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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。
OBJECTIVE: To prepare monoclonal antibody (mAb) against human apolipoprotein B100 (Apo B100) and to establish a sandwich ELISA for the detection of human Apo B100 double antibody. Methods: Mice were immunized with human Apo B100 antigen and the hybridoma cell lines were obtained by cell fusion and screening. The cell lines were expanded in serum-free medium and the supernatant was purified to obtain antibodies. The antibody affinity, subtype, specificity and epitope were determined. Finally, a sandwich ELISA method was established. Results: Four anti-human Apo B100 hybridoma cell lines (4-1-2, 4-2-2, 4-3-2 and 4-6-6) were obtained. The secreted antibodies did not cross other related proteins Reaction, affinity reached 1 × 109L / mol. The detection range of double antibody sandwich method with 4-3-2 and 4-6-3 was (1.3 ~ 80) ng / mL, the sensitivity was 1.24 ng / mL, the intra-assay CVs were less than 10% Less than 15%, the recovery rate of 90%. Conclusion: The anti-human Apo B100 mAb was successfully prepared and a double-antibody sandwich ELISA assay was established for the quantitative detection of human Apo B100, which laid the foundation for the detection of Apo B100 and the diagnosis of the disease.