發(fā)布時(shí)間：2018-06-26 14:49

  本文選題：單克隆抗體 + 純化　； 參考：《蘇州大學(xué)》2005年碩士論文

【摘要】：單克隆抗體(mAb)的純化是其理論研究和臨床應(yīng)用的基礎(chǔ),文獻(xiàn)報(bào)道的純化方法較多,包括各種沉淀法、離子交換層析法(IEX)、固定化金屬螯合親和層析法(IMAC)、凝膠層析法(GF)、羥基磷灰石層析法(CHT)、疏水作用層析法(HIC)和親和層析(AC)法等,但各種方法的適用性及其規(guī)律至今尚需進(jìn)一步研究。本文選擇gp130、CD28和CD80三種鼠抗人mAb作為研究對象,采用IEX、IMAC、CHT、GF、AC、(NH_4)_2SO_4沉淀、辛酸沉淀等方法對上述三種小鼠腹水來源的mAb的純化進(jìn)行了比較性研究,在此基礎(chǔ)上進(jìn)一步從純化后的抗體純度、回收率和生物學(xué)活性等方面對多種不同純化方法進(jìn)行了分析,并優(yōu)化了實(shí)驗(yàn)條件,從而建立了可獲得高純度、生物學(xué)活性好和純化過程易于放大的mAb純化方法,為其理論研究和臨床應(yīng)用提供了必要的實(shí)驗(yàn)基礎(chǔ)。具體如下: 1.離子交換一步法快速純化gP130激發(fā)型單克隆抗體B-S12的研究 采用中高壓層析儀(Biologic Duo-Flow)和陰離子交換層析柱(Uno Q1或Bio-Scale Q5),建立了從小鼠腹水中純化抗gp130 mAb B-S12的一步層析方法。 本論文分別研究了上樣pH值和離子強(qiáng)度梯度洗脫等條件對mAb B-S12純度的影響,結(jié)果表明:經(jīng)預(yù)處理后的樣品以Tris-HCl緩沖溶液(pH7.0,20mmol/l)稀釋10倍后上Uno Q1柱,0-0.5mol/l NaCl 10柱體積(CV)梯度洗脫,可獲得純度大于95%的mAb B-S12,抗體回收率達(dá)66%,純化后的抗體在體外對XG-2細(xì)胞有明顯的促增殖作用。同時(shí),采用Bio-Scale Q5層析柱對本方法的放大可行性進(jìn)行了研究,結(jié)果令人滿意。 在方法篩選、優(yōu)化過程中,比較了陰離子交換一步層析法(AIEX)、陽離子交換一步層析法(CIEX)以及ProteinG AC法在純度、回收率和生物學(xué)活性等方面的差異。結(jié)果表明:雖然CIEX一步層析法也能獲得純度較高的mAb B-S12(純度90%),但純度和回收率(52%)均不如AIEX一步法。而ProteinG AC法,雖然其純化后的抗體純度較高(90%),但其在體外對XG-2細(xì)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 魏晨曦;鄰苯二甲酸二丁酯的酶聯(lián)免疫吸附分析研究[D];華中師范大學(xué);2011年

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本文編號：2070740