人参皂苷Rb1单克隆抗体的制备与鉴定

用人参皂苷Rb1分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)反应合成了交联抗原GRb1-BSA(GRb1与BSA的结合比为10∶1)和GRb1-OVA(GRb1与OVA的结合比为8∶1)。以GRb1-BSA为免疫原,分5次免疫3只BALB/C小鼠,取免疫脾细胞与小鼠骨髓瘤细胞进行融合,Hat筛选,有限稀释法克隆。建立分泌单克隆抗体的杂交瘤细胞系。采用ELISA(酶联免疫吸附测定)间接法和竞争法检测抗体的特异性。结果获得了一只小鼠分泌抗体效价达1∶16000,并获得两株分泌GRb1的单克隆抗体的细胞系,分别命名为1F7和1G3,单抗检测显示在0.01~1μg/mL呈线性关系,GRb1的检测范围达50~350 ng/mL,两株单抗针对相同的抗原决定簇,制备的单克隆抗体可用于GRb1含量的检测和分离。

Preparation and identification of monoclonal antibody against ginsenoside Rb1

Cross-linking antigens GRb1-BSA(ratio 10∶1) and GRb1-OVA(ratio 8∶1) were produced by the reactions of ginsenoside Rb1 with serum albumin(BSA) and ovalbumin(OVA) respectively.Three BALB/C mice were immunized five times with GRb1-BSA as the immunogen.Using polyethylene glycol(PEG) method,the immunized splenocytes were isolated and fused with a hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line,SP2/0.Hybridomas that produce monoclonal antibody(McAb) reactive to GRb1 were cloned by limiting dilution method.Hybridoma cell line that secretes McAb was obtained.Indirect enzyme-linked immunosorbent assay(ELISA) was applied to determine the titer of antisera and competitive ELISA was used to detect McAb.As results,the titer of the antisera is over 1∶16 000 by ELISA,two cell lines(1F7,1G3) that secrete McAb are obtained,McAb detection shows linear relation in the range from 0.01 g/mL to 1 μg/mL.The full range of GRb1 detection is from 50 ng/mL to 350 ng/mL by competitive ELISA.The two McAbs are contraposed the same determining cluster and can be used to establish ELISA for GRb1 content detection and GRb1 isolation.