基于混合抗体的酶联免疫分析方法同时检测孔雀石绿和隐孔雀石绿

Simultaneous Detection of Malachite Green and Leucomalachite Green Based on Hybrid Antibody ELISA Analysis Method

孔雀石绿是'三致'物质,常被非法使用于渔业生产。水产品和环境中常以孔雀石绿和隐孔雀石绿两种形式同时存在,均具有强致癌性。但由于二者结构差异较大,现有的单克隆抗体只能分别检测样品中单一药物的残留量,难以客观地反映出孔雀石绿和隐孔雀石绿的总残留量。目前同时检测两种结构的免疫分析方法鲜有报道,本文分别制备了针对孔雀石绿和隐孔雀石绿的高特异性单克隆抗体,通过研究混合抗体模式建立了可以同时检测两种物质的酶联免疫分析方法,该方法对孔雀石绿和隐孔雀石绿混合物的半抑制浓度(50%inhibiting concentration,IC50)为3.27 ng/m L,检测线LOD(IC10)为0.24 ng/m L,线性范围为0.62~17.25 ng/m L。本方法的建立对保障水产品安全具有重要意义,也为混合抗体法同时检测两种或多种残留药物提供了方法借鉴。

Malachite green (MG) is a carcinogenic, teratogenic, and mutagenic chemical that is often illegally used in fishery production. The chemical residues mainly exist in two forms, as MG and its metabolite leucomalachite green (LMG), both of which have severe carcinogenicity, in aquatic products and the environment. Due to the significant structural differences of these two compounds, existing monoclonal antibodies are only able to detect one drug residue in a sample, and cannot accurately reflect the total amount of MG and LMG residues. To date, immunoassay methods for simultaneous detection of MG and LMG residues have rarely been reported owing to the structural differences between the two. Highly specific monoclonal antibodies of MG and LMG were prepared separately, while the indirect competitive enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection of MG and LMG was developed based on the hybrid antibody model. The half maximal inhibitory concentration (IC50) value of the established method on MG and LMG was 3.27 ng/mL, while the limit of detection (LOD) was 0.24 ng/mL and the linear range was 0.62~17.25 ng/mL. Thus, the establishment of the indirect competitive ELISA (icELISA) method is of great significance for ensuring the safety of aquatic food, in addition to providing a reference method to detect two or more residual chemicals simultaneously by the hybrid antibody assay. © 2015, South China University of Technology. All right reserved.