环孢素A单克隆抗体的研制
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摘要
目的:研究环孢素A完全抗原的合成及单克隆抗体制备的方法。
方法:室温搅拌条件下,在脱水剂1-乙基-(3-二甲丙胺基)碳二亚胺盐酸盐(EDCI)的作用下,将环孢素A与4-苯甲酰苯甲酸的共轭物(CsA-BBa)与聚L-赖氨酸(PL)按质量比为3.4:1的比例进行反应。反应产物用截留量1万的透析膜透析分离、纯化。在190-300nm范围内对合成抗原进行紫外图谱扫描鉴定;用5μg/ml的抗原包被酶标板,酶联免疫吸附法(ELISA)进行抗原特异性鉴定。用完全抗原免疫七周龄雌性BALB/C小鼠, ELISA法检测免疫小鼠血清抗体效价。在50%聚乙二醇(PEG)的作用下,将高效价小鼠脾细胞与骨髓瘤细胞进行融合(脾细胞:骨髓瘤细胞=10:1)并接种于24 h前准备的饲养层细胞培养板中,HAT培养液对融合细胞进行选择性培养,用ELISA法对杂交瘤细胞进行筛选、鉴定。
结果:环孢素A完全抗原(CsA-BBa-PL)紫外吸收峰峰位209.5nm,较合成原料物质CsA-BBa(212nm)及PL(203nm)发生了明显迁移,证实了产物的生成;环孢素A完全抗原ELISA特异性检测结果与阴性对照的比值为23.22。免疫BALB/C小鼠血清效价值为:3.35,3.43,3.37,3.31,3.38,空白对照、阴性对照、阳性对照值依次为0.080、0.42、3.43。免疫小鼠血清效价测定结果明显大于空白对照及阴性对照值,并与阳性对照值接近。所筛选的杂交瘤细胞能够稳定分泌环孢素A单克隆抗体。
结论:该实验方法能够有效的合成环孢素A完全抗原;合成抗原能够刺激BALB/C小鼠机体产生免疫应答反应并产生相应环孢素A抗体;实验所采用杂交瘤技术能够成功的制备环孢素A单克隆抗体。
OBJECTIVE: To study the methodology of immunogen synthesis and the monoclonal antibody preparation of Cyclosporine A.
METHODS: With the stirring at room temperature and dehydrolyzing agent 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide (EDCI ), the antigen of cyclosporine A(CsA-BBa-PL) was synthesized through the reaction between the conjugate of cyclosporine A and 4-benzoulbenzoic (CsA-BBa) and poly-L-lysine(PL), the weight proportion was 3.4 to 1. The products were isolated and purified by dialysis, the specification of the dialysis-membrane was 10,000. It was identified by UV spectra within 190 to 300 nm. The specificity of the antigen of CsA was identified by enzyme-linked immunosorbent assay (ELISA), the concentration of antigen used for the identification was 5μg/ml. The female BALB/C mice, seven weeks old, were immunized with the antigen of cyclosporine A, the blood serum potencies were measured by indirect enzyme-linked immunosorbent assay. With the help of fusogenic agent polyethylene glycol (PEG), the concentration of which is 50%, the spleen cells, which had higher blood serum potencies, were fused with SP2/0 myeloma cells (the ratio is ten to one). Then the fused cells were cultured in the cell culture plates which had owned feeder layer cells for 24 hours. The selected solution HAT was used to culture the fusion cells so as to select hybridoma cells. The hybridoma cells was selected and identified by ELISA.
RESULTS: The UV spectra peaks of CsA-BBa and PL and the antigen of CsA were 212nm, 203nm, 209.5nm respectively, compared to the materials used to synthesize antigen of cyclosporine A, the UV spectra of the antigen changed obviously, which proved that the conjugate was formed through the reaction. The positive to negative ratio of the specificity identification was 23.22. The blood serum potencies of the immune mice were 3.35, 3.43, 3.37, 3.31, and 3.38 respectively. The blank control and negative control and positive control were 0.080, 0.42 and 3.43 respectively. The potencies of the mice were obviously higher than the blank control and negative control, which were close to the positive control. The selected hybridoma cells were able to secrete monoclonal antibodies of cyclosporine A stably. CONCLUSIONS: The antigen of cyclosporine A was able to synthetized by this reaction. The antigen was able to induce the immunological reaction of mice and made the body form the antibodies accordingly. The hybridoma technique was able to prepare monoclonal antibody of Cyclosporine A successfully.
方法:室温搅拌条件下,在脱水剂1-乙基-(3-二甲丙胺基)碳二亚胺盐酸盐(EDCI)的作用下,将环孢素A与4-苯甲酰苯甲酸的共轭物(CsA-BBa)与聚L-赖氨酸(PL)按质量比为3.4:1的比例进行反应。反应产物用截留量1万的透析膜透析分离、纯化。在190-300nm范围内对合成抗原进行紫外图谱扫描鉴定;用5μg/ml的抗原包被酶标板,酶联免疫吸附法(ELISA)进行抗原特异性鉴定。用完全抗原免疫七周龄雌性BALB/C小鼠, ELISA法检测免疫小鼠血清抗体效价。在50%聚乙二醇(PEG)的作用下,将高效价小鼠脾细胞与骨髓瘤细胞进行融合(脾细胞:骨髓瘤细胞=10:1)并接种于24 h前准备的饲养层细胞培养板中,HAT培养液对融合细胞进行选择性培养,用ELISA法对杂交瘤细胞进行筛选、鉴定。
结果:环孢素A完全抗原(CsA-BBa-PL)紫外吸收峰峰位209.5nm,较合成原料物质CsA-BBa(212nm)及PL(203nm)发生了明显迁移,证实了产物的生成;环孢素A完全抗原ELISA特异性检测结果与阴性对照的比值为23.22。免疫BALB/C小鼠血清效价值为:3.35,3.43,3.37,3.31,3.38,空白对照、阴性对照、阳性对照值依次为0.080、0.42、3.43。免疫小鼠血清效价测定结果明显大于空白对照及阴性对照值,并与阳性对照值接近。所筛选的杂交瘤细胞能够稳定分泌环孢素A单克隆抗体。
结论:该实验方法能够有效的合成环孢素A完全抗原;合成抗原能够刺激BALB/C小鼠机体产生免疫应答反应并产生相应环孢素A抗体;实验所采用杂交瘤技术能够成功的制备环孢素A单克隆抗体。
OBJECTIVE: To study the methodology of immunogen synthesis and the monoclonal antibody preparation of Cyclosporine A.
METHODS: With the stirring at room temperature and dehydrolyzing agent 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide (EDCI ), the antigen of cyclosporine A(CsA-BBa-PL) was synthesized through the reaction between the conjugate of cyclosporine A and 4-benzoulbenzoic (CsA-BBa) and poly-L-lysine(PL), the weight proportion was 3.4 to 1. The products were isolated and purified by dialysis, the specification of the dialysis-membrane was 10,000. It was identified by UV spectra within 190 to 300 nm. The specificity of the antigen of CsA was identified by enzyme-linked immunosorbent assay (ELISA), the concentration of antigen used for the identification was 5μg/ml. The female BALB/C mice, seven weeks old, were immunized with the antigen of cyclosporine A, the blood serum potencies were measured by indirect enzyme-linked immunosorbent assay. With the help of fusogenic agent polyethylene glycol (PEG), the concentration of which is 50%, the spleen cells, which had higher blood serum potencies, were fused with SP2/0 myeloma cells (the ratio is ten to one). Then the fused cells were cultured in the cell culture plates which had owned feeder layer cells for 24 hours. The selected solution HAT was used to culture the fusion cells so as to select hybridoma cells. The hybridoma cells was selected and identified by ELISA.
RESULTS: The UV spectra peaks of CsA-BBa and PL and the antigen of CsA were 212nm, 203nm, 209.5nm respectively, compared to the materials used to synthesize antigen of cyclosporine A, the UV spectra of the antigen changed obviously, which proved that the conjugate was formed through the reaction. The positive to negative ratio of the specificity identification was 23.22. The blood serum potencies of the immune mice were 3.35, 3.43, 3.37, 3.31, and 3.38 respectively. The blank control and negative control and positive control were 0.080, 0.42 and 3.43 respectively. The potencies of the mice were obviously higher than the blank control and negative control, which were close to the positive control. The selected hybridoma cells were able to secrete monoclonal antibodies of cyclosporine A stably. CONCLUSIONS: The antigen of cyclosporine A was able to synthetized by this reaction. The antigen was able to induce the immunological reaction of mice and made the body form the antibodies accordingly. The hybridoma technique was able to prepare monoclonal antibody of Cyclosporine A successfully.
引文
[1] Abecassis M, Bartlett ST, Collins AJ,et al. Kidney Transplantation as Primary Therapy for End-Stage Renal Disease: A National Kidney Foundation/Kidney Disease Outcomes Quality Initiative (NKF/KDOQITM) Conference[J]. Clin J Am Soc Nephrol,2008,3(2):471-480.
[2] Jiang Y, Lv LZ, Cai QC,et al. Liver transplant for 70 patients with end-stage liver diseases[J]. Hepatobiliary Pancreat Dis Int,2007,6(1):24-28.
[3] Terasaki PI, Cecka JM, Gjertson DW,et al. Risk rate and long-term kidney transplant survival[J]. Clin Transpl,1996,443-458.
[4] Malaise J, De Roover A, Squifflet JP,et al. Immunosuppression in pancreas transplantation: the Euro SPK trials and beyond[J]. Acta Chir Belg,2008,108(6):673-678.
[5]唐振乡,魏建英.环孢霉素A的临床应用于安全性分析[J].现代中西医结合杂志,2008,17(35):5553-5556.
[6] Ready A. Experience with cyclosporine[J]. Transplant Proc, 2004,36(2 Suppl):135S-138S.
[7]朱斌.肾移植受者环孢素A血药浓度监测的回顾分析[J].现代医院,2003,3(4):20-22.
[8]胡婷婷,姜云平,陆国庆等.环孢霉素制剂和血药浓度监测的临床意义[J].西南军医,2005,7(6):61-63.
[9] Kandaswamy R, Humar A, Casingal V,et al. Stable kidney function in the second decade after kidney transplantation while on cyclosporine-based immunosuppression[J]. Transplantation,2007,83(6):722-726.
[10] Diana Faren L , Goa PB.Cyclosporin A review of itspharmacodynamic and phamacokinetic properties and the rapeutic use immunoregulatory disorders[J].Drugs Evaalustion,1993,45(6):953-1040.
[11]沈景芬.环孢素A血药浓度监测方法的研究进展[J].安徽医药,2008,13(1):83-86.
[12]沈景芬.环孢素A血药浓度监测方法的研究进展[J].安徽医药, 2009,13(1):83-85.
[13]杨薇,顾健,李玉.荧光偏振免疫法在环孢素A血药浓度监测中的质控评价[J].中国医院药学杂志,2005,25(9):820-822.
[14]储小曼.治疗药物监测内部质量控制评价[J].中国医院药学杂志,2005,25(3):265-267.
[15] Zucchelli GC, Pilo A, Clerico A,et al. The TDx assay for cyclosporine and its metabolites in blood samples compared with HPLC and RIA methods [J]. Drugs Exp Clin Res, 1989,15(4):185-188.
[16]李月越,陈杭薇,王萍,等.胶体金免疫层析法快速检测流感病毒应用研究[J].第四军医大学学报,2008,29(18):1652-1654.
[17]李欣,赵立子,黄民.治疗药物监测概况及研究进展[J].现代食品与药品杂志, 2006,16(5):60-64.
[18] Kohler G,Milstein C.Continuous culture of fused cells secreting antibody of predefined specificity[J].Nature,1975,256(5517):495-497.
[19] Anthony V. Recent Developments in Polymer Research[M]. Nova Publishers,2007,201-203.
[20] Cavallaro G, Palumbo FS, Licciardi M,et al. Novel cationic copolymers of a polyasparthylhydrazide: synthesis and characterization[J]. Drug Deliv,2005,12(6):377-384.
[21] Davis LE,Roth SA,Anderson B. Antisera specificities to 1-ethyl-3-(3-dimethylam- inopropyl) carbodiimide adducts of proteins[J]. Immunology, 1984,53(3):435-441.
[22] Beuvery EC, Speijers GJ, Lutz BI,et al. Analytical, toxicological and immunological consequences of the use of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide as coupling reagent for the preparation of meningococcal group C polysaccharide-tetanus toxoid conjugate as vaccine for human use[J]. Dev Biol Stand, 1986,63:117-28.
[23]郑劼，张焜,赵肃清.双酚A人工抗原的光谱表征及免疫鉴定研究[J].光谱学与光谱分析,2008,28(7):1583-1586.
[24]俎晶晶,屈凌波,孙武勇.甲硝唑人工抗原的制备及其EUSA检测[J].动物医学进展,2007,28(1):11-13.
[25] Wang Y,Wei D,Yang H,et al. Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples[J]. Talanta,2008,77(5):1783-1789.
[26]李玉珍,林亲录,肖怀秋.酶联免疫吸附技术及其在食品安全检测中的应用研究进展[J].中国食品添加剂,2006(3):108-l1O.
[27]余冰宾.生物化学实验指导[M].清华大学出版社,2004,120-122.
[28] Tang L, Zheng L, Qi CJ, Ma HB,et al. Preparation of a novel functional anti-human CD40 monoclonal antibody and identification of its bioactivity[J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2007,23(9):841-843.
[29]潘赛贻周洁娴黄健康.华支睾吸虫可溶性抗原对BALB/C鼠免疫效果的研究[J].广东卫生防疫,1998,24(3):1-3.
[30] Donald J.The basis of toxicity testing[M]. CRC Press,1977,168.
[31] Bantroch S, Buhler T, and Lam JS. Appropriate coating methods and other conditions for enzyme-linked immunosorbent assay of smooth, rough, and neutral lipopolysaccharides of pseudomonas aeruginosa[J]. Clin Diag Lab Immunol,1994,1(1):55-62.
[32] Fuller SA, Takahashi M, Hurrell JG. Preparation of mouse feeder cells for fusion and cloning[M]. Curr Protoc Mol Biol, 2001, Chapter 11:Unit11.6.
[33]徐志凯.实用单克隆抗体技术[M].陕西科学技术出版社,2006:1-29.
[34] Xuelin Zhou, Hu Zhao, Xiangdong Gao. Preparation and Characterization of Monoclonal Antibodies Against a Polysaccharide from Flammulina velutipes Mycelium[J]. Hybridoma, 2008, 27(6): 439-444.
[35] Zhao PC, Yuan DW, Zhu R,et al. Preparation of monoclonal antibody against sulfadiazine and development of an ELISA kit[J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2004,20(2):191-194.
[36] Yang T, Zhong P, Qu L, et al. Preparation and identification of anti-2, 4-dinitroph- enyl monoclonal antibodies[J].J Immunol Methods 2006,313(1–2):20-28.
[37] Su LJ, Xiao L, Zhan T, Chen L,et al. Preparation and identification of monoclonal antibodies against snake venom C-type lectin like protein Agkisacutacin[J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2007,23(11):1031-1033.
[38]张彩,田志刚.人NK细胞克隆化培养条件的初步探讨[J].细胞生物学杂志,2001,107-109.
[39]李刚,赵静,王保民,等.甘草有效成分甘草酸人工抗原的光谱鉴定研究[J].Special Wild Economic Animal and Plant Research,2005,27(2):17-18.
[40]赵肃清,蔡燕飞,雷红涛,等.农药甲胺磷人工抗原的光谱鉴定研究[J].光谱学与光谱分析,2004,24(2):207.
[41] Maria-Pilar Marco,Shirley Gee,Bruce D.Hammock,Immunochemical Techniques for Environmental Ana1ysis II.Antibody Production and immunoassay Development. Trends in An alytical Chemistry,1995,14(8):415.
[42]刘辉.免疫学与免疫学检验[M].人民军医出版社,2006,55-56.
[43]贲亚琍,朱德锐,刘德立.农药人工抗原合成方法的设计与优选[J].农药,2008,47(1):10-14.
[44]王兰兰.临床免疫学和免疫学检验[M].人民卫生出版社,2004,23-24.
[45]刘一超,戴晓雁,黄文才等.环孢菌素A人工抗原的制备[J] .四川大学学报,2001,38(3):407-410.
[46] Goka AK,Farthing MJ. The use of 3,3',5,5'-tetramethylbenzidine as a peroxidase substrate in microplate enzyme-linked immunosorbent assay[J]. J Immunoassay,1987,8(1):29-41.
[47]陈玉社,杨明峰,公丕欣,等.实验性变态反应性脑脊髓炎模型[J].泰山医学院学报,2006,27(1):72-74.
[48] Yu CY, Whitacre CC. Sex, MHC and complement C4 in autoimmune diseases[J]. Trends Immunol, 2004, 25(12): 694-699.
[49]楚电峰,杜元钊.免疫佐剂的应用与研究进展[J].中国动物检疫,2007,24(10):43-44.
[50] Witte P L,Ber R.Improved efficiency of hybridoma ascites production by intrasplenic inoculation in mice[J]. 1983,70(3):575-577.
[1] Kohler G,Milstein C.Continuous culture of fused cells secreting antibody of predefined specificity[J].Nature,1975,256(5517):495-497.
[2] M ike Clark.Antibody humanization:a case of the‘Emperor’S new clothes’[J]. Special issue of Immunology Today, 2000,21(8):394-402.
[3] Hughes D.Therapeutic antibodies make a comeback[J].Drug Discovery Today,1998,3(10):439-442.
[4]罗萍,邹全明,毛旭虎.治疗性单克隆抗体研究进展及临床应用现状[J].第三军医大学学报,2004,26(5):454-456.
[5] Presta LG. Engineering of therapeutic antibodies to minimize immunogenicity and optimize function[J].Adv Drug Deliv Rev,2006,58(5-6):640-656.
[6] Qian W, Wang L, Li B, et al. Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity[J]. Biochem Biophys Res Commun,2008, 367(2):497-502.
[7] Carter P. Improving the efficacy of antibody—based cancer therapies[J].Nat Rev Cancer,2001,2(11):118-129.
[8] Gabriel HM, Oliveira EI. Role of abciximab in the treatment of coronary artery disease[J]. Expert Opin Biol Ther,2006,6(9):935-942.
[9]葛彦.人源化抗体研制策略分析及应用研究[J].国外医学免疫学分册,2004,27(5):271-274.
[10] Ishida T, Tmai K. The expression technology of chimeric and humanized antibodies[J]. Nippon Rinsho,2002,60(3):439-444.
[11] Riechmann L,Clark M,Waldmann H,et a1.Reshaping human antibodies for therapy[J].Nature,1988,332(6162):323-327.
[12]王秀红,周素芳.基因工程抗体研究进展[J].生物技术通讯,2007,18(2):304-306.
[13] Caldas C, Coelho V, Kalil J, et al. Humanizaion of the anti-18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen[J]. Mol Immunol, 2003,39(15):941-952.
[14] Nakanishi T,Tsumoto K,Yokota A,et al. Critical contribution of VH-VL interaction to reshaping of an antibody: the case of humanization of anti-lysozyme antibody, HyHEL-10[J]. Protein Sci,2008,17(2):261-270.
[15]朱啸林,程枫,钱关祥.基于互补决定区移植的单抗人源化进展[J].上海交通大学学报:医学版,2007,27(11):1382-1384.
[16] Wu H, Nie Y, Huse W D, et al. Hummanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues[J]. J Mol Biol, 1999, 294:151-162.
[17]贾建桃,马丽菊,王秦秦.治疗性单克隆抗体研究新进展[J].医学综述,2007,13(5):325-327.
[18]彭如意,王捷.人源化治疗性单克隆抗体的研究进展[J].实用医学杂志,2007,23(5):753-754.
[19] Lonberg N. Fully human antibodies from transgenic mouse and phage display platforms[J]. Curr Opin Immunol,2008,20(4):450-459.
[20]刘松友,王长太.单克隆抗体治疗制剂研究与应用[J].国外医学(预防、诊断、治疗用生物制品分册),2005,28(4):177-179.
[21]陈立慧,宋海峰,刘秀文.治疗性人源化单克隆抗体研究进展[J].中国新药杂志,2004,13(7) :590-594.
[22]吴永强,董关木.人源化单克隆抗体研究进展[J].微生物学免疫学进展,2008,36(2):73-77.
[23] Lonberg N. Human monoclonal antibodies from transgenic mice[J]. Handb Exp Pharmacol,2008,181:69-97.
[24] Foon KA, Yang XD, Weiner LM,et al. Preclinical and clinical evaluations of ABX-EGF,a fully human anti-epidermal growth factor receptor antibody[J].Int J Radiat Oncol Biol Phys,2004,58(3):984-990.
[25] Willemsen R,Chames P,Schooten E,et al. Selection of human antibody fragmentsdirected against tumor T-cell epitopes for adoptive T-cell therapy[J]. Cytometry A, 2008,73(11):1093-1099.
[26]杜阳峰,罗荣城,李贵平,等.抗乙型肝炎表面抗原Fab片段联合抗细胞核单抗为载体的肝癌放射免疫治疗实验研究[J].南方医科大学学报,2008,28(3):460-462.
[27] Gafner V,Trachsel E,Neri D. An engineered antibody-interleukin-12 fusion protein with enhanced tumor vascular targeting proteins[J]. Int J Cancer,2006,119(9):2205-2212.
[28]龚明霞.基因工程抗体的研究[J].医学综述,2003,9(2) :69-71.
[29]卢筱华,杨冬华,汤绍辉,等.错配PCR和DNA改组技术提高抗肝癌单链抗体亲和力[J].中国病理生理杂志,2006,22(6):1203-1206.
[30]吴小平,间锡蕴,杨东玲,等.人单链抗体亲和力成熟研究[J].中国病理生理杂志,2001,21(2):156-159.
[31]崔华清,王清明.基于重链抗体构建的单域抗体研究进展[J].生物工程学报,2005,21:497-501.
[32] Caldas C, Coelho V, Kalil J, et al. Humannization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen[J]. Mol Immunol,2003,39(15):941-952.
[33] Müller D, Kontermann RE. Recombinant bispecific antibodies for cellular cancer immunotherapy[J]. Curr Opin Mol Ther,2007,9(4):319-26.
[34] Asano R, Kawaguchi H, Watanabe Y,et al. Diabody-based recombinant formats of humanized IgG-like bispecific antibody with effective retargeting of lymphocytes to tumor cells[J]. J Immunother,2008,31(8):752-761.
[35]刘辉.免疫学与免疫学检验[M].北京:人民军医出版社,2006,69.
[36]戴克胜.双特异性单链抗体[J].国外医学免疫学分册,2003,26(3) :123-125.
[37]沈备奋.治疗性抗体的改造[J]. Med Mol Biol, 2006,3(4) :245-249.
[38]胡晓林,张春明.基因工程抗体亲和力成熟的策略[J].免疫学杂志,2006,22(6): 702-704.
[39] Chowdhury P S. Engineering hot spots for affinity enchancement of antibodies[J].Methods Mol Biol, 2003, 207:179-196.
[40] Puja s, Rhona S. Anti-CD74 antibody doxorubicin conjugate, IMMU-11O,in a human multiple myelona xenograft and in monkeys[J].Clinical Cancer Research,2005,11:5257-5264.
[41]朱洁,王绮如.单克隆抗体药物在肿瘤靶向治疗中的应用与展望[J].中国医师杂志,2007,9(7):1006-1007.
[42]张寒,邹丽敏,郑胡镛.急性淋巴细胞白血病最新治疗策略(中)[J].中国小儿血液与肿瘤杂志,2007,12(5):233-235.
[43] Walsh G. Biopharmaceutical bienchmarks-2003[J]. Nat Biotechnol, 2003,21(8):865-870.
[44]刘兢.生物技术和药物研究进展[J].中国科学技术大学出版社,2008,38(8):905-908.
[45] Laffly E, Sodoyer R. Monoclonal antibodies, 30 years of success[J] .J Soc Biol,2006,200(4):325-343.
[2] Jiang Y, Lv LZ, Cai QC,et al. Liver transplant for 70 patients with end-stage liver diseases[J]. Hepatobiliary Pancreat Dis Int,2007,6(1):24-28.
[3] Terasaki PI, Cecka JM, Gjertson DW,et al. Risk rate and long-term kidney transplant survival[J]. Clin Transpl,1996,443-458.
[4] Malaise J, De Roover A, Squifflet JP,et al. Immunosuppression in pancreas transplantation: the Euro SPK trials and beyond[J]. Acta Chir Belg,2008,108(6):673-678.
[5]唐振乡,魏建英.环孢霉素A的临床应用于安全性分析[J].现代中西医结合杂志,2008,17(35):5553-5556.
[6] Ready A. Experience with cyclosporine[J]. Transplant Proc, 2004,36(2 Suppl):135S-138S.
[7]朱斌.肾移植受者环孢素A血药浓度监测的回顾分析[J].现代医院,2003,3(4):20-22.
[8]胡婷婷,姜云平,陆国庆等.环孢霉素制剂和血药浓度监测的临床意义[J].西南军医,2005,7(6):61-63.
[9] Kandaswamy R, Humar A, Casingal V,et al. Stable kidney function in the second decade after kidney transplantation while on cyclosporine-based immunosuppression[J]. Transplantation,2007,83(6):722-726.
[10] Diana Faren L , Goa PB.Cyclosporin A review of itspharmacodynamic and phamacokinetic properties and the rapeutic use immunoregulatory disorders[J].Drugs Evaalustion,1993,45(6):953-1040.
[11]沈景芬.环孢素A血药浓度监测方法的研究进展[J].安徽医药,2008,13(1):83-86.
[12]沈景芬.环孢素A血药浓度监测方法的研究进展[J].安徽医药, 2009,13(1):83-85.
[13]杨薇,顾健,李玉.荧光偏振免疫法在环孢素A血药浓度监测中的质控评价[J].中国医院药学杂志,2005,25(9):820-822.
[14]储小曼.治疗药物监测内部质量控制评价[J].中国医院药学杂志,2005,25(3):265-267.
[15] Zucchelli GC, Pilo A, Clerico A,et al. The TDx assay for cyclosporine and its metabolites in blood samples compared with HPLC and RIA methods [J]. Drugs Exp Clin Res, 1989,15(4):185-188.
[16]李月越,陈杭薇,王萍,等.胶体金免疫层析法快速检测流感病毒应用研究[J].第四军医大学学报,2008,29(18):1652-1654.
[17]李欣,赵立子,黄民.治疗药物监测概况及研究进展[J].现代食品与药品杂志, 2006,16(5):60-64.
[18] Kohler G,Milstein C.Continuous culture of fused cells secreting antibody of predefined specificity[J].Nature,1975,256(5517):495-497.
[19] Anthony V. Recent Developments in Polymer Research[M]. Nova Publishers,2007,201-203.
[20] Cavallaro G, Palumbo FS, Licciardi M,et al. Novel cationic copolymers of a polyasparthylhydrazide: synthesis and characterization[J]. Drug Deliv,2005,12(6):377-384.
[21] Davis LE,Roth SA,Anderson B. Antisera specificities to 1-ethyl-3-(3-dimethylam- inopropyl) carbodiimide adducts of proteins[J]. Immunology, 1984,53(3):435-441.
[22] Beuvery EC, Speijers GJ, Lutz BI,et al. Analytical, toxicological and immunological consequences of the use of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide as coupling reagent for the preparation of meningococcal group C polysaccharide-tetanus toxoid conjugate as vaccine for human use[J]. Dev Biol Stand, 1986,63:117-28.
[23]郑劼，张焜,赵肃清.双酚A人工抗原的光谱表征及免疫鉴定研究[J].光谱学与光谱分析,2008,28(7):1583-1586.
[24]俎晶晶,屈凌波,孙武勇.甲硝唑人工抗原的制备及其EUSA检测[J].动物医学进展,2007,28(1):11-13.
[25] Wang Y,Wei D,Yang H,et al. Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples[J]. Talanta,2008,77(5):1783-1789.
[26]李玉珍,林亲录,肖怀秋.酶联免疫吸附技术及其在食品安全检测中的应用研究进展[J].中国食品添加剂,2006(3):108-l1O.
[27]余冰宾.生物化学实验指导[M].清华大学出版社,2004,120-122.
[28] Tang L, Zheng L, Qi CJ, Ma HB,et al. Preparation of a novel functional anti-human CD40 monoclonal antibody and identification of its bioactivity[J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2007,23(9):841-843.
[29]潘赛贻周洁娴黄健康.华支睾吸虫可溶性抗原对BALB/C鼠免疫效果的研究[J].广东卫生防疫,1998,24(3):1-3.
[30] Donald J.The basis of toxicity testing[M]. CRC Press,1977,168.
[31] Bantroch S, Buhler T, and Lam JS. Appropriate coating methods and other conditions for enzyme-linked immunosorbent assay of smooth, rough, and neutral lipopolysaccharides of pseudomonas aeruginosa[J]. Clin Diag Lab Immunol,1994,1(1):55-62.
[32] Fuller SA, Takahashi M, Hurrell JG. Preparation of mouse feeder cells for fusion and cloning[M]. Curr Protoc Mol Biol, 2001, Chapter 11:Unit11.6.
[33]徐志凯.实用单克隆抗体技术[M].陕西科学技术出版社,2006:1-29.
[34] Xuelin Zhou, Hu Zhao, Xiangdong Gao. Preparation and Characterization of Monoclonal Antibodies Against a Polysaccharide from Flammulina velutipes Mycelium[J]. Hybridoma, 2008, 27(6): 439-444.
[35] Zhao PC, Yuan DW, Zhu R,et al. Preparation of monoclonal antibody against sulfadiazine and development of an ELISA kit[J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2004,20(2):191-194.
[36] Yang T, Zhong P, Qu L, et al. Preparation and identification of anti-2, 4-dinitroph- enyl monoclonal antibodies[J].J Immunol Methods 2006,313(1–2):20-28.
[37] Su LJ, Xiao L, Zhan T, Chen L,et al. Preparation and identification of monoclonal antibodies against snake venom C-type lectin like protein Agkisacutacin[J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2007,23(11):1031-1033.
[38]张彩,田志刚.人NK细胞克隆化培养条件的初步探讨[J].细胞生物学杂志,2001,107-109.
[39]李刚,赵静,王保民,等.甘草有效成分甘草酸人工抗原的光谱鉴定研究[J].Special Wild Economic Animal and Plant Research,2005,27(2):17-18.
[40]赵肃清,蔡燕飞,雷红涛,等.农药甲胺磷人工抗原的光谱鉴定研究[J].光谱学与光谱分析,2004,24(2):207.
[41] Maria-Pilar Marco,Shirley Gee,Bruce D.Hammock,Immunochemical Techniques for Environmental Ana1ysis II.Antibody Production and immunoassay Development. Trends in An alytical Chemistry,1995,14(8):415.
[42]刘辉.免疫学与免疫学检验[M].人民军医出版社,2006,55-56.
[43]贲亚琍,朱德锐,刘德立.农药人工抗原合成方法的设计与优选[J].农药,2008,47(1):10-14.
[44]王兰兰.临床免疫学和免疫学检验[M].人民卫生出版社,2004,23-24.
[45]刘一超,戴晓雁,黄文才等.环孢菌素A人工抗原的制备[J] .四川大学学报,2001,38(3):407-410.
[46] Goka AK,Farthing MJ. The use of 3,3',5,5'-tetramethylbenzidine as a peroxidase substrate in microplate enzyme-linked immunosorbent assay[J]. J Immunoassay,1987,8(1):29-41.
[47]陈玉社,杨明峰,公丕欣,等.实验性变态反应性脑脊髓炎模型[J].泰山医学院学报,2006,27(1):72-74.
[48] Yu CY, Whitacre CC. Sex, MHC and complement C4 in autoimmune diseases[J]. Trends Immunol, 2004, 25(12): 694-699.
[49]楚电峰,杜元钊.免疫佐剂的应用与研究进展[J].中国动物检疫,2007,24(10):43-44.
[50] Witte P L,Ber R.Improved efficiency of hybridoma ascites production by intrasplenic inoculation in mice[J]. 1983,70(3):575-577.
[1] Kohler G,Milstein C.Continuous culture of fused cells secreting antibody of predefined specificity[J].Nature,1975,256(5517):495-497.
[2] M ike Clark.Antibody humanization:a case of the‘Emperor’S new clothes’[J]. Special issue of Immunology Today, 2000,21(8):394-402.
[3] Hughes D.Therapeutic antibodies make a comeback[J].Drug Discovery Today,1998,3(10):439-442.
[4]罗萍,邹全明,毛旭虎.治疗性单克隆抗体研究进展及临床应用现状[J].第三军医大学学报,2004,26(5):454-456.
[5] Presta LG. Engineering of therapeutic antibodies to minimize immunogenicity and optimize function[J].Adv Drug Deliv Rev,2006,58(5-6):640-656.
[6] Qian W, Wang L, Li B, et al. Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity[J]. Biochem Biophys Res Commun,2008, 367(2):497-502.
[7] Carter P. Improving the efficacy of antibody—based cancer therapies[J].Nat Rev Cancer,2001,2(11):118-129.
[8] Gabriel HM, Oliveira EI. Role of abciximab in the treatment of coronary artery disease[J]. Expert Opin Biol Ther,2006,6(9):935-942.
[9]葛彦.人源化抗体研制策略分析及应用研究[J].国外医学免疫学分册,2004,27(5):271-274.
[10] Ishida T, Tmai K. The expression technology of chimeric and humanized antibodies[J]. Nippon Rinsho,2002,60(3):439-444.
[11] Riechmann L,Clark M,Waldmann H,et a1.Reshaping human antibodies for therapy[J].Nature,1988,332(6162):323-327.
[12]王秀红,周素芳.基因工程抗体研究进展[J].生物技术通讯,2007,18(2):304-306.
[13] Caldas C, Coelho V, Kalil J, et al. Humanizaion of the anti-18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen[J]. Mol Immunol, 2003,39(15):941-952.
[14] Nakanishi T,Tsumoto K,Yokota A,et al. Critical contribution of VH-VL interaction to reshaping of an antibody: the case of humanization of anti-lysozyme antibody, HyHEL-10[J]. Protein Sci,2008,17(2):261-270.
[15]朱啸林,程枫,钱关祥.基于互补决定区移植的单抗人源化进展[J].上海交通大学学报:医学版,2007,27(11):1382-1384.
[16] Wu H, Nie Y, Huse W D, et al. Hummanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues[J]. J Mol Biol, 1999, 294:151-162.
[17]贾建桃,马丽菊,王秦秦.治疗性单克隆抗体研究新进展[J].医学综述,2007,13(5):325-327.
[18]彭如意,王捷.人源化治疗性单克隆抗体的研究进展[J].实用医学杂志,2007,23(5):753-754.
[19] Lonberg N. Fully human antibodies from transgenic mouse and phage display platforms[J]. Curr Opin Immunol,2008,20(4):450-459.
[20]刘松友,王长太.单克隆抗体治疗制剂研究与应用[J].国外医学(预防、诊断、治疗用生物制品分册),2005,28(4):177-179.
[21]陈立慧,宋海峰,刘秀文.治疗性人源化单克隆抗体研究进展[J].中国新药杂志,2004,13(7) :590-594.
[22]吴永强,董关木.人源化单克隆抗体研究进展[J].微生物学免疫学进展,2008,36(2):73-77.
[23] Lonberg N. Human monoclonal antibodies from transgenic mice[J]. Handb Exp Pharmacol,2008,181:69-97.
[24] Foon KA, Yang XD, Weiner LM,et al. Preclinical and clinical evaluations of ABX-EGF,a fully human anti-epidermal growth factor receptor antibody[J].Int J Radiat Oncol Biol Phys,2004,58(3):984-990.
[25] Willemsen R,Chames P,Schooten E,et al. Selection of human antibody fragmentsdirected against tumor T-cell epitopes for adoptive T-cell therapy[J]. Cytometry A, 2008,73(11):1093-1099.
[26]杜阳峰,罗荣城,李贵平,等.抗乙型肝炎表面抗原Fab片段联合抗细胞核单抗为载体的肝癌放射免疫治疗实验研究[J].南方医科大学学报,2008,28(3):460-462.
[27] Gafner V,Trachsel E,Neri D. An engineered antibody-interleukin-12 fusion protein with enhanced tumor vascular targeting proteins[J]. Int J Cancer,2006,119(9):2205-2212.
[28]龚明霞.基因工程抗体的研究[J].医学综述,2003,9(2) :69-71.
[29]卢筱华,杨冬华,汤绍辉,等.错配PCR和DNA改组技术提高抗肝癌单链抗体亲和力[J].中国病理生理杂志,2006,22(6):1203-1206.
[30]吴小平,间锡蕴,杨东玲,等.人单链抗体亲和力成熟研究[J].中国病理生理杂志,2001,21(2):156-159.
[31]崔华清,王清明.基于重链抗体构建的单域抗体研究进展[J].生物工程学报,2005,21:497-501.
[32] Caldas C, Coelho V, Kalil J, et al. Humannization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen[J]. Mol Immunol,2003,39(15):941-952.
[33] Müller D, Kontermann RE. Recombinant bispecific antibodies for cellular cancer immunotherapy[J]. Curr Opin Mol Ther,2007,9(4):319-26.
[34] Asano R, Kawaguchi H, Watanabe Y,et al. Diabody-based recombinant formats of humanized IgG-like bispecific antibody with effective retargeting of lymphocytes to tumor cells[J]. J Immunother,2008,31(8):752-761.
[35]刘辉.免疫学与免疫学检验[M].北京:人民军医出版社,2006,69.
[36]戴克胜.双特异性单链抗体[J].国外医学免疫学分册,2003,26(3) :123-125.
[37]沈备奋.治疗性抗体的改造[J]. Med Mol Biol, 2006,3(4) :245-249.
[38]胡晓林,张春明.基因工程抗体亲和力成熟的策略[J].免疫学杂志,2006,22(6): 702-704.
[39] Chowdhury P S. Engineering hot spots for affinity enchancement of antibodies[J].Methods Mol Biol, 2003, 207:179-196.
[40] Puja s, Rhona S. Anti-CD74 antibody doxorubicin conjugate, IMMU-11O,in a human multiple myelona xenograft and in monkeys[J].Clinical Cancer Research,2005,11:5257-5264.
[41]朱洁,王绮如.单克隆抗体药物在肿瘤靶向治疗中的应用与展望[J].中国医师杂志,2007,9(7):1006-1007.
[42]张寒,邹丽敏,郑胡镛.急性淋巴细胞白血病最新治疗策略(中)[J].中国小儿血液与肿瘤杂志,2007,12(5):233-235.
[43] Walsh G. Biopharmaceutical bienchmarks-2003[J]. Nat Biotechnol, 2003,21(8):865-870.
[44]刘兢.生物技术和药物研究进展[J].中国科学技术大学出版社,2008,38(8):905-908.
[45] Laffly E, Sodoyer R. Monoclonal antibodies, 30 years of success[J] .J Soc Biol,2006,200(4):325-343.