,制备供试样品,点样于聚醚砜膜上,以乙腈:水:冰醋酸按25∶75∶1的比例展开;NaIO4处理,与BSA结合,使G-Rb1固定于膜上。将PES膜上的G-Rb1与抗G-Rb1 mAb反应后,加过氧化物酶标记的羊抗小鼠IgG,用含0.03%H2O2的1mg/ml4-氯-1-萘酚磷酸缓冲液显色。结果在聚醚砜膜上,在标准品和供试样品的相同位置可清晰地观察到一个青蓝色G-Rb1斑点,该方法检测限为0.25μg。结论这种G-Rb1的免疫检识方法较薄层层析具有更好的专一性和可靠性,灵敏度接近高效液相色谱,且不需要昂贵的仪器,便于操作。

Objective To establish a novel immunoassay for qualitative detection of ginsenoside Rb1 in rat serum. Methods Anti-G-Rb1 monoclonal antibody （mAb） was through a hybfidoma approach. Rat serum containing G-Rbl was deproteinized with methanol to prepare the sample for testing, which was loaded onto polyethersulfone （PES） membrane and developed in the mixture of acetonitrile, water and acetic acid （25：75：1）. After treatment with NaIO4, the membrane was transferred to 1% BSA solution for immobilization of G-Rbl. The membrane was subsequently treated with anti-G-Rb1 mAb solution, followed by addition ofperoxidase-labeled goat anti-mouse IgG and color development using 4-chloro-l-naphthol-0.03% H2O2. Results On the PES membrane, a clear blue spot representing G-Rbl occurred where the rat serum for testing and the standard G-Rbl samples were blotted. The limit of this immunodetection was 0.25μg. Conclusion This immunoassay has greater specificity and reliability than thin-layer chromatography with a sensitivity similar to that of high-performance liquid chromatography, and does not require sophisticated equipment for convenient G-Rb1 detection in rat serum.