Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand.
School of Pharmacy, Walailak University, Nakhon Si Thammarat, Thailand.
Phytochem Anal. 2024 Apr;35(3):483-492. doi: 10.1002/pca.3305. Epub 2023 Nov 15.
The stem of the plant species Derris scandens (Roxb.) Benth. (DS) contains genistein-7-O-[α-rhamnopyranosyl-(1→6)]-β-glucopyranoside (GTG), which is a unique marker. Previous analyses of GTG using antibody-based immunoassays were compromised because of their high cross-reactivity with structurally related compounds of DS, thereby limiting their applicability in DS quality control.
Conjugation of GTG with carrier proteins was achieved using the Mannich reaction to produce a highly specific monoclonal antibody (mAb) targeting GTG (anti-GTG mAb).
The anti-GTG mAb was generated using hybridoma technology and characterised using an indirect competitive enzyme-linked immunosorbent assay (icELISA). Both lateral-flow immunoassay (LFIA) and icELISA were developed to detect and quantify GTG in DS raw materials and associated products.
icELISA using the anti-GTG mAb showed 100% specificity for GTG, with only 1.77% cross-reactivity with genistin and less than 0.01% cross-reactivity with other compounds. icELISA demonstrated a linear range for GTG determination between 62.5 and 2000 ng/mL. The limits of detection (LOD) and quantification were 49.68 and 62.50 ng/mL for GTG, respectively. The precision of the analysis ranged from 1.28% to 4.20% for repeatability and from 1.03% to 7.05% for reproducibility. The accuracy of the analysis ranged from 101.97% to 104.01% for GTG recovery. GTG levels determined via icELISA were consistent with those confirmed via high-performance liquid chromatography (HPLC) (R = 0.9903). Moreover, the LOD of LFIA for GTG was 500 ng/mL.
Immunoassays utilising specific anti-GTG mAbs were successfully developed, including LFIA for rapid GTG detection and icELISA for GTG quantification.
植物物种鸡血藤（Roxb.）Benth.（DS）的茎含有染料木素-7-O-[α-鼠李吡喃糖基-(1→6)]-β-吡喃葡萄糖苷（GTG），这是一种独特的标志物。先前使用基于抗体的免疫分析对 GTG 进行的分析受到其与 DS 中结构相关化合物高度交叉反应性的影响，从而限制了它们在 DS 质量控制中的适用性。
使用曼尼希反应将 GTG 与载体蛋白缀合，以产生针对 GTG 的高度特异性单克隆抗体（抗 GTG mAb）。
使用杂交瘤技术生成抗 GTG mAb，并使用间接竞争酶联免疫吸附测定（icELISA）进行表征。开发了侧向流动免疫分析（LFIA）和 icELISA 来检测和定量 DS 原料药和相关产品中的 GTG。
icELISA 使用抗 GTG mAb 对 GTG 具有 100%的特异性，与染料木苷的交叉反应性仅为 1.77%，与其他化合物的交叉反应性小于 0.01%。icELISA 显示 GTG 测定的线性范围在 62.5 和 2000ng/mL 之间。GTG 的检测限（LOD）和定量限分别为 49.68 和 62.50ng/mL。分析的精密度范围为重复性 1.28%至 4.20%，再现性 1.03%至 7.05%。GTG 回收率分析的准确度范围为 101.97%至 104.01%。icELISA 测定的 GTG 水平与高效液相色谱（HPLC）确认的水平一致（R=0.9903）。此外，LFIA 测定 GTG 的 LOD 为 500ng/mL。
成功开发了利用特异性抗 GTG mAb 的免疫分析，包括用于快速 GTG 检测的 LFIA 和用于 GTG 定量的 icELISA。
