血清抗體提取工藝及抗體凍干工藝的研究
發(fā)布時(shí)間：2018-10-19 13:14
【摘要】：抗體（Antibody）又稱免疫球蛋白，存在于人和動物血液（血清）、組織液及其他外分泌液中�？贵w是機(jī)體對抗原物質(zhì)產(chǎn)生免疫應(yīng)答的重要物質(zhì)，具有各種免疫功能，在疾病爆發(fā)時(shí)可通過被動免疫方式用于緊急治療。但是目前為止提取抗體的生產(chǎn)工藝依然是傳統(tǒng)的硫酸銨鹽析法，此方法由于所提抗體得率低、提取過程繁瑣致使抗體價(jià)格昂貴而得不到廣泛應(yīng)用。因此尋找新的提取工藝代替?zhèn)鹘y(tǒng)鹽析法來大量提取抗體、降低生產(chǎn)成本是解決抗體價(jià)格昂貴的根本方法。 為尋找適合提取血清中抗體的新工藝，，本研究以雙水相系統(tǒng)為對象，篩選并優(yōu)化出適合提取血清中抗體的雙水相系統(tǒng)。對雙水相系統(tǒng)提取血清中抗體的得率、純度及擴(kuò)大后的穩(wěn)定行進(jìn)行檢測。通過對比傳統(tǒng)工藝分析雙水相系統(tǒng)的優(yōu)越性，探討雙水相系統(tǒng)的應(yīng)用價(jià)值。最后對提取的抗體進(jìn)行凍干保存。根據(jù)抗體的特性篩選出適合抗體液凍干的最佳保護(hù)劑，將提取的抗體進(jìn)行凍干保存。方便了抗體的運(yùn)輸，保證了抗體的長期存放。本研究主要包括以下幾個(gè)方面： 1.萃取血清抗體的雙水相系統(tǒng)建立與優(yōu)化。首先對雙水相系統(tǒng)的組成成分進(jìn)行篩選，得出最佳聚合物和親液鹽種類。然后優(yōu)化雙水相系統(tǒng)萃取條件，主要包括鹽水相濃度及中性鹽的濃度、pH大小等條件的優(yōu)化。得出雙水相系統(tǒng)的組成為50%（w/w）PEG6000、20%（w/w）硫酸銨、pH=7.0、加入5%（w/w）氯化鈉后效果更佳。對雙水相系統(tǒng)與血清添加比例進(jìn)行篩選。得出雙水相系統(tǒng)與血清的最佳比例為聚合物水相體積：鹽水相體積：血清體積=3：3：1。 2.利用雙水相系統(tǒng)工業(yè)化生產(chǎn)血清中抗體。通過擴(kuò)大500倍(500mL)、10000(10000mL)倍，所提抗體得率及純度均不變，工藝穩(wěn)定性好。本研究還從生產(chǎn)成本、人力、物力、生產(chǎn)周期、所提抗體純度及得率等方面與目前用于工業(yè)生產(chǎn)的硫酸銨鹽析法進(jìn)行對比分析。此工藝可提高得率近1倍，且工藝簡單、成本低、節(jié)省時(shí)間、人力和物力。 3.抗體凍干條件的篩選。本研究對不同凍干保護(hù)劑以及保護(hù)劑的濃度進(jìn)行篩選，確定適合保護(hù)抗體液凍干的保護(hù)劑為蔗糖。為確保凍干后抗體的物理性狀良好且效價(jià)無降低，在反復(fù)篩選后得出抗體最佳凍干保護(hù)劑為5%（w/w）的蔗糖。根據(jù)篩選的凍干曲線加入適當(dāng)濃度保護(hù)劑凍干后抗體常溫放置一年后效價(jià)無影響。 雙水相系統(tǒng)的研究為血清中抗體的提取提供新工藝，為替換傳統(tǒng)工藝提供依據(jù)�？贵w凍干工藝的研究為抗體的保存與運(yùn)輸提供方法。也為今后抗體的廣泛應(yīng)用提供條件。
[Abstract]:Antibody (Antibody), also called immunoglobulin, is present in human and animal blood (serum), tissue fluid and other exocrine fluids. Antibody is an important substance that produces immune response to antigens. It has various immune functions and can be used in emergency treatment by passive immunization when the disease breaks out. However, the traditional ammonium sulfate salting-out method is still used in the production of the antibody, which is not widely used because of the low yield of the antibody and the complicated extraction process, which results in the high price of the antibody. Therefore, to find a new extraction process instead of the traditional salt-out method to extract a large number of antibodies and reduce the production cost is the fundamental method to solve the high price of antibodies. In order to find a new technology for extracting antibody from serum, the two-phase system was selected and optimized. The extraction rate, purity and expanded stability of antibody from serum were determined by aqueous two-phase system. By comparing the advantages of two-phase water system with traditional technology, the application value of two-phase water system is discussed. Finally, the extracted antibody was freeze-dried and preserved. According to the characteristics of antibody, the best protective agent for freeze-drying of antibody solution was selected, and the extracted antibody was freeze-dried and preserved. It facilitates the transport of antibodies and ensures the long-term storage of antibodies. This research mainly includes the following aspects: 1. Establishment and Optimization of Aqueous Two-phase system for extraction of Serum Antibodies. Firstly, the composition of aqueous two-phase system was screened, and the best polymer and hydrophilic salt were obtained. Then the extraction conditions of aqueous two-phase system were optimized, including the concentration of brine phase and neutral salt, the size of pH and so on. The results show that the composition of aqueous two-phase system is 50% (w / w) PEG6000,20% (w / w) ammonium sulfate, and the effect of adding 5% (w / w) sodium chloride to pH=7.0, is better. The ratio of two-phase water system to serum was screened. The results show that the optimum ratio of aqueous phase system to serum is polymer aqueous phase volume: brine phase volume: serum volume = 3: 3: 1.2. The antibody in serum was produced by using two-phase aqueous system. By expanding 500-fold (500mL) and 10000 (10000mL) times, the yield and purity of the antibody were unchanged, and the process stability was good. In this study, the production cost, manpower, material resources, production cycle, purity and yield of antibody were compared with ammonium sulfate salting-out method used in industrial production. This process can increase the yield by nearly 1 times, and the process is simple, low cost, save time, manpower and material resources. 3. Screening of freeze-drying conditions for antibodies. In this study, sucrose was chosen as the protective agent suitable for freeze-drying of protective antibody by screening different concentration of freeze-drying protectant and protective agent. In order to ensure that the physical properties of freeze-dried antibodies were good and their titers were not decreased, the optimum freeze-dried sucrose with 5% (w / w) sucrose was obtained after repeated screening. According to the selected freeze-drying curve, the titer of antibody was not affected after a year's storage at room temperature. The study of aqueous two-phase system provides a new process for the extraction of antibodies in serum and a basis for replacing the traditional process. The study of antibody freeze-drying technology provides a method for the preservation and transportation of antibody. It also provides conditions for the wide application of antibodies in the future.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.5
本文編號：2281224
[Abstract]:Antibody (Antibody), also called immunoglobulin, is present in human and animal blood (serum), tissue fluid and other exocrine fluids. Antibody is an important substance that produces immune response to antigens. It has various immune functions and can be used in emergency treatment by passive immunization when the disease breaks out. However, the traditional ammonium sulfate salting-out method is still used in the production of the antibody, which is not widely used because of the low yield of the antibody and the complicated extraction process, which results in the high price of the antibody. Therefore, to find a new extraction process instead of the traditional salt-out method to extract a large number of antibodies and reduce the production cost is the fundamental method to solve the high price of antibodies. In order to find a new technology for extracting antibody from serum, the two-phase system was selected and optimized. The extraction rate, purity and expanded stability of antibody from serum were determined by aqueous two-phase system. By comparing the advantages of two-phase water system with traditional technology, the application value of two-phase water system is discussed. Finally, the extracted antibody was freeze-dried and preserved. According to the characteristics of antibody, the best protective agent for freeze-drying of antibody solution was selected, and the extracted antibody was freeze-dried and preserved. It facilitates the transport of antibodies and ensures the long-term storage of antibodies. This research mainly includes the following aspects: 1. Establishment and Optimization of Aqueous Two-phase system for extraction of Serum Antibodies. Firstly, the composition of aqueous two-phase system was screened, and the best polymer and hydrophilic salt were obtained. Then the extraction conditions of aqueous two-phase system were optimized, including the concentration of brine phase and neutral salt, the size of pH and so on. The results show that the composition of aqueous two-phase system is 50% (w / w) PEG6000,20% (w / w) ammonium sulfate, and the effect of adding 5% (w / w) sodium chloride to pH=7.0, is better. The ratio of two-phase water system to serum was screened. The results show that the optimum ratio of aqueous phase system to serum is polymer aqueous phase volume: brine phase volume: serum volume = 3: 3: 1.2. The antibody in serum was produced by using two-phase aqueous system. By expanding 500-fold (500mL) and 10000 (10000mL) times, the yield and purity of the antibody were unchanged, and the process stability was good. In this study, the production cost, manpower, material resources, production cycle, purity and yield of antibody were compared with ammonium sulfate salting-out method used in industrial production. This process can increase the yield by nearly 1 times, and the process is simple, low cost, save time, manpower and material resources. 3. Screening of freeze-drying conditions for antibodies. In this study, sucrose was chosen as the protective agent suitable for freeze-drying of protective antibody by screening different concentration of freeze-drying protectant and protective agent. In order to ensure that the physical properties of freeze-dried antibodies were good and their titers were not decreased, the optimum freeze-dried sucrose with 5% (w / w) sucrose was obtained after repeated screening. According to the selected freeze-drying curve, the titer of antibody was not affected after a year's storage at room temperature. The study of aqueous two-phase system provides a new process for the extraction of antibodies in serum and a basis for replacing the traditional process. The study of antibody freeze-drying technology provides a method for the preservation and transportation of antibody. It also provides conditions for the wide application of antibodies in the future.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.5
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