双抗体夹心ELISA定量检测IFNβ-HSA融合蛋白分析方法的建立
摘要
β干扰素(IFNβ)是干扰素家族中的一员,具有抗病毒、抗增殖和免疫调节作用。雷楗勇等构建了人β干扰素与人血清白蛋白融合蛋白(IFNβ-HSA),将其在毕赤酵母系统中进行表达。这种融合的方法增大了蛋白药物的分子量,减少了异源蛋白的免疫原性,从而减少肾小球滤过率和体内清除率,有效提高了IFNβ在体内的半衰期,具有广阔的生产和临床应用前景。当前IFNβ-HSA融合蛋白的研究还处在发酵优化提高产量,摸索蛋白纯化工艺提高得率的研究阶段,并且很快将进入药代动力学和临床应用研究中。在这些研究中迫切需要一种能够从介质中区分IFNβ-HSA融合蛋白及其结构类似物、降解片段等,而定量检测IFNβ-HSA融合蛋白原型药物的分析方法。
目前蛋白多肽药物的检测分析方法主要有理化分析法、生物检定法和免疫分析法。酶联免疫吸附测定(ELISA)技术是1971年由Engvall和Perlman建立的一种生物活性物质微量测定技术,以其灵敏度高、特异性好等优点,在生命科学各领域得到广泛应用。本课题旨在建立一种双抗体夹心ELISA定量检测IFNβ-HSA融合蛋白原型药物的分析方法。
首先从雷楗勇博士构建的IFNβ-HSA融合蛋白表达菌株出发,通过发酵培养、蛋白纯化得到一定纯度的IFNβ-HSA融合蛋白作为抗原标准参考品。经过定量分析,抗原浓度为166μg/mL。选取了两株针对IFNβ-HSA融合蛋白两端多肽的单克隆抗体,建立了双抗体夹心ELISA定量检测方法,可以定量检测IFNβ-HSA融合蛋白原型药物。经过方法学考核。该方法的灵敏度为10 ng/mL,测量范围51.88 ng/mL～3320 ng/mL,不同时间不同批次对1660 ng/mL、415 ng/mL、103.75 ng/mL三个浓度点测定,批内变异系数为4.86±1.05%,批间变异系数8.03±1.08%。与IFNβ、IFNα、HSA、IFNα2b-HSA等IFNβ-HSA融合蛋白结构类似物基本无交叉反应。发酵液上清液中回收率分别为101.2±9.2%、101.9±8.0%、98.7±6.8%。稳定性试验表明有效期在3个月以上。
本方法可以应用到发酵优化和分离纯化研究中,用以跟踪测定IFNβ-HSA融合蛋白的诱导表达,测定纯化得率等。并且为后期药代动力学、临床研究提供了新思路和备选方法。
The interferon (IFN) is one member of the potent multifunctional cytokines family,which possesses a wide range of anti-viral, immunoregulatory and growth inhibitory properties. As a member of interferon family, interferonβhas been applied to treat many diseases, such as MS and hepatitis. To prolong the half-life of IFNβin vivo, Lei Jianyong developed a long-acting IFNβ(IFNβ-HSA) by albumin fusion technology. The fused gene IFNβ-HSA was expressed in pichia pastoris.The fusion protein IFNβ-HSA has broad application prospects in the future.
At present, the detection analysis of protein peptide drugs mainly adopt physicochemical analysis techniques, bioassay and immunoassay.Enzyme-linked immunosorbent assay (ELISA) , one of immunoassay, developed in 1971 by Engvall and Perlman, was widely used in various fields with its high sensitivity and specificity advantage. This subject’s aim was to establish a method for quantitative analysis of IFNβ-HSA fusion protein drug.
We developed a double antibody sandwich ELISA for quantitative analysis of recombinant fusion protein IFNβ-HSA by using a anti-IFNβmonoclonal antibody for capture and a HRP-labeled conjugate of another anti-HSA monoclonal antibody for detection antibody to construct a sandwich ELISA. The optimal concentration of the first coating antibody and detection antibody are both 2μg/mL. The standard protein curve regression equation was: y=0.248x-0.861; its linear detection was from 51.88 to 3320 ng/mL (R2=0.991). And recoveries ranged from 91.9 to 110.4% in typical matrixes, while the intra- and inter-assay precisions were 4.86% and 8.08% respectively. The sensitivity of the IFNβ-HSA ELISA with a limit of detection was 10 ng/mL.
A sensitive, repeatable double antibody sandwich ELISA is established for quantitative analysis of recombinant fusion protein IFNβ-HSA. It can be used in the research of fermentation , purification and clinical diagnosis.
目前蛋白多肽药物的检测分析方法主要有理化分析法、生物检定法和免疫分析法。酶联免疫吸附测定(ELISA)技术是1971年由Engvall和Perlman建立的一种生物活性物质微量测定技术,以其灵敏度高、特异性好等优点,在生命科学各领域得到广泛应用。本课题旨在建立一种双抗体夹心ELISA定量检测IFNβ-HSA融合蛋白原型药物的分析方法。
首先从雷楗勇博士构建的IFNβ-HSA融合蛋白表达菌株出发,通过发酵培养、蛋白纯化得到一定纯度的IFNβ-HSA融合蛋白作为抗原标准参考品。经过定量分析,抗原浓度为166μg/mL。选取了两株针对IFNβ-HSA融合蛋白两端多肽的单克隆抗体,建立了双抗体夹心ELISA定量检测方法,可以定量检测IFNβ-HSA融合蛋白原型药物。经过方法学考核。该方法的灵敏度为10 ng/mL,测量范围51.88 ng/mL～3320 ng/mL,不同时间不同批次对1660 ng/mL、415 ng/mL、103.75 ng/mL三个浓度点测定,批内变异系数为4.86±1.05%,批间变异系数8.03±1.08%。与IFNβ、IFNα、HSA、IFNα2b-HSA等IFNβ-HSA融合蛋白结构类似物基本无交叉反应。发酵液上清液中回收率分别为101.2±9.2%、101.9±8.0%、98.7±6.8%。稳定性试验表明有效期在3个月以上。
本方法可以应用到发酵优化和分离纯化研究中,用以跟踪测定IFNβ-HSA融合蛋白的诱导表达,测定纯化得率等。并且为后期药代动力学、临床研究提供了新思路和备选方法。
The interferon (IFN) is one member of the potent multifunctional cytokines family,which possesses a wide range of anti-viral, immunoregulatory and growth inhibitory properties. As a member of interferon family, interferonβhas been applied to treat many diseases, such as MS and hepatitis. To prolong the half-life of IFNβin vivo, Lei Jianyong developed a long-acting IFNβ(IFNβ-HSA) by albumin fusion technology. The fused gene IFNβ-HSA was expressed in pichia pastoris.The fusion protein IFNβ-HSA has broad application prospects in the future.
At present, the detection analysis of protein peptide drugs mainly adopt physicochemical analysis techniques, bioassay and immunoassay.Enzyme-linked immunosorbent assay (ELISA) , one of immunoassay, developed in 1971 by Engvall and Perlman, was widely used in various fields with its high sensitivity and specificity advantage. This subject’s aim was to establish a method for quantitative analysis of IFNβ-HSA fusion protein drug.
We developed a double antibody sandwich ELISA for quantitative analysis of recombinant fusion protein IFNβ-HSA by using a anti-IFNβmonoclonal antibody for capture and a HRP-labeled conjugate of another anti-HSA monoclonal antibody for detection antibody to construct a sandwich ELISA. The optimal concentration of the first coating antibody and detection antibody are both 2μg/mL. The standard protein curve regression equation was: y=0.248x-0.861; its linear detection was from 51.88 to 3320 ng/mL (R2=0.991). And recoveries ranged from 91.9 to 110.4% in typical matrixes, while the intra- and inter-assay precisions were 4.86% and 8.08% respectively. The sensitivity of the IFNβ-HSA ELISA with a limit of detection was 10 ng/mL.
A sensitive, repeatable double antibody sandwich ELISA is established for quantitative analysis of recombinant fusion protein IFNβ-HSA. It can be used in the research of fermentation , purification and clinical diagnosis.
引文
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[29]田旺,李正赫,杨文博等.绿色荧光蛋白α2b-干扰素融合蛋白的表达与活性研究[J].南开大学学报(自然科学版),2005,(5).
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[32]闫萍,杨琦,王惠珍.改良Lowry法和Bradford法测定蚯蚓提取物中蛋白质含量的比较[J].山西医科大学学报,2006,(1)
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[35]唐宇龙.检测结核分枝杆菌CFP10-ESAT6分泌蛋白的方法建立和应用研究[D].湖南农业大学:2007
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[54] Bhargava M,Kumar R.Estimation of ferritin by conventional ELISA & its avidin-biotin modification[J].India J Med Res.1986,84:445—447
[2] Isaacs A,Lindenmann J.Virus interference.I. The interferon.Proc. R.Soc.Lond.B, 1957, 147:258—67
[3]孙志宏,陈正礼.干扰素研究进展[J].延安大学学报:自然科学版,2000,19(1):76—79
[4]屈艾,汪承润.干扰素及其研究进展[J].徐州师范大学学报:自然科学版,2002,20(2): 57—60
[5] Ganes CS,Peter L. The Interferon System:A Bird's eye view of It's Biothecmistry [J], 1992, 267(8):5017—5020
[6]张惠展主编.基因工程概论[M].上海:华东理工大学出版社,1999.274—276.
[7]刘鸿丽,常明,哈斯.干扰素研究及应用进展[J].生物技术通讯,2004,15(5):506—508
[8]何诚.干扰素的分子生物学研究进展[J].国外医学:微生物学分册,1994,17(4):145—147,155
[9] Wills R J,Ferraiolo B L.The role of pharmacokinetics in the developnent of biotechnologically derivedagents[J].Clin Pharmacokinet,1992,23(6):406—414
[10]朱玉贤,李毅.现代分子生物学[M].北京:高等教育出版社,1985
[11] Ringenberg Q S,Anderson P C.Intrferons in the treatment of skin disease [J].Int Dennatol,1986,25(5):273
[12] Rissanen A,Myllyla G,cantell K,et al.Interferon therapy for ALS[J].Ann Nerrol, 1980,7(4):392
[13] Keeffe E B,Hollinger F B.Therapy of hepatitis ciconsensus interfon trials[J]. Hepatokogy, 1997,26:101
[14] Walsh G.Biopharmaceutical benchmark[J].Nature Biotechnology,2001,18:813
[15] Ruiz-Moreno M, Fernandez P, Leal A. Pilot interferon-βtrial in children with chronic hepatitis B who had previously not responed to interferonαtherapy[J]. Pediatrics, 1997, 99: 222—225
[16]郭涛.新编药物动力学[M].北京:中国科学技术出版社,2005,428
[17]杨彩铃,张海霞,刘满仓.胰岛素的色谱分析方法[J].分析科学学报,2006, 22(3): 349—354
[18]方春生,顾月清.蛋白多肽类药物体内动力学分析方法的研究新进展[J].药学进展, 2007,31(10):433—438
[19]宋清.定量分析中的误差和数据评价[M].北京:高等教育出版社,1984,22
[20]曾经泽,黄仁玉.药物分析方法的质量控制[J].中国药学杂志,1996,31(12):707
[21]盼秀荣.分析化学准确度的保证和评价[M].北京:计量出版社,1985,40
[22]汤仲明.生物技术药物药代动力学[M].北京:化学工业出版社,2004,20—21
[23] Engvall E,Perlmann P.Immunochemistry[M],1971,8(7):871
[24]焦奎.酶联免疫分析技术及应用[M].北京:北京化学工业出版社,2004
[25] Wrightt T G著.ELISA在临床检验中的应用.郑永晨等译.吉林:吉林科学技术出版社,1992
[26]陶义训.免疫学和免疫学检验[M].北京:人民卫生出版社,1988
[27]蒋成.酶免疫测定法[M].北京:人民卫生出版社,1984
[28]王燕,窦恒利,陆慧琦,韩焕兴.人源抗HBS-Fab-IFN-α融合蛋白制备和纯化[J].陕西医学杂志,2005,(11)
[29]田旺,李正赫,杨文博等.绿色荧光蛋白α2b-干扰素融合蛋白的表达与活性研究[J].南开大学学报(自然科学版),2005,(5).
[30]张龙翔,张庭芳,李令媛编.生化实验方法和技术[M].第二版.北京:高等教育出版社,1997,34-140
[31]程君生,毛开荣,丁家波等.微量Bradford法测定提纯禽结核菌素蛋白含量[J].中国兽药杂志,2007,(6)
[32]闫萍,杨琦,王惠珍.改良Lowry法和Bradford法测定蚯蚓提取物中蛋白质含量的比较[J].山西医科大学学报,2006,(1)
[33] Suter M,Butler JE.The immunochemistry of sandwich ELISAs II.A novel systerm prevents the denaturation of capture antibodies[J]. Immunol Lett.1986,13(6):313—316
[34]庞秋霞.抗AIBI单克隆抗体1A_2E_1和4B_9F_(12)的制备、鉴定及双抗体夹心ELISA试剂盒的构建[D].四川大学:2007
[35]唐宇龙.检测结核分枝杆菌CFP10-ESAT6分泌蛋白的方法建立和应用研究[D].湖南农业大学:2007
[36]李艳平.硫酸软骨素化学发光法的快速测定及其ELISA双抗体夹心法的建立[D].郑州大学:2007
[37]余玲.双单克隆抗体夹心ELISA检测IFN-α方案建立及临床应用的探讨[D].四川大学:2004
[38] Asturias, J. A., M. C. Arilla, et al.Quantification of profilins by a monoclonal antibody- based sandwich ELISA[J]. Journal of Immunological Methods. 1999, 229(1-2): 61—71
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