抗链霉素单克隆抗体杂交瘤细胞株的建立及其免疫学特性鉴定

GENERATION AND IMMUNOLOGICAL TRAIT OF MONOCLONAL ANTIBODY FOR STREPTOMYCIN

用EDC一步法和戊二醛法将SM偶联于载体蛋白牛血清白蛋白(BSA)和OVA,合成免疫原BSA-SM和包被原OVA-SM。用紫外线扫描、SDS-PAGE进行鉴定;用BSA-SM免疫BALB/C小鼠,用杂交瘤技术建立分泌SM mAb的细胞株3F9-C4;对SM mAb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,BSA-SM人工抗原分子结合比为1∶25;筛选出3F9-C4敏感特异的杂交瘤细胞1株,间接ELISA测定细胞培养上清,效价为1∶3.2×102,同种型为IgG2a/κ;腹水的亲和常数(Ka)为8.4×1011L/mol;IC50为8.99μg/L,与双氢链霉素交叉反应为109.6%,与其他SM结构相似物和功能近似物无交叉反应性。本试验获得了抗SM高价、敏感、特异的mAb,可用于SM的残留检测。

The coating antigen OVA-SM and immunogen BSA-SM synthesized with SM was coupled with BSA and OVA by EDC-method and glutaraldehyde method. Ultraviolet and SDS-PAGE were used to identify SM artificial antigen. BALB/C mice were immunized by BSA-SM, the hybridoma lines that secrete SM mAb were established by using monoclonal antibody hybridoma technology. The immunological traits such as titer, affinity, sensitivity and specificity of this mAb were characterized. The results showed that the conjugation ratio of SM to BSA in SM artificial antigen was about 25 ： 1. The titer of supernatant hybridoma cell lines of 3F9-C4 was 1：3.2× 10^2 by indirect ELISA. The isotypes in ascites was IgG2a/K, the affinity constant （Ka） was 8.4 × 10^11 L/mol, IC50 was 8.99μg/L, cross-reactivity to dihydrostreptomycin was 109.6% and little cross-reactivity to other compounds. The experiment generated SM mAb was high-titer,sensitivity and specificity. And this mAb can be used to detecte SM residues in food.