金属螯合亲和层析法纯化抗乙肝核心抗原单克隆抗体

Purification of anti-HBcAg monoclonal antibodies using immobilized metal ion affinity chromatography

摘要:

采用金属螯合亲和层析法, 纯化了小鼠腹水来源的抗乙肝核心抗原单克隆抗体, 对上样缓冲液的pH和离子强度、洗脱液种类和洗脱方式进行优化。结果表明, 采用降低pH分步洗脱时, 最佳上样缓冲液为pH 8.0, 20 mmol/L PB + 0.5 mol/L NaCl, 抗体在pH 5.0被洗脱下来, 抗体回收率80%, 纯度85%。采用咪唑浓度梯度洗脱时, 最佳的上样缓冲液为pH 8.0, 20 mmol/L PB + 5 mmol/L咪唑, 抗体纯度大于95%, 回收率65%; 在上样缓冲液中不添加NaCl而添加少量的咪唑, 更有利于抗体分离。以上洗脱方式都能较好地保持mAb的生物学活性, 为该抗体的应用提供了必要的实验基础。

Abstract:

Anti-HBcAg monoclonal antibodies from mouse ascites were purified by using immobilized metal ion affinity chromatography. We optimized the conditions of sample loading and elution. The results showed that when the pH stepwise elution was used, the best solution for sample loading was 20 mmol/L phosphate buffer containing 0.5 mol/L sodium chloride at pH 8.0 and the mAb was eluted at pH 5.0. The purity of obtained mAb was more than 85% and recovery reached 80%. When the adsorbed proteins were eluted by using gradient elution of an imidazole, the best solution for loading condition was 20 mmol/L phosphate buffer containing 5 mmol/L imidazole at pH 8.0. The purity and recovery of antibody were up to 95%.