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目的进行人参总皂苷(ginseng total saponins,GTS)多克隆抗体的制备,并用不同方法进行验证。方法首先在D101大孔吸附树脂纯化的基础上,采用阴阳离子交换树脂的方法进行人参皂苷高纯提取物的制备,香草醛-硫酸比色法估测人参总皂苷的含量;然后通过过碘酸钠法构建人参总皂苷和牛血清白蛋白(BSA)的偶联物作为抗原制备得到人参总皂苷多抗;使用Protein A/G琼脂糖纯化和抗原柱双纯化抗体;最后用免疫细胞化学和生物膜层干涉(biological membrane layer interference,BLI)技术验证抗体。结果得到高纯度的人参总皂苷;SDS-PAGE表明抗原GTS-BSA偶联物制备成功;ELISA法检测受试组效价为(29.52±4.31),亲和树脂纯化后得到较纯多抗;生物膜层干涉技术表明人参总皂苷多抗与人参总皂苷、人参皂苷Re、Rg1、Rh1、Rh2、原人参二醇、F1、化合物K、原人参三醇的亲和力分别为3.99×10~(-7)、4.69×10~(-9)、2.59×10~(-9)、2.40×10~(-6)、1.62×10~(-4)、2.64×10~(-7)、1.66×10~(-10)、1.08×10~(-7)和1.32×10~(-6);免疫细胞化学(ICC)从细胞学水平上验证了抗体的有效性。结论人参总皂苷多抗的效价和特异性较高,可为下一步研究人参总皂苷在脑中的分布做好铺垫。
Objective To prepare polyclonal antibody of ginseng total saponins (GTS) and validate it by different methods. Methods Firstly, based on the purification of macroporous resin D101, the extract of ginsenosides was purified by anion-cation exchange resin. The content of ginsenosides was estimated by vanillin-sulfuric acid colorimetry. Then, Sodium method to construct a conjugate of ginsenosides and bovine serum albumin (BSA) as an antigen to obtain total ginsenoside polyclonal antibody; using Protein A / G agarose purification and double purification of antigen column; and finally using immunocytochemistry and biofilm Biological membrane layer interference (BLI) technique to validate antibodies. The results showed that the purity of ginsenosides was high. SDS-PAGE showed that the conjugate of GTS-BSA was successfully prepared. The ELISA titer of test group was (29.52 ± 4.31) Membrane-layer interference technique showed that the affinity of total ginsenoside anti-ginseng with total ginsenoside, ginsenoside Re, Rg1, Rh1, Rh2, protopanaxadiol, F1, compound K, protopanaxatriol were 3.99 × 10-7 ), 4.69 × 10 -9, 2.59 × 10 -9, 2.40 × 10 -6, 1.62 × 10 -4, 2.64 × 10 -7, 1.66 × 10 ~ (-10), 1.08 × 10 ~ (-7) and 1.32 × 10 ~ (-6) respectively. The immunocytochemistry (ICC) validated the antibody’s cytotoxicity. Conclusion Ginseng total saponin polyclonal antibody titer and specificity higher for the next step to study the distribution of ginseng saponin in the brain well pave the way.