莱克多巴胺单克隆抗体非亲和层析法纯化研究

Ractopamine monoclonal antibody purification based on non-affinity purification techniques

抗体纯化对免疫测定和生物偶联的灵敏度、重现性和稳定性具有重要影响。本文对硫酸铵沉淀法(Ammonium sulfate precipitation,AS)及AS分别与阴离子交换层析法(Anion exchange chromatography,AEC)、阳离子交换层析法(Cation exchange chromatography,CEC)、疏水作用层析法(Hydrophobic interaction chromatography,HIC)和陶瓷羟基磷灰石法(Ceramic hydroxyapatite,CHT)联合应用纯化腹水来源的莱克多巴胺单克隆抗体(Ractopamine monoclonal antibody,RAC-mAb)进行了研究。优化缓冲液pH、盐浓度及层析介质,分别采用Bradford法、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(Sodium dodecyl-sulfate polyacrylamide gel electrophoresis,SDS-PAGE)、间接竞争酶联免疫吸附试验(Indirect competitive enzyme-linked immunosorbent assay,icELISA)和胶体金免疫层析技术(Colloidal gold immunochromatography,CGIC)对抗体纯化后回收率、纯度及生物学活性进行评估。结果显示,采用AS+AEC法纯化抗体回收率高、纯度和生物学活性好,抗体回收率为40.1%,纯度为75.1%,经icELISA平行测定3次所得灵敏度(IC_(50))最佳为1.73±0.22 ng/mL,CGIC测定其在PBS及猪尿样本添加cut-off值分别为5 ng/mL和60 ng/mL。本研究为单克隆抗体纯化提供了理论参考和必要的实验基础。

Antibody purification is vitally important to the sensitivity,reproducibility and stability of immunoassays and bioconjugation.In this study,ammonium sulfate precipitation(AS),the combination between AS and anion exchange chromatography(AEC),cation exchange chromatography(CEC),hydrophobic interaction chromatography(HIC)or ceramic hydroxyapatite(CHT)were employed to purify the ractopamine monoclonal antibody(RAC-mAb)from ascites.Optimized the buffer pH,salt ion concentration and chromatography medium,the recovery,purity and biological activity of the antibodies after purification were evaluated by Bradford,sodium dodecyl-sulfate polyacrylamide gel electrophoresis(SDS-PAGE),indirect competitive enzyme-linked immunosorbent assay(icELISA)and colloidal gold immunochromatography(CGIC)methods,respectively.The results indicated that AS+AEC was most recommended because of its high recovery rate,good purity and biological activity,the recovery and purity of mAbs purified were 40.1%and 75.1%.The sensitivity(IC_(50))of the purified antibody by AS+AEC were 1.73±0.22 ng/mL,which were determined by icELISA(n=3);and the cut-off values in PBS and pig urine sample addition were 5 ng/mL and 60 ng/mL(AS+AEC),which were measured by CGIC.This study provided theoretical reference and necessary experimental basis for the purification of monoclonal antibodies.