摘要
【目的】制备抗庆大霉素(gentamicin,GM)的高亲和力特异性单克隆抗体,并鉴定其免疫学特性,为进一步研究GM快速检测试剂盒和试纸条打下基础。【方法】用EDC法将BSA和OVA分别和GM偶联作为免疫原或包被原,并经聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定。用合成的BSA-GM免疫Balb/c小鼠,经过4次免疫后,用间接ELISA和阻断ELISA选择细胞融合备用鼠,选择高效价、敏感的小鼠进行抗原超强免疫;取其脾细胞应用杂交瘤技术与骨髓瘤细胞建立分泌GM单克隆抗体(monoclonal antibody,mAb)的杂交瘤细胞株;用体内诱生腹水法制备GM mAb,对GM mAb的效价、敏感性和特异性等免疫学特性进行鉴定,;应用阻断ELISA试验原理组装GM-Kit,并对鲜奶中添加的GM标准样品进行测定。【结果】SDS-PAGE电泳鉴定表明BSA-GM人工抗原偶联成功;免疫的3只小鼠血清抗体效价均达到10-3;其中2号小鼠血清GM抑制效价较高且IC50最低,达17.28 ng.mL-1,融合后筛选出5E2-D7、1A3-A9、5E2-A12和2A6-B5共6株敏感特异的杂交瘤细胞,其细胞培养上清液效价分别为1﹕1600、1﹕800、1﹕800和1﹕800,腹水效价分别为1﹕2.05×107、1﹕5.12×106、1﹕2.56×106和1﹕2.56×106,5E2-D7株对GM的IC50为0.30 ng.mL-1,与链霉素、卡那霉素等其他氨基糖苷类抗生素无交叉反应性;检测鲜牛奶样的平均添加回收率分别为96.0%,平均变异系数均低于15%。【结论】本试验获得了高效价、敏感、特异的抗GM mAb,为GM残留检测奠定了坚实的基础。
【Objective】 The aim of the study is to prepare high sensitivity and specificity monoclonal antibody（mAb） against Gentamicin（GM） and identifiy its immunological characteristics.The monoclonal antibody against GM would lay a foundation for development of GM ELISSA kit and strip.【Method】 The GM was conjugated to carrier protein BSA and OVA by EDC to obtain the artificial immunogen and reactionogen and SDS-PAGE was used to identify these artificial antigens.Balb/c mice were immunized with BSA-GM.The titer of polyclonal antibody（pAb） was detected by indirect ELISA and blocking ELISA after three times immunization.The high titer,sensitivity and specificity mouse was selected for cell fusing.The hybridoma lines that secrete GM mAb were established by using monoclonal antibody hybridoma technology and the immunological characteristics such as titer,sensitivity and specificity of the mAb were characterized.A rapid test ELISA Kit was established and identified by blocking ELISA and has been used for the GM standard samples in milk detection.【Result】 SDS-PAGE results showed that GM artificial antigen was synthesized successfully.Three BALB/c mice indirect ELISA titer against GM were above 10-3 and the IC50 of No.2 mice was 17.28 ng.mL-1.Four hybridoma cell lines,5E2-D7,1A3-A9,5E2-A12,and 2A6-B5,were screened for specificity to GM after cell fusing,the indirect ELISA titer of the mAb were 1﹕1600,1﹕800,1﹕800,and 1﹕800 in supernatant,1﹕1.02×107,1﹕5.12×106,1﹕2.56×106,and 1﹕2.56×106 in ascites,the mAb of 5E2-D7 showed good sensitivity with an IC50 of 0.30 ng.mL-1 to GM,no cross-reactivity to other compounds.The recovery of GM spiked in milk was 96.0% and the coefficient variation was below 15%.【Conclusion】 The high-titer,sensitivity and specificity GM mAb has been prepared and laid a foundation for GM residue detection.
出处
《中国农业科学》
CAS
CSCD
北大核心
2010年第12期2584-2589,共6页
Scientia Agricultura Sinica
基金
"十一五"国家科技支撑计划(2006BAK02A21)
关键词
庆大霉素
单克隆抗体
杂交瘤
初步应用
gentamicin
monoclonal antibody
hybridoma
preliminary application
