Zhang Bo, Zhan Zhi-lai, Kang Li-ping, Yuan Yuan, Huang Lu-qi, Ding Feng-wei, Nan Tie-gui
Zhongguo Zhong Yao Za Zhi. 2015 Nov;40(22):4359-63.
Lonicerae Japonicae Flos was one of the most widely used traditional Chinese medicine for its special biological activities. The content of luteoloside, one of its major compounds, was an important standard for the quantity control of Lonicerae Japonicae Flos. The major method used for the detection of luteoloside was instrumental analysis. Compared with the ELISA method, instrumental analysis was time-consuming, complex pretreatment and low-throughout. Thus, it was significantly important to develop an enzyme-linked immunosorbent assay (ELISA) for luteoloside analysis. Here, the conjugates of luteoloside-bovine (LG-BSA) and luteoloside-ovalbumin (LG-OVA) were produced as the immunogen and coating antigen by the carbodiimide ( CDI) method, respectively. The conjugation ratio of carrier protein and the hapten in the conjugate were determined by UV-Vis spectrophotometry (UV). LG-BSA conjugate was used to immunize Bal b/c mice to produce antiserum. The titer and specificity of antiserum were detected by ELISA. The conjugation ratio of hapten and carries protein were 3. 7: 1 (LG-BSA) and 1. 0: 1 (LG-OVA). The antiserum titer was higher than 2 000 with the linear range of 18.4-4 852.4 μg x L(-1), R2 = 0.988 4 and IC50 = 298.7 μg x L(-1). The result showed that the conjugate antigen LG-BSA was synthesized successfully and the mice can produce specific antiserum injected with artificial antigen.
金银花是一种因其特殊生物活性而被广泛应用的传统中药。木犀草苷是其主要成分之一，其含量是金银花质量控制的重要标准。检测木犀草苷的主要方法是仪器分析。与酶联免疫吸附测定（ELISA）方法相比，仪器分析耗时、预处理复杂且通量低。因此，开发一种用于木犀草苷分析的酶联免疫吸附测定法具有重要意义。在此，分别通过碳二亚胺（CDI）法制备了木犀草苷-牛血清白蛋白（LG-BSA）和木犀草苷-卵清蛋白（LG-OVA）偶联物作为免疫原和包被抗原。通过紫外可见分光光度法（UV）测定偶联物中载体蛋白与半抗原的偶联比。用LG-BSA偶联物免疫Bal b/c小鼠以产生抗血清。通过ELISA检测抗血清的效价和特异性。半抗原与载体蛋白的偶联比分别为3.7:1（LG-BSA）和1.0:1（LG-OVA）。抗血清效价高于2000，线性范围为18.4 - 4852.4 μg·L⁻¹，R² = 0.9884，IC50 = 298.7 μg·L⁻¹。结果表明，偶联抗原LG-BSA成功合成，注射人工抗原的小鼠能够产生特异性抗血清。
