磺胺二甲氧嘧啶单克隆抗体的制备及其免疫学特性鉴定

PREPARATION AND IMMUNOLOGICAL TRAIT OF MONOCLONAL ANTIBODY FOR SULFADIMETHOXINE

用重氮化法将SDM(磺胺二甲氧嘧啶)偶联于载体蛋白BSA和OVA,合成免疫原BSA-SDM和包被原OVA-SDM,并用紫外扫描(UV)、SDS-PAGE进行鉴定;用BSA-SDM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SDM mAb细胞株,用体内诱生腹水法制备SDM mAb;对SDM 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-SDM人工抗原制备成功,分子结合比约为1∶12.1;筛选出1B43、D9、4E1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∶3.2×102、1∶5.12×102、1∶8.1×102,腹水分别为1∶2.56×105、1∶2.56×1051、∶5.12×105,4E1亲和常数(Ka)为1.25×1010L/mol;4E1株对SDM的IC50为16.64ng/ml,与磺胺-6-甲氧嘧啶交叉反应率为5%,与其他磺胺类药物无交叉反应性。本试验获得了抗SDM高价、敏感、特异的mAb,可用于SDM残留检测的免疫学试验。

Diazotization was used to conjugate sulfadimethoxine （SDM） to carrier protein BSA and obtained immunizing antigen. The coating antigen OVA-SDM was obtained in the same way, ultraviolet（UV）and SDS-PAGE were used to identify SDM artificial antigen. BALB/C mice were immunized with BSA-SDM, the titre of polyclonal antibody（pAb） was detemined by indirect ELISA and blocking ELISA. The hybridoma lines that secrete SDM mAb were established with using monoclonal antibody hybridoma technology. The immunological traits such as titer, affinity, sensitivity and specificity of the mAb were characterized. The results showed that SDM artificial antigen was synthesized successfully and its conjugation ratio of SDM to BSA was about 12.1：1, three hybridoma ceil lines of 1B4,3D9,4E1 were screened for specificity to SDM ,the indirect ELISA titer of the mAb were 1：3.2 × 10^2, 1：5.12 × 10^2, 1：8.10 × 10^2 in supernatant; 1：2.56 × 10^5, 1：2.56 × 10^5, 1：5.12 × 10^5 in ascites, the affinity constant（Ka）of 4E1 was about 1.25 × 10^10 L/mol, the mAb of 4E1 showed good sensitivity with the IC50 of 16.64ng/ml to SDM, 5 % cross-reactivity to sulfarnonomethoxine and little or no cross-reactivity to other compounds. SDM mAb of high-titer, sensitivity and specificity had been generated and made it possible to establish immunoassay of SDM residues in feed and animal food.