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目的建立抗巴比妥单克隆抗体杂交瘤细胞株,制备抗巴比妥单克隆抗体,并对其免疫学特性进行鉴定。方法将巴比妥分子环状丙二酰脲环上氨基引入活性羧基,然后通过缩合反应分别与匙孔血蓝蛋白(KLH)和牛血清白蛋白(BSA)偶联,得到完全抗原BAR-KLH和BAR-BSA,并用紫外分光光度计鉴定。用BAR-KLH免疫Balb/C小鼠,利用细胞融合技术建立稳定分泌抗巴比妥单克隆抗体的杂交瘤细胞株;采用体内诱生腹水法制备巴比妥单克隆抗体,饱和硫酸铵法、亲和层析法纯化,并进行免疫学特性鉴定。结果完全抗原偶联成功,其偶联率为30∶1;筛选出1株杂交瘤细胞命名为5C6,其产生的单抗效价为1∶6.4×104,属于IgG1/kappa,抗体亲和力常数为2.27×108L/moL。纯化后的巴比妥单克隆抗体用ELISA法检测其灵敏度为130ng/mL,并与其他7种镇静催眠类药物交叉反应率小于1%。结论本研究制备的杂交瘤细胞株5C6分泌的抗体具有高特异性和灵敏度。
OBJECTIVE To establish a hybridoma cell line against monoclonal antibody to barbitub and to prepare a monoclonal antibody against barbiturates and to identify its immunological properties. Methods The amino groups on the cyclic valinamide of barbiturates were introduced into activated carboxyl groups and then conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) through condensation reaction to obtain the complete antigen BAR-KLH and BAR-BSA and identified by UV spectrophotometer. Balb / C mice were immunized with BAR-KLH, and hybridoma cell lines stably secreting anti-barbiturate monoclonal antibodies were established by cell fusion technique. The in vivo induced ascites monoclonal antibody, saturated ammonium sulfate method, Purification by affinity chromatography and immunological characterization. Results The complete antigen conjugation was successful with a coupling ratio of 30:1. One hybridoma cell line was screened as 5C6 and its titer was 1: 6.4 × 104, which belonged to IgG1 / kappa. The antibody affinity constant was 2.27 × 108 L / moL. The purified monoclonal antibody to barbiturates showed the sensitivity of 130 ng / mL by ELISA and less than 1% cross-reaction with other sedative and hypnotic drugs. Conclusion The antibody secreted by hybridoma cell line 5C6 prepared in this study has high specificity and sensitivity.