乙氧基磷酸酯类有机磷农药单克隆抗体的制备与鉴定

制备针对乙氧基磷酸酯类有机磷农药的单克隆抗体，以此为基础建立该类农药的快速免疫筛选检测方法．以二乙基磷酸乙酸为通用结构半抗原，分别使之与牛血清白蛋白和鸡卵清蛋白共价偶联，合成免疫原和包被原并对其进行结构鉴定．偶联成功后的免疫原用于免疫Balb/c小鼠．将免疫成功小鼠的脾细胞与小鼠SP2/0骨髓瘤细胞融合，筛选能稳定分泌抗乙氧基磷酸酯类有机磷农药单克隆抗体的杂交瘤细胞株．获得的小鼠腹水用辛酸-硫酸铵法纯化，所得纯化抗体以琼脂双扩散法鉴定其免疫球蛋白类型，间接竞争ELISA方法测定其对半抗原的灵敏度、特异性和亲和性．结果表明，该抗体分泌IgG1亚类的单克隆抗体，且与二乙基磷酸乙酸的亲和性较高(1.4×10⁷ L/mol)，所得抗体对毒死蜱、对硫磷、丙溴磷、氧化乐果、除线磷、二嗪农、溴硫磷、辛硫磷、喹硫磷、三唑磷等农药有特异性反应．该检测技术可用于上述农药的快速定性或定量检测．

Preparation and Identification of a Monoclonal Antibody Specific to a Broad Spectrum of O, O-diethyl Organophosphorus Pesticides

The objective of this study was to develop an enzyme-linked immunosorbent assay (ELISA) with specific monoclonal antibodies for quick detection and screening of a broad spectrum of O,O-diethyl organophosphorus pesticides. Diethylphosphono acetic acid was used as a hapten and conjugated with bovine sera albumin (BSA) or ovalbumin (OVA) to prepare immunogen and coating antigen, respectively. Two hybridoma cell lines that produce monoclonal antibodies against organophosphorus pesticides have been obtained by fusing mouse myeloma cells SP2/0 and splenocytes from Balb/c mice immunized with the immunogen.The immunoglobulin class was analyzed by double immunodiffusion and the affinity and specificity of McAbs were determinated with indirect enzyme-linked immunosorbent assay (ELISA). Results indicated that the immunoglobulin class of McAb was IgG1, McAbs have a high affinity for diethylphosphono acetic acid (1.4×10⁷ L/mol) and are able to cross-react to Chlorpyifos, Parathion, Profenofos, Omethoate, Dichlofenthion, Diazinon, Bromophos, Phoxim. This immunoassay is therefore capable of detecting 8 different organophosphorus pesticides. It is concluded that this assay is a qualified screening tool for quantitative or semi-quantitative detection of the 8 organophosphorous pesticides.