Zhang Yue, Cao Peng, Lu Fang, Yan Xin, Jiang Bingqian, Cheng Jinjun, Qu Huihua
School of Life Science, Beijing University of Chinese Medicine.
Third Affiliated Hospital, Beijing University of Chinese Medicine.
J Vis Exp. 2019 Apr 6(146). doi: 10.3791/57116.
The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (<1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.
食品和天然产物中生物活性成分的分析已成为包括中药以及食品安全/毒理学在内的许多领域中一个热门的研究方向。许多传统分析技术需要昂贵的设备和/或专业知识。值得注意的是，酶联免疫吸附测定（ELISA）已成为分析食品和天然产物的一种新兴方法。该方法基于抗体介导的目标成分检测。然而，由于天然产物中的许多生物活性成分分子量较小（<1000 Da）且不会引发免疫反应，因此针对它们制备单克隆抗体（mAb）通常很困难。在本方案中，我们详细说明了针对目标分子生成mAb以及创建相关间接竞争（ic）ELISA以快速分析多个样品中化合物所需的步骤。该程序描述了人工抗原（即半抗原-载体偶联物）的合成、免疫、细胞融合、单克隆杂交瘤制备、mAb的表征以及基于ELISA的mAb应用。通过高碘酸钠法合成半抗原-载体偶联物，并通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）进行评估。免疫后，从抗体效价最高的免疫小鼠中分离脾细胞，并使用基于聚乙二醇（PEG）的方法与次黄嘌呤-氨基蝶呤-胸腺嘧啶核苷（HAT）敏感的小鼠骨髓瘤细胞系Sp2/0 -Ag14进行融合。通过icELISA筛选分泌对目标抗原具有反应性的mAb的杂交瘤的特异性和交叉反应性。此外，应用有限稀释法制备单克隆杂交瘤。最终的mAb通过icELISA进一步表征，然后用于基于ELISA的应用，以快速方便地检测天然产物中的示例半抗原（柚皮苷（NAR））。
