一种新的沙丁胺醇人工抗原的合成及单克隆抗体的制备

Synthesis of a newly designed artificial antigen and preparation of a monoclonal antibody against salbutamol

【目的】为证明采用4-溴丁酸乙酯半抗原合成人工抗原可制备灵敏度高、特异性好的单克隆抗体.【方法】将硫酸沙丁胺醇(SAL)与4-溴丁酸乙酯反应制备半抗原(SAL-HS);通过N-羟基琥珀酰亚胺活化酯法合成免疫原与包被原,用聚丙烯酰胺凝胶电泳、紫外光谱和质谱方法对沙丁胺醇免疫原及包被原进行鉴定.免疫原免疫Balb/c小鼠,采用融合技术产生杂交瘤细胞株,体内诱生腹水法制备单克隆抗体,并鉴定其免疫学特性.【结果和结论】筛选出1株杂交瘤细胞株,其细胞培养上清液效价达1∶128 000,腹水效价达1∶2 560 000,免疫球蛋白亚型鉴定为IGg1;抗体对沙丁胺醇的半数抑制浓度(IC50)为:0.746 ng/mL;与其他同类结构药物的交叉反应率均小于0.015%.

[ Objective ] To prove that the artificial antigen which was synthesised by 4-bromine ethyl buty- rate was successful, and that the monoclonal antibody had a high sensitivity and specificity. [ Method ] Salbutamol sulfate was connect with 4-bromine ethyl butyrate, then converted into hapten（SAL-HS）. The immunogen SAL-BSA （salbutamol-bovine serum albumin ） and coating antigen SAL-OVA （ovalbumin） were synthesized by the N-Hydroxy-succinimide active ester method. The conjugations were identified by SDS-PAGE, UV and mass spectrum. Balb/c mice were immunized by immunogen; splenocytes were fused with SP2/0 myeloma cells ; hybridomas cells were produced for a monoelonal antibody by ascites in vivo to identify immunological characteristics. [ Result and conclusion ] Cultural supernatants titer of hybridomas cells and ascites were 1：128 000 and 1：2 560 000 respectively; the subclasses of monoclonal antibody was IGgl ; the IC50 of standard curve was 0. 746 ng/mL, with other drugs of similar structure showing cross-reactivity affinities being less than 0. 015%.