School of Pharmacy, Chengdu Medical College, Chengdu 610500, China; College of Life Science and Technology, Southwest Minzu University, Chengdu 6a0041, China.
College of Life Science and Technology, Southwest Minzu University, Chengdu 6a0041, China; Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China.
Fitoterapia. 2020 Apr;142:104488. doi: 10.1016/j.fitote.2020.104488. Epub 2020 Jan 28.
A new strategy for the hapten design of natural glycoside and application for the preparation of antibody is reported in this work. With astragaloside IV (AGS-IV) as an example, C6"-CHOH on a glucosyl group was selectively oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation to C6"-COOH, which was subsequently condensed with -NH on bovine serum albumin to get artificial antigen. Then, the successful preparation of artificial antigen was verified by TCL, SDS-PAGE, UV, and MALDI-TOF-MS. Finally, rabbits were immunized with artificial antigen to obtain an antibody against AGS-IV. After tests of the titer, IC, and cross-reactivity, the results showed that the antibody prepared by TEMPO oxidation in this work had higher specificity than that the antibody prepared by conventional sodium periodate (NaIO) oxidation. The hapten, as a carboxylic acid derivative of AGS-IV, has better water solubility than AGS IV, which is more suitable for the synthesis of the hapten-carrier protein conjugate in aqueous phase, achieving another virtue of TEMPO oxidation over NaIO oxidation. This new strategy provides new ideas for the design of haptens of other natural glycosides, as well as the preparation of their antibodies.
本工作报道了一种设计天然糖苷半抗原的新策略，并将其应用于抗体的制备。以黄芪甲苷（AGS-IV）为例，通过 2,2,6,6-四甲基哌啶-1-氧自由基（TEMPO）氧化，选择性地将葡萄糖基上的 C6"-CHOH 氧化为 C6"-COOH，随后与牛血清白蛋白上的 -NH 缩合，得到人工抗原。然后，通过 TCL、SDS-PAGE、UV 和 MALDI-TOF-MS 验证了人工抗原的成功制备。最后，用人工抗原免疫兔子，得到抗 AGS-IV 的抗体。经过效价、IC 和交叉反应性测试，结果表明，与传统的高碘酸钠（NaIO）氧化法相比，本工作中通过 TEMPO 氧化法制备的抗体具有更高的特异性。半抗原作为 AGS-IV 的羧酸衍生物，其水溶性优于 AGS-IV，更适合在水相合成半抗原-载体蛋白缀合物，这是 TEMPO 氧化相对于 NaIO 氧化的另一个优点。该新策略为其他天然糖苷半抗原的设计以及它们的抗体的制备提供了新的思路。
