抗脱氧雪腐镰刀菌烯醇单克隆抗体的制备及鉴定  

Preparation and characterization of monoclonal antibodies against deoxynivalenol

作　　者：赵丽[1] 温红玲[1] 侯霄煜[2] 蒲双双[1] 宋艳艳[1] 许洪芝[1] 李凤琴[3] 

机构地区：[1]山东大学公共卫生学院微生物检验所,济南250012 [2]济南铁路疾病预防控制中心,济南250012 [3]中国疾病预防控制中心营养与食品安全所,北京100052

出　　处：《山东大学学报（医学版）》2010年第12期146-149,共4页Journal of Shandong University:Health Sciences

基　　金：国家高技术研究发展计划(863计划)资助课题(2007AA10Z423)

摘　　要：目的制备抗脱氧雪腐镰刀菌烯醇(DON)单克隆抗体(McAb)并对其进行初步鉴定。方法采用小剂量长周期的免疫方案,以DON-牛血清白蛋白(BSA)偶联物免疫雌性BALB/c小鼠,采用细胞融合的方法制备杂交瘤细胞株,酶联免疫吸附试验(ELISA)检测上清中McAb,对抗体分泌阳性的细胞株进行克隆化,直至抗体阳性率100%,用竞争抑制ELISA法进一步检测McAb的特异性,腹水诱生法制备大量McAb,并用饱和硫酸铵对其进行纯化,用抗体亚类鉴定试剂盒对该McAb亚类进行鉴定,BCA蛋白测定试剂盒测定蛋白含量,ELISA检测McAb的滴度、参考工作稀释度和亲和常数。结果 DON-BSA免疫的BALB/c小鼠血清效价为1∶256000,与BSA有强烈的交叉反应。细胞融合后,ELISA筛选抗体分泌阳性的杂交瘤细胞株,经3轮克隆化,建立了1株能稳定分泌抗DON-BSAMcAb的杂交瘤细胞株3G5,腹水诱生法制备了大量的McAb。该McAb属IgG1,IgG含量为6.06g/L,抗体滴度为1∶500000,参考工作稀释度为1∶64000,抗体亲和常数为1.62×109。用间接竞争抑制ELISA测得校正曲线线性范围9.8～10000ng/mL,线性方程Y=-0.2726X+0.2259(r=0.9309)。结论获得了分泌抗DON-McAb的杂交瘤细胞株,该单克隆抗体灵敏度高,特异性强,可用于制备高质量国产DON-ELISA检测试剂盒。Objective To prepare monoclonal antibodies（McAbs）against deoxynivalenol（DON）and characterize their properties.Methods Using a low-dose and long-cycle immunization scheme,female BALB/c mice were immunized with DON coupled to bovine serum albumin（DON-BSA）.Hybridoma cell lines were obtained by cell fusion.McAbs in the supernatant were determined by ELISA.Hybridoma cells which secreted McAbs to DON-BSA were cloned again and again until the McAb-positive rate reached 100%,and the specificity of McAbs to DON was further confirmed by competition-inhibition ELISA,and a large amount of McAbs was made by ascites in liquid paraffin-primed mice and purified by saturated ammonium sulfate.The subtype of the McAb was tested by a subtype identification ELISA kit.The protein concentration was tested by a BCA kit.The titer,work concentration for reference and affinity constant of the McAb were measured by ELISA.Results The titer of antibodies in sera from Balb/c mice immunized by DON-BSAwas 1∶256 000 and all had a strong cross-reaction with BSA.Hybridoma cells which secreted McAbs against DON-BSA were screened by ELISA after cell fusion.A hybridoma cell line secreting McAb against DON-BSA was established by 3 rounds of cloning and named 3G5.A large amount of McAbs was obtained by ascites production.The subtype of the McAb belongs to IgG1,the concentration of IgG was 6.06 g/L;the McAb titer was 1∶ 500 000,the McAb work concentration for reference was 1∶64 000 and the McAb affinity constant was 1.62×109.The linear range of the competitive inhibition ELISA calibration curve was from 9.8 to 10 000 ng/mL,and the linear regression equation was Y=-0.272 6X＋0.225 9（r=0.930 9）.Conclusion The hybridoma cell line secreting McAbs against deoxynivalenol was obtained,and the prepared antibody showed high sensitivity and specificity to DON,so it can be used to produce a homemade DON-ELISA kit of high quality.

关 键 词：脱氧雪腐镰刀菌烯醇 单克隆抗体 特性鉴定 亲和常数 

分 类 号：R117[医药卫生—公共卫生与预防医学] R155.5