几种单克隆抗体纯化方法的比较性研究
发布时间:2018-06-26 14:49
本文选题:单克隆抗体 + 纯化 ; 参考:《苏州大学》2005年硕士论文
【摘要】:单克隆抗体(mAb)的纯化是其理论研究和临床应用的基础,文献报道的纯化方法较多,包括各种沉淀法、离子交换层析法(IEX)、固定化金属螯合亲和层析法(IMAC)、凝胶层析法(GF)、羟基磷灰石层析法(CHT)、疏水作用层析法(HIC)和亲和层析(AC)法等,但各种方法的适用性及其规律至今尚需进一步研究。本文选择gp130、CD28和CD80三种鼠抗人mAb作为研究对象,采用IEX、IMAC、CHT、GF、AC、(NH_4)_2SO_4沉淀、辛酸沉淀等方法对上述三种小鼠腹水来源的mAb的纯化进行了比较性研究,在此基础上进一步从纯化后的抗体纯度、回收率和生物学活性等方面对多种不同纯化方法进行了分析,并优化了实验条件,从而建立了可获得高纯度、生物学活性好和纯化过程易于放大的mAb纯化方法,为其理论研究和临床应用提供了必要的实验基础。具体如下: 1.离子交换一步法快速纯化gP130激发型单克隆抗体B-S12的研究 采用中高压层析仪(Biologic Duo-Flow)和阴离子交换层析柱(Uno Q1或Bio-Scale Q5),建立了从小鼠腹水中纯化抗gp130 mAb B-S12的一步层析方法。 本论文分别研究了上样pH值和离子强度梯度洗脱等条件对mAb B-S12纯度的影响,结果表明:经预处理后的样品以Tris-HCl缓冲溶液(pH7.0,20mmol/l)稀释10倍后上Uno Q1柱,0-0.5mol/l NaCl 10柱体积(CV)梯度洗脱,可获得纯度大于95%的mAb B-S12,抗体回收率达66%,纯化后的抗体在体外对XG-2细胞有明显的促增殖作用。同时,采用Bio-Scale Q5层析柱对本方法的放大可行性进行了研究,结果令人满意。 在方法筛选、优化过程中,比较了阴离子交换一步层析法(AIEX)、阳离子交换一步层析法(CIEX)以及ProteinG AC法在纯度、回收率和生物学活性等方面的差异。结果表明:虽然CIEX一步层析法也能获得纯度较高的mAb B-S12(纯度90%),但纯度和回收率(52%)均不如AIEX一步法。而ProteinG AC法,虽然其纯化后的抗体纯度较高(90%),但其在体外对XG-2细胞的促增殖作用均低于
[Abstract]:The purification of monoclonal antibody (mAb) is the basis of its theoretical research and clinical application. There are many purification methods reported in the literature, including various precipitation methods, ion exchange chromatography (IEX), immobilized metal chelating affinity chromatography (IMAC), gel chromatography (GF), hydroxyl phosphite chromatography (CHT), hydrophobic interaction chromatography (HIC) and affinity chromatography (AC) method, etc. However, the applicability and regularity of various methods are still needed to be further studied. In this paper, three anti human mAb mice, gp130, CD28 and CD80, were selected as the research object. The purification of the three mouse ascites derived from IEX, IMAC, CHT, GF, AC, (NH_4) _2SO_4 precipitation, and octanoic acid precipitation were compared. The purity, recovery and biological activity of the purified antibody were analyzed, and the experimental conditions were optimized. The mAb purification method, which could obtain high purity, good biological activity and purify process, was established, which provided the necessary experimental basis for its theoretical research and clinical application. As follows:
Rapid purification of gP130 activated monoclonal antibody B-S12 by 1. ion exchange one-step method
A one-step chromatography method for purification of anti gp130 mAb B-S12 from mouse ascites was established by using Biologic Duo-Flow and anion exchange chromatography column (Uno Q1 or Bio-Scale Q5).
In this paper, the effects of pH value and ionic strength gradient elution on the purity of mAb B-S12 were studied. The results showed that the pre treated samples were diluted 10 times with Tris-HCl buffer solution (pH7.0,20mmol/l) and Uno Q1 column, 0-0.5mol/l NaCl 10 column volume (CV) gradient elution, and the purity of mAb B-S12 was greater than 95%, and the recovery rate of antibody was obtained. Up to 66%, the purified antibody could promote the proliferation of XG-2 cells in vitro. At the same time, the Bio-Scale Q5 chromatography column was used to study the feasibility of this method, and the results were satisfactory.
In the process of selection and optimization, the differences in purity, recovery and biological activity of the anion exchange one-step chromatography (AIEX), the cation exchange one-step chromatography (CIEX) and the ProteinG AC method were compared. The results showed that the purity and recovery of mAb B-S12 (purity 90%) could also be obtained by CIEX step chromatography, but the purity and recovery rate (5) were obtained. 2%) it is not as good as AIEX one step method, while ProteinG AC method, although its purified antibody has higher purity (90%), but its proliferation promoting effect on XG-2 cells is lower than in vitro.
【学位授予单位】:苏州大学
【学位级别】:硕士
【学位授予年份】:2005
【分类号】:R392
【引证文献】
相关硕士学位论文 前1条
1 魏晨曦;邻苯二甲酸二丁酯的酶联免疫吸附分析研究[D];华中师范大学;2011年
,本文编号:2070740
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