巴比妥单克隆抗体制备及其免疫学特性鉴定

Production and characterization of specific and high-affinity monoclonal antibodies for barbital

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 the hybridoma cell line secreting highly specific monoclonal antibodies against barbital and identify its immunological characteristics. Methods An active carboxyl group was introduced to the barbital molecule at 1 or 3 position of heterocyclic stem nucleus, The barbital hapten then was conjugated to KLH and BSA by EDC method. The conjugations was evaluated by ultraviolet absorbtion. The monoclonal antibodies against barbital was made by immunization of Balb/C mice with BAR- KLH. The stable hybridoma cell line secreting anti-barbital monoclonal antibody was established by cell fusion technology. Purified monoclonal antibodies （McAbs） were obtained by saturated ammonium sulfate precipitation and affinity chromatography purification of the mice ascites. The sensitivity, affinity and specificity of the barbital monoclonal antibody have been characterized. Results The barbital hapten was successfully conjugated to KLH and BSA, and its coupling ratio was 30：1. The hybridoma cell line named as 5C6 secreting antibodies against barbital with the titration at 1：6.4 x 10^4. The monoclonal antibody subtype was confirmed as IgG1/kPa with affinity constant 2. 27 x 10^8L/tooL. Blocking ELISA method results showed the sensitivity of the purified anti-BAR McAb was 130ng/mL. There are less cross reaction for 5C6 to other sedatives （CR 〈 1.0）. Conclusion The anti-barbital monoclonal antibody from hybridoma cell line 5C6 was of high sensitivity and specificity against barbital.