抗重金属Cr^(3+)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

作　　者：王亚楠[1] 丁菡[1] 王晓斐[2] 雷壮[1] 牛琳琳[1] 王自良[1] 

机构地区：[1]河南科技学院动物科学学院,河南新乡453003 [2]河南科技学院新科学院,河南新乡453003

出　　处：《西北农业学报》2016年第11期1597-1602,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金："十二五"国家科技支撑计划(2011BAK10B01-15)~~

摘　　要：旨在研制敏感、特异、稳定的抗Cr^(3+)单克隆抗体(Cr^(3+)mAb)。用异硫氰酸苄基乙二胺四乙酸(ITCBE)鳌合Cr^(3+)形成Cr^(3+)-ITCBE半抗原,异硫氰酯法制备人工免疫原Cr^(3+)-ITCBE-BSA和检测抗原Cr^(3+)-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(ICPAES)进行免疫原鉴定;用Cr^(3+)-ITCBE-BSA免疫Balb/C小鼠,细胞融合技术建立Cr^(3+)mAb杂交瘤细胞株,体内诱生腹水法制备Cr^(3+)mAb,并对其免疫学特性进行分析。结果表明,免疫原制备成功,Cr^(3+)-ITCBE-BSA中Cr^(3+)和BSA的质量浓度分别为140.8mg/L和5.81g/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∶(2.56×103)、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Cr^(3+)的半数抑制质量浓度(IC50)为16.6μg/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Cr^(3+)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 Cr^3＋（Cr^3＋mAb）.Artificial hapten Cr^3＋-ITCBE was synthesized to chelate chromium ion（Cr^3＋）using ITCBE.The method of isothiocyanate（ITC）was used to conjugate Cr^3＋-ITCBE to bovine serum albumin（BSA）and artificial immunogen Cr^3＋-ITCBE-BSA was obtained,the detecting antigen Cr^3＋-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 Cr^3＋-ITCBE-BSA.Balb/C mice were immunized with Cr^3＋-ITCBE-BSA and hybridoma lines that secrete monoclonal antibody against Cr^3＋（Cr^3＋mAb）were established through cell fusion,Cr^3＋mAb were induced from in vivo method and and their immunological properties of Cr^3＋mAb were identified.The results showed that Cr^3＋-ITCBE-BSA was synthesized successfully and its mass concentration of Cr^3＋and BSA was 140.8 mg/L and 5.81g/L respectively.Three hybridoma lines of 2A3C11,2A3D9,2A11G5 were screened and the best one was 2A11G5 with indirect ELISA titers of 1∶（2.56×10^3）in supernatant and 1∶（5.12×10^5）in ascites.The isotype of the mAb was IgG1/κand its affinity constant（Ka）was 2.69×10^9 L/mol.The mAb showed good sensitivity with an IC50 of 16.6μg/L to Cr^3＋and little or no cross-reactivity with other compounds.This study had successfully developed the Cr^3＋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.

关 键 词：CR^3+ 人工免疫原 单克隆抗体 免疫学特性 

分 类 号：TS207.51[轻工技术与工程—食品科学]