抗人Apo B_(100)单克隆抗体的制备及夹心ELISA检测方法的建立

Preparation of monoclonal antibodies against human Apo B_(100) and establishment of a sandwich ELISA system

目的:制备抗人载脂蛋白B100(Apo B100)单克隆抗体(mAb),建立人Apo B100双抗体夹心ELISA检测方法。方法:将人Apo B100抗原免疫小鼠,通过细胞融合、筛选后得到杂交瘤细胞株。将细胞株用无血清培养基扩大培养并纯化上清获得抗体,测定抗体亲和力、亚型、特异性及表位,最后建立双抗体夹心ELISA方法。结果:获得4株抗人Apo B100的杂交瘤细胞株(4-1-2、4-2-2、4-3-2、4-6-3),其分泌的抗体不与其他相关蛋白交叉反应,亲和力达到1×109L/mol。用4-3-2和4-6-3建立的双抗体夹心法的检测范围为(1.3～80)ng/mL,灵敏度1.24 ng/mL,批内变异系数均小于10%,批间变异系数均小于15%,回收率在90%以上。结论:成功制备了抗人Apo B100mAb,建立了定量检测人Apo B100的双抗体夹心ELISA方法,为Apo B100检测及疾病的诊断奠定基础。

AIM： To prepare the monoclonal antibodies （mAbs） against human Apo B100 and establish a doubleantibody sandwich ELISA system for human Apo B100 detection. METHODS： The BALB/c mice were immunized with human Apo B100 antigen. After cell fusion and screening, the hybridoma cells were cultured in serum-free medium and the supernatant was purified to obtain mAbs using protein A. The affinity, subtype, specificity and epitope of the mAbs were characterized and the sandwich ELISA system was established. RESULTS： Four hybridoma cell lines 4-1-2, 4-2-2, 4-3-2 and 4-6-3 were obtained. The affinity of the anti- Apo B100 mAbs was up to 1 × 10^9 L/mol and nearly had no cross reaction with other relevant proteins. Linear detection of the sandwich ELISA system using 4-3-2 and 4-6-3 covered a range from （1.3 -80） ng/mL, and its sensitivity was/.24 ng/rnL. The intra-assay coefficient of variation （CV） and inter-assay CV were less than 10% and 15%, respectively, and the recovery rate was above 90%. CONCLUSION： The mAbs against human Apo B100 have been prepared and a sandwich ELISA system for human Apo B100 detection has been established successfully, which provide a basis for human Apo B100 detection and disease diagnosis.