新霉素单克隆抗体的制备及其免疫学特性鉴定

Preparation and Immunological Properties of Neomycin Monoclonal Antibody

用碳二亚胺(EDC)法将新霉素(NEO)偶联于载体蛋白BSA和OVA,合成免疫原BSA-NEO和包被原OVA-NEO,用红外扫描(IR)、SDS-PAGE进行鉴定;用BSA-NEO免疫BALB/c小鼠,间接ELISA和阻断ELISA选择细胞融合备用鼠;应用杂交瘤技术建立分泌NEOmAb细胞株,用体内诱生腹水法制备NEOmAb;对NEOmAb的效价、亲和性、敏感性和特异性等免疫学特性进行鉴定。结果表明,成功制备了BSA-NEO人工抗原;筛选出1E9、4E8、1G1共3株敏感特异的杂交瘤细胞,间接ELISA效价细胞培养上清分别为1∶2.56×103、1∶1.28×103、1∶5.12×103,腹水效价分别为1∶5.12×105、1∶2.56×105、1∶1.02×106,1G1亲和常数(Ka)为3.75×1010(L/mol);1G1株对NEO的IC50为2.57 ng/mL,NEO mAb对庆大霉素、链霉素、土霉素、环丙沙星、二氟沙星等无交叉反应。试验获得了抗NEO高价、敏感、特异的mAb,可用于NEO残留检测的免疫学试验。

Neomycin was conjugated to cartier protein BSA and OVA by the method of EDC to form the immunizing antigen BSA-NEO and the coating antigen OVA-NEO. IR and SDS-PAGE were used to identify the NEO artificial antigen. Balb/c mice were immunized with BSA-NEO. The titre of polyclonal antibody was detected by indirect ELISA and blocking ELISA. The hybfidoma lines that secrete NEO mAb were established with monoclonal antibody hybridoma tech- nology. The immunological traits such as titer, affinity, sensitivity and specificity of the mAb were characterized. The resuits showed that NEO artificial antigen was synthesized successfully. Three hybridoma cell lines of 1E9,4E8 and 1G1 were screened for specificity to NEO. The indirect ELISA titer of the mAb were 1 ： 2.56 × 10^3, 1 ： 1.28 × 10^3 and 1 ： 5.12 × 10^3 in supernatant, and 1 ： 5.12× 10^5, 1 ： 2.56× 10^5 and 1 ： 1.02× 10^6 in ascites. The affinity constant（Ka） of 1G1 was about 3.75 × 10^10（L/mol） .The mAb of 1G1 showed good sensitivity with an IC50 of 2.57 ng/mL to NEO.No cross-reactivity to other compounds was detected, sncb as Gentamicin, Streptomycin, Oxytetracycline, Ciprofloxacin, Difloxacin,etc .The NEOmAb with high-titer, sensitivity and specificity had been generated, and could be used to establish the immunoassay of NEO residues in feed and animal food.