Data_Sheet_1_Development of an ic-CLEIA for precise detection of 3-CQA in herbs and patent medicines: ensuring quality control and therapeutic efficacy.docx

Background3-caffeoylquinic acid (3-CQA), a member of the chlorogenic acid family, possesses diverse pharmacological properties, such as scavenging, antioxidant, and antiapoptotic activity, rendering substantial value to alimentary consumables and therapeutic substances. However, the pervasiveness of non-standard practices, notably the misuse and abuse of indigenous botanicals, coupled with the inherent susceptibility of 3-CQA to degradation under light and heat exposure, engenders discernible disparateness in the quality profiles of the same kinds of herbs. Consequently, precise quantification of 3-CQA becomes imperative.
MethodsIn this context, an artificial antigen was synthesized as a specific conjugate of 3-CQA and bovine serum albumin (3-CQA-BSA), followed by the generation of a monoclonal antibody (mAb) against the conjugate. Through optimization, a mAb-based indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) was developed.
ResultsIt demonstrated an IC50 and the calibration range of 2.97 ng/mL and 0.64–13.75 ng/mL, respectively, outperforming the conventional enzyme-linked immunosorbent assay (ELISA). Notably, the ic-CLEIA displayed 10.71% cross-reactivity with 3,5-dicaffeoylquinic acid, alongside minimal cross-reactivity toward other isomeric counterparts and analogs. Validation experiments on herbs and Chinese patent medicines using ic-CLEIA, confirmed by high-performance liquid chromatography (HPLC) analysis, revealed a robust correlation coefficient of 0.9667 between the two modalities.
ConclusionThese findings unequivocally demonstrated that the proposed ic-CLEIA represents a viable and reliable analytical method for 3-CQA determination. This method holds significant potential for ensuring the quality control and therapeutic efficacy germane to herbs and patent medicines, spanning diverse therapeutic milieus and applications.

背景
3-咖啡酰奎宁酸（3-CQA）属于绿原酸家族，具备清除、抗氧化及抗细胞凋亡等多样药理活性，在食用消费品与治疗性药物中具有重要应用价值。然而，本土植物药误用滥用等不规范操作普遍存在，加之3-CQA本身在光照与热暴露条件下易发生降解，导致同类草药的质量特征存在显著差异。因此，精准定量3-CQA成为迫切需求。

方法
在此背景下，研究人员合成了3-CQA与牛血清白蛋白（BSA）的特异性结合物作为人工抗原（3-CQA-BSA），随后制备了针对该结合物的单克隆抗体（mAb）。通过工艺优化，建立了基于mAb的间接竞争化学发光酶联免疫分析法（ic-CLEIA）。

结果
该方法的半抑制浓度（IC₅₀）为2.97 ng/mL，校准范围为0.64~13.75 ng/mL，性能优于传统酶联免疫吸附测定（ELISA）。值得注意的是，ic-CLEIA与3,5-二咖啡酰奎宁酸的交叉反应率为10.71%，而与其他同分异构体及类似物的交叉反应极低。采用ic-CLEIA对草药及中成药开展验证实验，并经高效液相色谱（HPLC）分析确证，两种检测方法的相关系数达0.9667，相关性优异。

结论
本研究结果明确证实，所构建的ic-CLEIA是一种可行且可靠的3-CQA定量分析方法。该方法在草药及中成药的质量控制与疗效保障领域具备重要应用潜力，可适配多种治疗场景与应用方向。