Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, China.
Anal Chim Acta. 2012 Jul 29;736:85-91. doi: 10.1016/j.aca.2012.05.033. Epub 2012 May 28.
A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V(H) and V(L)) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V(H) and V(L) genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25±0.03 and 0.02±0.004 ng mL(-1), respectively, and the linear response range extended from 0.05 to 1.45 ng mL(-1). The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The results showed a good correlation between the data of dc-CLEIA and HPLC-MS (R(2)>0.99), indicating that the assay was an efficient analytical method for monitoring food safety.
建立了一种基于单链可变片段(scFv)-碱性磷酸酶(AP)融合蛋白的莱克多巴胺(RAC)快速、灵敏的化学发光酶免疫分析(CLEIA)方法。scFv 基因是通过从分泌针对 RAC 抗体的杂交瘤细胞系 AC2 中克隆重链和轻链可变区基因(V(H)和 V(L))，并通过拼接重叠延伸聚合酶链反应用连接子将 V(H)和 V(L)基因组装而成。所得 scFv 基因插入含有 AP 的表达载体 pLIP6/GN 中，在大肠杆菌 BL21 菌株中产生融合蛋白。纯化的 scFv-AP 融合蛋白用于开发用于检测 RAC 的直接竞争 CLEIA(dcCLEIA)方案。结合的 50%抑制所需的平均浓度和检测限分别为 0.25±0.03 和 0.02±0.004 ng mL(-1)，线性响应范围从 0.05 扩展到 1.45 ng mL(-1)。该测定比基于相同融合蛋白的相应酶联免疫吸附测定(ELISA)灵敏 10 倍。交叉反应研究表明，融合蛋白与 RAC 类似物没有交叉反应。dcCLEIA 用于分析 RAC 加标猪肉样品，并用高效液相色谱-串联质谱法(HPLC-MS)进行验证。结果表明，dc-CLEIA 数据与 HPLC-MS 之间具有良好的相关性(R(2)>0.99)，表明该测定是监测食品安全的有效分析方法。
