中国医药生物技术
中國醫藥生物技術
중국의약생물기술
CHINESE MEDICINAL BIOTECHNOLOGY
2013年
6期
425-428
,共4页
肖增鸿%黄昭亮%林月霞%董斌%田素娟
肖增鴻%黃昭亮%林月霞%董斌%田素娟
초증홍%황소량%림월하%동빈%전소연
抗体,单克隆%分步沉淀%色谱法%离子交换
抗體,單剋隆%分步沉澱%色譜法%離子交換
항체,단극륭%분보침정%색보법%리자교환
Antibody,monoclonal%Fractional precipitation%Chromatography%ion exchange
目的：比较不同纯化方法对腹水型抗体的纯化效果，探索出最适合实验室抗体生产的纯化方法。方法制备的 IgG1单克隆抗体腹水（4-D10）分别利用两步硫酸铵沉淀法（AS 法）、辛酸-硫酸铵沉淀法（CA-AS法）、辛酸-硫酸铵沉淀法联合 DEAE 阴离子交换层析法与辛酸-硫酸铵沉淀法联合蛋白 G 亲和层析法纯化处理后，经蛋白纯度、回收率、抗体免疫活性鉴定分析后，比较不同的纯化方法对腹水型抗体的纯化效果。结果① AS 法的蛋白纯度为25%，低于 CA-AS 法（44%）；AS 法回收率为63%，高于 CA-AS 法（41.8%）；比较抗体效价表明，AS 法（1.024×106）与 CA-AS 法（1.024×106）两者相当。②粗提后纯化效果表明，蛋白 G亲和层析法回收率明显降低，DEAE 纯品虽纯度略低，但回收率较高，且效价（5.12×105）高于蛋白 G 亲和层析法（1.28×105）。结论 CA-AS 联合 DEAE 离子交换法是适合实验室生产单抗的低成本、纯化效果和回收效果较佳的方法。
目的：比較不同純化方法對腹水型抗體的純化效果，探索齣最適閤實驗室抗體生產的純化方法。方法製備的 IgG1單剋隆抗體腹水（4-D10）分彆利用兩步硫痠銨沉澱法（AS 法）、辛痠-硫痠銨沉澱法（CA-AS法）、辛痠-硫痠銨沉澱法聯閤 DEAE 陰離子交換層析法與辛痠-硫痠銨沉澱法聯閤蛋白 G 親和層析法純化處理後，經蛋白純度、迴收率、抗體免疫活性鑒定分析後，比較不同的純化方法對腹水型抗體的純化效果。結果① AS 法的蛋白純度為25%，低于 CA-AS 法（44%）；AS 法迴收率為63%，高于 CA-AS 法（41.8%）；比較抗體效價錶明，AS 法（1.024×106）與 CA-AS 法（1.024×106）兩者相噹。②粗提後純化效果錶明，蛋白 G親和層析法迴收率明顯降低，DEAE 純品雖純度略低，但迴收率較高，且效價（5.12×105）高于蛋白 G 親和層析法（1.28×105）。結論 CA-AS 聯閤 DEAE 離子交換法是適閤實驗室生產單抗的低成本、純化效果和迴收效果較佳的方法。
목적：비교불동순화방법대복수형항체적순화효과，탐색출최괄합실험실항체생산적순화방법。방법제비적 IgG1단극륭항체복수（4-D10）분별이용량보류산안침정법（AS 법）、신산-류산안침정법（CA-AS법）、신산-류산안침정법연합 DEAE 음리자교환층석법여신산-류산안침정법연합단백 G 친화층석법순화처리후，경단백순도、회수솔、항체면역활성감정분석후，비교불동적순화방법대복수형항체적순화효과。결과① AS 법적단백순도위25%，저우 CA-AS 법（44%）；AS 법회수솔위63%，고우 CA-AS 법（41.8%）；비교항체효개표명，AS 법（1.024×106）여 CA-AS 법（1.024×106）량자상당。②조제후순화효과표명，단백 G친화층석법회수솔명현강저，DEAE 순품수순도략저，단회수솔교고，차효개（5.12×105）고우단백 G 친화층석법（1.28×105）。결론 CA-AS 연합 DEAE 리자교환법시괄합실험실생산단항적저성본、순화효과화회수효과교가적방법。
Objective To compare different methods for purification of antibodies from mouse ascites fluid in order to explore the most suitable laboratory method for purifying antibody. Methods Ammonium sulfate precipitation (AS) and caprylic acid-ammonium sulfate precipitation (CA-AS) methods were used for purification of crude IgG1 antibodies, respectively. The crude samples were further purified by protein G chromatography and DEAE ion exchange chromatography, respectively. Results The purity of antibody purified by AS method was 25%, lower than that of 44%purified by CA-AS method. The antibody recovery by AS method was 63%, higher than that of the CA-AS method 41.8%. The antibody titers obtained by AS method and CA-AS method were almost the same, 1.024 × 106. Further antibody purification by protein G chromatography and DEAE chromatography were performed. The recovery of antibody by protein G chromatography was lower than that by DEAE chromatography. Conclusion CA-AS combined with DEAE chromatography method is the most suitable method for the small scale purification of monoclonal antibody from mouse ascites fluid due to its better recovery, higher purity and lower cost.
