抗重金属Cr3+单克隆抗体的研制及其免疫学特性分析
Preparation of Anti-Chromium Ion Monoclonal Antibodies and Analysis of Immunological Properties

旨在研制敏感、特异、稳定的抗Cr3+单克隆抗体（Cr3+ mAb）。用异硫氰酸苄基乙二胺四乙酸（ITCBE）鳌合Cr3+形成Cr3+-ITCBE半抗原，异硫氰酯法制备人工免疫原Cr3+-ITCBE-BSA和检测抗原Cr3+-ITCBE-OVA，紫外扫描法（UV）、凝胶电泳法（SDS-PAGE）和电感耦合等离子体原子发射光谱法（ICP-AES）进行免疫原鉴定；用Cr3+-ITCBE-BSA免疫Balb/C小鼠，细胞融合技术建立Cr3+ mAb杂交瘤细胞株，体内诱生腹水法制备Cr3+ mAb，并对其免疫学特性进行分析。结果表明，免疫原制备成功，Cr3+-ITCBE-BSA中Cr3+和BSA的质量浓度分别为140.8 mg/L和5.81 g/L，筛选出2A3C11、2A3D9、2A11G5 3株杂交瘤细胞，其中最好的2A11G5间接ELISA效价细胞培养上清为1∶（2.56×10）、腹水为1∶（5.12×105），同种型为IgG1/κ，亲和常数（Ka）为2.69×109 L/mol，对Cr3+的半数抑制质量浓度（IC50）为16.6 μg/L，与其他重金属离子无交叉反应。表明成功研制亲和力高、特异性强、稳定性好的Cr3+ mAb，为铬离子残留免疫学检测方法的建立奠定基础。
To develop sensitive, specific and stable monoclonal antibody against Cr3+( Cr3+ mAb). Artificial hapten Cr3+-ITCBE was synthesized to chelate chromium ion(Cr3+) using ITCBE. The method of isothiocyanate(ITC) was used to conjugate Cr3+-ITCBE to bovine serum albumin (BSA) and artificial immunogen Cr3+-ITCBE-BSA was obtained, the detecting antigen Cr3+-ITCBE-OVA was obtained in the same way. Ultraviolet (UV), sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and inductively coupled plasma atomic emission spectroscopy (ICP-AES) were used to identify Cr3+-ITCBE-BSA. Balb/C mice were immunized with Cr3+-ITCBE-BSA and hybridoma lines that secrete monoclonal antibody against Cr3+( Cr3+ mAb) were established through cell fusion, Cr3+ mAb were induced from in vivo method and and their immunological properties of Cr3+ mAb were identified. The results showed that Cr3+-ITCBE-BSA was synthesized successfully and its mass concentration of Cr3+ and BSA was 140.8 mg/L and 5.81 g/L respectively. Three hybridoma lines of 2A3C11, 2A3D9, 2A11G5 were screened and the best one was 2A11G5 with indirect ELISA titers of 1∶(2.56×10) in supernatant and 1∶(5.12×105) in ascites. The isotype of the mAb was IgG1/κ and its affinity constant(Ka) was 2.69×109 L/mol. The mAb showed good sensitivity with an IC50 of 16.6 μg/L to Cr3+ and little or no cross-reactivity with other compounds. This study had successfully developed the Cr3+ mAb, which had high affinity, specificity and stability, and provided a potency of establishing immunoassay methods for determining Chromium ion residues in environment and food.