西马特罗杂交瘤细胞株的建立及其单克隆抗体制备和鉴定

ESTABLISHMENT OF HYBRIDOMA CELL LINES SECRETING ANTI-CIMATEROL MONOCLONAL ANTIBODY AND PREPARATION,IDENTIFICATION OF THEIR IMMUNOLOGICAL PROPERTIES

用重氮化法将牛血清白蛋白(BSA)和卵清蛋白(OVA)分别与西马特罗(CIM)偶联作为免疫原或包被原,用BSA-CIM免疫BALB/c小鼠,经过3次免疫后,OVA-CIM包被后用间接ELISA和阻断ELISA选择细胞融合备用鼠,选择高效价、高敏感性和高特异性的小鼠进行抗原超强免疫;取脾细胞应用杂交瘤技术与骨髓瘤细胞建立分泌CIM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CIM mAb,对CIM mAb的效价、敏感性和特异性等免疫学特性进行鉴定。结果显示免疫的6只小鼠血清抗体效价均达到10-3;融合后筛选出3B11-H4、2A11-G11和4F5-F11共3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∶800、1∶1600和1∶1600,腹水效价分别为1∶2.56×106、1∶1.02×107和1∶2.56×107,3B11-H4株对CIM的IC50为0.40ng/ml,与瘦肉精、莱克多巴胺等其他β2激动剂交叉反应性小于0.2%。本试验获得了高效价、敏感、特异的抗CIM mAb,为CIM残留ELISA检测试剂盒和试纸条的建立奠定了坚实的基础。

Artificial antigen BSA-CIM and OVA-CIM were synthesized using diazotization by linking carrier proteins BSA and OVA to CIM.The titer of polyclonal antibody（pAb） was detected by indirect ELISA and blocking ELISA after three times immunization by BSA-CIM.The high titer,sensitivity and specificity mouse was selected for cell fusing.The hybridoma lines that secrete CIM mAb were established using monoclonal antibody hybridoma technology and the immunological characteristics such as titer,sensitivity and specificity of mAb were characterized.The results showed that six BALB/c mice indirect ELISA titer against CIM were above 1 × 10^-3 and three hybridoma cell lines of 3B11-H4,2A11-G11 and 4F5-F11were screened for specificity to CIM,the indirect ELISA titer of the mAb were 1 ∶ 800,1 ∶ 1600and 1∶ 1600 in supernatant,1 ∶ 2.56 × 10^6,1 ∶ 1.02 × 10^7 and 1 ∶ 2.56 × 10^7 in ascites,the mAb of 3B11-H4 showed good sensitivity with an IC50 of 0.40ng/ml to CIM and had less than 0.2% cross-reactivity to other compounds.The results showed the high-titer,sensitivity and specificity CIM mAb has been generated and made it possible to establish immunoassay of CIM residues.