A型肉毒神经毒素单克隆抗体的制备及ELISA方法的建立  

Preparation of monoclonal antibody against botulinum neurotoxin type A and establishment of ELISA methed

在线阅读下载全文

作  者:叶婷 陆俭 李小娟 李晨昊 刘子嫣 张雪平 赵涛 朱衍志 YE Ting;LU Jian;LI Xiaojuan;LI Chenhao;LIU Ziyan;ZHANG Xueping;ZHAO Tao;ZHU Yanzhi(Lanzhou Biotechnology Development Co,Ltd,Lanzhou,Gansu 730046,China)

机构地区:[1]兰州生物技术开发有限公司质量检定室,兰州730046

出  处:《国际检验医学杂志》2024年第S02期68-71,共4页International Journal of Laboratory Medicine

摘  要:目的筛选高亲和力的抗A型肉毒神经毒素单克隆抗体,建立A型肉毒神经毒素快速检测方法。方法以A型肉毒神经毒素类毒素为免疫原免疫小鼠获得单克隆抗体,对所得单克隆抗体进行效价检测、类和亚类鉴定以及特异性相关检测。利用交叉搭配试验筛选最佳标记抗体,并确定兔多抗的包被浓度和标记抗体的稀释倍数,建立夹心酶联免疫吸附试验(ELISA)方法,对该法的专属性、精密度、准确度和线性进行初步研究。结果本研究共获得6株单克隆抗体,9C_(2)和3F6的效价最高;5D3、9C_(2)、1B5和6D6均为IgG1类抗体且显示A型特异性;9C_(2)、1B5和7G11对A型肉毒神经毒素的型特异阻断能力强。以5μg/mL兔多抗为包被抗体、1∶2000稀释的9C_(2)为酶标二抗建立方法,A型肉毒神经毒素蛋白浓度在6~100 ng/mL范围内呈现线性关系(R=0.995),重复性CV值为4.4%;中间精密度CV值为2.6%;回收率均在85%~118%之间。结论初步建立了快速检测A型肉毒神经毒素的夹心ELISA方法。Objective To select high-affinity anti-botulinum neurotoxin type A monoclonal antibodies and establish rapid detection method for botulinum neurotoxin type A.To screen high affinity monoclonal antibod-ies against type A botulinum neurotoxin and establish a rapid detection method for type A botulinum neuro-toxin.Methods Monoclonal antibodies were obtained by immunizing mice with type A botulinum neurotoxin like toxins as immunogens,and the resulting monoclonal antibodies were subjected to potency testing,class and subclass identification,and specificity related testing.Cross matching experiments were used to screen for the best labeled antibodies,and the coating concentration and dilution ratio of the labeled antibodies were de-termined.A sandwich enzyme-linked immunosorbent assay(ELISA)method was established to preliminarily study the specificity,precision,accuracy,and linearity of the method.Results The study were obtained 6 mon-oclonal antibodies.Monoclonal antibodies 9C_(2)and 3F6 had the highest titers.5D3,9C_(2),1B5,6D6 were IGg1 an-tibodies and had Type A specificity.9C_(2),1B5,7G11 showed strongly specific blocking ability to botulinum neu-rotoxin type A.Using 5μg/mL rabbit polyantibody as coating antibody and 1∶2000 dilution of 9C_(2)as en-zyme-labeled secondary antibody,the concentration of botulinum neurotoxin type A protein showed a linear in the range of 6-100 ng/mL(R=0.995).The repeatability CV was 4.4%.The intermediate precision CV was 2.6%.The recoveries were 85%-118%.Conclusion A sandwich ELISA method for rapid detection of type A botulinum neurotoxin is preliminarily established.

关 键 词:A型肉毒神经毒素 杂交瘤 单克隆抗体 酶联免疫吸附测定 

分 类 号:Q785[生物学—分子生物学]

 

参考文献:

正在载入数据...

 

二级参考文献:

正在载入数据...

 

耦合文献:

正在载入数据...

 

引证文献:

正在载入数据...

 

二级引证文献:

正在载入数据...

 

同被引文献:

正在载入数据...

 

相关期刊文献:

正在载入数据...

相关的主题
相关的作者对象
相关的机构对象