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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pH8.0,20mmol/LPB+0.5mol/LNaCl,抗体在pH5.0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pH8.0,20mmol/LPB+5mmol/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。
Monoclonal antibodies against HBV core antigen purified from mouse ascites were purified by metal chelate affinity chromatography, and the pH and ionic strength of the loading buffer, type of eluate and elution mode were optimized. The results showed that the optimum loading buffer was pH8.0, 20mmol / LPB + 0.5mol / LNaCl, the antibody was eluted at pH5.0, the recovery rate of the antibody was 80% and the purity was 85 %. When using imidazole concentration gradient elution, the optimal loading buffer was pH8.0, 20mmol / LPB and 5mmol / L imidazole. The purity of the antibody was more than 95% and the recovery was 65%. No NaCl was added in the loading buffer Add a small amount of imidazole, more conducive to antibody separation. The above elution methods can better maintain the biological activity of mAb, which provides the necessary experimental basis for the application of the antibody.