多环芳烃-菲单克隆抗体制备及鉴定

为快速、准确检测环境中的菲(Phe),研究制备了抗菲单克隆抗体,并分析了抗菲单克隆抗体的免疫学特性。采用活泼酯法将2-菲丁酸与载体蛋白(BSA、OVA)共价连接制备免疫抗原(Phe-BSA)和检测抗原(Phe-OVA)。通过非变性聚丙烯酰胺凝胶电泳、紫外扫描图谱分析及计算偶联比验证偶联结果。采用足垫法免疫Balb/C小鼠,利用细胞融合技术、阳性杂交瘤筛选及克隆制备出抗菲单克隆抗体。结果表明:完全抗原制备成功,获得1株能稳定分泌抗Phe抗体的杂交瘤细胞株1G7,纯化后抗体效价为1∶6.4×105,亲和力常数为3.29×108L/mol。该单克隆抗体亚型为Ig G1,对Phe的IC50值为17.82 ng/m L,与5种多环芳烃具有交叉反应,可用于对环境中多种PAHs的广谱检测。

Preparation and Identification of Monoclonal Antibody against Polycyclic Aromatic Hydrocarbons-phenanthrene

In order to rapidly and accurately detect phenanthrene (Phe) in the environment,a monoclonal antibody (McAb) against Phe was prepared,and the immunological properties of the McAb were analyzed.2-phenanthrene butanoic acid was conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) for immunogen (Phe-BSA) and coating antigen (Phe-OVA) by active ester method.The conjugates were confirmed by nondenaturing agarose gel electrophoresis and ultraviolet spectrophotometry,respectively.Balb/C mice were immunized by footpad immunization method.After cell fusion,hybridoma selection and cloning,McAb against Phe was prepared.The results indicated that complete antigens of Phe were synthesized successfully.1 G7 cell line that stably secreted McAb against Phe was obtained.Titer,average affinity,IC5oand isotype of the McAb were 1 ∶ 6.4 × 105,3.29×108 L/mol,17.82 ng/mL and IgG1,respectively.The results of cross-reactivity showed that five PAHs had high CR value with the McAb,demonstrating that McAb has the potential to be used for broad spectrum assay of Phe and other homologous PAHs.