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High throughput monoclonal antibody generation by immunizing multiple antigens

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Science China(Life Sciences) 2014年第7期57卷 710-717页

作者： LIU Ying WANG YunDan LIU Jing ZUO Wei HAO Lu ZHANG LiLi ZHEN Bei State Key Laboratory of Proteomics beijing proteome research center Beijing Institutes of Life Science Chinese Academy of Sciences Anhui Medical University

Recognizing proteins via the production of highly specific monoclonal antibodies(mabs)is crucial to identifying proteins for proteomic ***,traditional mab generation is time-consuming with low *** this study,we assessed the high throughput method of producing mabs by immunizing mice with multiple antigens in order to obtain hybridomas against these multiple antigens in one cell *** selected eight proteins that play important roles in human physiological or pathological *** proteins were mixed and simultaneously administered to one *** observed the immunizing period for 10 d,and determined the effect of liquid medium and semi-solid medium in hybridoma *** a result,all eight immunogens induced antibodies in the immunized mouse in one cell fusion,we obtained hybridomas specific to all eight proteins by enzyme-linked immuno sorbent assay(ELISA)screening,hybridomas against five out of eight showed specific positive in Western-blotting *** indicates that we generated mabs specific to eight proteins in one cell fusion,greatly increasing the efficiency of mab ***,we observed that hybridomas selected from the liquid medium and semi-solid medium showed different reactivity to *** study established high-throughput and time-saving methods for production of *** results provide alternative approaches for increasing the efficacy of mab generation.

关键词： high throughput mab multiple antigen immunization ELISA

Production and Characterization of Monoclonal Antibodies of Shrimp White Spot Syndrome Virus Envelope Protein VP28

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中国病毒学 2007年第1期22卷 21-25页

作者： Wan-gang GU Jun-fa YUAN Ge-lin XU Li-juan LI Ni LIU Cong ZHANG Jian-hong ZHANG Zheng-li SHI State Key Laboratory of Virology Wuhan Institute of Virology Chinese Academy of Sciences Wuhan 430071 China Wuhan Institute of Biological Products Ministry of Health Wuhan 430060 China College of Life Science Central China Normal University Wuhan 430079 China

BALB/c 老鼠与净化的白点症候群病毒(WSSV ) 被使免疫。六根 monoclonal 抗体房间线被 ELISA 选择， VP28 蛋白质在 E 表示了。coli。在 vitro 中立化，实验证明他们中的 4 个能在喇蛄禁止病毒感染。西方污点建议所有这些 monoclonal ... 详细信息

BALB/c 老鼠与净化的白点症候群病毒(WSSV ) 被使免疫。六根 monoclonal 抗体房间线被 ELISA 选择， VP28 蛋白质在 E 表示了。coli。在 vitro 中立化，实验证明他们中的 4 个能在喇蛄禁止病毒感染。西方污点建议所有这些 monoclonal 抗体对 VP28 的 conformational 结构。monoclonal 抗体 7B4 用胶体的金粒子被标记并且过去常由标记的 immunogold 在病毒信封上定位 VP28。这些 monoclonal 抗体能被用来为 WSSV 感染开发免疫学的诊断方法。关键词怀特点症候群病毒(WSSV )- mab - 信封 - 本地化 - 中立化 CLC 数字 S945.

关键词： White spot syndrome virus (WSSV) mab Envelope Localization Neutralization

mabHIM_(70)对人NpNADPH氧化酶的调节(Ⅱ)

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科学技术与工程 2002年第3期2卷 34-37页

作者：法祥光杨琳中国医学科学院天津300020 中国协和医科大学血液学研究所天津300020

在发现 mab HIM_(70)对 Np NADPH 氧化酶胞浆组分p47^(phox)、p67^(phox)和 Rac2的表达和参与 NADPH 氧化酶活化均有降调节作用基础上,进一步探索了 mab HIM_(70)对该降调节作用的机理。发现 mab HIM_(70)可能对 NADPH 氧化酶上游 PKC、... 详细信息

在发现 mab HIM_(70)对 Np NADPH 氧化酶胞浆组分p47^(phox)、p67^(phox)和 Rac2的表达和参与 NADPH 氧化酶活化均有降调节作用基础上,进一步探索了 mab HIM_(70)对该降调节作用的机理。发现 mab HIM_(70)可能对 NADPH 氧化酶上游 PKC、PTK 及 P13K 信号传递途径的活化水平有降调节作用,该单抗对 NADPH 氧化酶活化有降调节的作用可能与此有关。

关键词： mab HIM70 调节人体细胞 NADPH氧化酶 p47^phox p67^phox Rac2 中性粒细胞激酶抑制剂降调节作用

对位取代苯甲酸酯晶体结构研究——Ⅱ.对氨基苯甲酸甲酯p-（H2N）C6H4CO2CH3的晶体结构

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结构化学 1983年第3期 219-223页

作者：林贤梯中国科学院福建物质结构研究所

对氨基苯甲酸甲酯（简写为p—mab）晶体结构已用X射线衍射法测定。晶体属单斜晶系,空间群为C2/c,晶胞参数a=16.242（2）,b=8.113（2）,c=12.724（2）,β=69.17（1）°;V=1567.13。ρobs=1.284gcm-3(ρcal=1.281gcm-3),Z=8。使用CAD4四圆衍射仪和MOKα射线收集强度数据,晶体结构用直接法解出,经全矩阵最小二乘修正,对于1215个I>3σ（I）的衍射点最后偏离因子R=0.050。

关键词：晶体结构直接法 mab 射线衍射结晶构造固体结构 H2N)C6H4CO2CH3 苯环平面晶架群空间群晶胞参数 CH CO

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STUDY OF RADIOIMMUNOIMAGING WITH MONOCLONAL ANTIBODY AGAINST THE PATIENTS WITH SCLC

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Chinese Journal of Cancer Research 1991年第4期3卷 59-60页

作者：李斯德洪锦心田培坤莫忠根苗积生谈立松吴姚裘章英剑蒋尚英蒋茂松娄葆金 Shanghai Cancer Institute Shanghai First Pneumology Hospital Cancer Hospital Shanghai Medical University Hua Dong Hospital Shanghai

It is an Innovative way to diagnose with cancer with monoclonal antibody (mab) in vivo. 1-3 After proving mab 2F7 (IgG2a) had a higher affinity and better specificity to human small cell lung cancer cells in vitro, 4.5 and gave a very clear cut γ- camera pictures for the xenografts of human small cell lung cancer (SCLC) born by nude mice, 6 the possibility of clinical radioimmunoimaging was studied with the mab 2F7 and its fragment F (ab') 2.

关键词： STUDY OF RADIOIMMUNOIMAGING WITH MONOCLONAL ANTIBODY AGAINST THE PATIENTS WITH SCLC ab than mab

RADIOIMMUNOLOCALIZATION OF XENOGRAFTED HUMAN GASTRIC ADENOCARCINOMA WITH ^(131)I-LABELED MONOCLONAL ANTIBODY RWS_(4) IN NUDE MICE

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Chinese Journal of Cancer Research 1990年第3期2卷 50-53页

作者：吴裕炘金道友余春仙江绍基朱承谟史爱蓝李培勇张素胤 Rui Jin Hospital Shanghai Second Medical University Shanghai Institute of Digestive Disease Shanghai Institute of Materia Medica Academia Sinicahe monoclonal antibody (MAb) RWS4 specific to membrane-associated antigen of human gastric adenocarcinoma was purified by protein A-Sepharose 4B affinity chromatography and labeled with 131I by chloramine-T method. 131-RWS was injected (65 μCi/10μg/0.2 ml intraperitoneally) into the stomach cancer-bearing nude mice (solid tumor about 1 cm in diameter) and its biodistribution was studied by SPECT and gamma-counter over a peroid of 7 days. A clear image of transplanted tumor was observed on the 4th day and the image became more clear on the 6th day. After SPECT scanning the animals were killed on the 3rd to 7th day separately and radioactivity was detected in various organs. The ratios of T/NT were calculated. The results were shown as follows: tumor/blood was 3.41±0.29 on the 6th day and the tumor/other organs (liver spleen stomach lung heart kidney and brain etc.) were>3. The specificity of the 131I-RWS4 was 7.74±0.65.

The monoclonal antibody (mab) RWS4 specific to membrane-associated antigen of human gastric adenocarcinoma was purified by protein A-Sepharose 4B affinity chromatography and labeled with 131I by chloramine-T method. 131-RWS,, was injected (65 μCi/10μg/0.2 ml, intraperitoneally) into the stomach cancer-bearing nude mice (solid tumor about 1 cm in diameter), and its biodistribution was studied by SPECT and gamma-counter over a peroid of 7 days. A clear image of transplanted tumor was observed on the 4th day, and the image became more clear on the 6th day. After SPECT scanning, the animals were killed on the 3rd to 7th day separately and radioactivity was detected in various organs. The ratios of T/NT were calculated. The results were shown as follows: tumor/blood, was 3.41±0.29 on the 6th day and the tumor/other organs (liver, spleen, stomach, lung, heart, kidney and brain etc.) were>3. The specificity of the 131I-RWS4 was 7.74±0.65.

关键词： IgG IN NUDE MICE I-LABELED MONOCLONAL ANTIBODY RWS mab SPECT

抗hnRNPB1单克隆抗体结合^(131)I对肺癌小鼠靶向治疗的实验研究

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四川大学学报（医学版） 2007年第5期38卷 766-769页

作者：陈明勇李为民徐丹陈文彬四川大学华西医院呼吸内科成都610041 四川大学华西医院呼吸病研究室

目的观察131I-抗hnRNPB1单克隆抗体(131I-抗hnRNPB1 mab)、抗hnRNPB1 mab对肺癌小鼠移植瘤的靶向治疗作用。方法用常规氯胺-T法合成131I-抗hnRNPB1 mab,30只C57BL/6小鼠皮下接种Lewis肺癌细胞7 d后,将荷瘤鼠随机分成5组,每组6只。分别... 详细信息

目的观察131I-抗hnRNPB1单克隆抗体(131I-抗hnRNPB1 mab)、抗hnRNPB1 mab对肺癌小鼠移植瘤的靶向治疗作用。方法用常规氯胺-T法合成131I-抗hnRNPB1 mab,30只C57BL/6小鼠皮下接种Lewis肺癌细胞7 d后,将荷瘤鼠随机分成5组,每组6只。分别经尾静脉注射,组:131I-抗hnRNPB1 mab 11.1 MBq/鼠单次治疗,组:131I-抗hnRNPB1 mab 11.1 MBq/鼠强化治疗(给药2次,每次给药间隔3 d),组:Na131I 11.1MBq/鼠,组:抗hnRNPB1 mab 50μg/鼠,组(对照组):生理盐水0.12 mL/鼠。治疗开始后每周测量1次小鼠肿瘤的长径及短径,4周后处死小鼠,分离肿瘤并称其质量,计算抑瘤率,肿瘤组织作常规HE染色检查。结果治疗结束时,、、、、组小鼠的平均肿瘤体积分别为(3869±192)mm3、(1987±149)mm3、(6922±532)mm3、(6962±509)mm3、(6597±521)mm3,其中、组分别与对照组相比差异具有统计学意义(Pmab结合131I对肺癌小鼠移植瘤的生长具有明显抑制作用,且抑制作用呈量效关系。

关键词：肺癌抗hnRNPB1 mab ^131I 放射免疫治疗

抗B7-H3单克隆抗体抑制雨蛙肽诱导的小鼠急性胰腺炎炎症反应和组织损伤

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细胞与分子免疫学杂志 2016年第3期32卷 323-327页

作者：庄晓惠许春芳苏州大学附属第一医院消化内科江苏苏州215006

目的探究抗B7-H3单克隆抗体(m Ab)在雨蛙肽诱导的急性胰腺炎(AP)中的作用。方法将小鼠随机分为对照组、AP组、抗B7-H3 m Ab治疗组。采用腹腔注射雨蛙肽的方法制备小鼠AP模型,治疗组于造模前1 h皮下注射抗B7-H3 m Ab,于造模后6、12、24 h... 详细信息

目的探究抗B7-H3单克隆抗体(m Ab)在雨蛙肽诱导的急性胰腺炎(AP)中的作用。方法将小鼠随机分为对照组、AP组、抗B7-H3 m Ab治疗组。采用腹腔注射雨蛙肽的方法制备小鼠AP模型,治疗组于造模前1 h皮下注射抗B7-H3 m Ab,于造模后6、12、24 h,收集各组小鼠血液、胰腺及肺组织。采用Western blot法和免疫组织化学染色检测对照组及AP组小鼠胰腺组织B7-H3蛋白表达。应用全自动生化免疫分析仪检测各组小鼠血清淀粉酶及脂肪酶活性;胰腺组织湿干比评估各组小鼠胰腺组织水肿变化;HE染色评估各组小鼠胰腺及肺组织病理变化,利用ELISA检测各组小鼠血清中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-1β水平。结果 B7-H3蛋白在AP模型小鼠胰腺组织高表达,且在12 h左右达到高峰;AP组血清淀粉酶及脂肪酶明显高于对照组,抗B7-H3 m Ab处理后明显降低;AP组胰腺及肺组织出现明显炎症反应,治疗组炎症反应明显减弱,胰腺组织湿干比大幅降低;AP组TNF-α、IL-6、IL-1β水平随时间延长而升高,12 h达到高峰后开始下降,而治疗组促炎因子水平相对降低。结论 B7-H3在雨蛙肽诱导的AP模型中存在高表达,抗B7-H3 m Ab可以减弱炎症反应,缓解胰腺及肺组织的损伤。

关键词：抗B7-H3 mab 急性胰腺炎肿瘤坏死因子α 白细胞介素6(IL-6) IL-1β

抗藤黄微球菌Rpf结构域单克隆抗体的制备与特性鉴定

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细胞与分子免疫学杂志 2008年第5期24卷 484-487页

作者：樊爱琳师长宏苏明权马静柏银兰程晓东徐志凯郝晓柯第四军医大学西京医院检验科陕西西安710032 第四军医大学训练部实验动物中心陕西西安710032 第四军医大学西京医院中医科陕西西安710032 第四军医大学基础部微生物学教研室陕西西安710032

目的:克隆、表达藤黄微球菌Rpf结构域多肽,并制备针对该多肽的单克隆抗体(mab)。方法:采用聚合酶链反应(PCR)藤黄微球菌基因组中扩增出藤黄微球菌Rpf结构域基因,用限制性内切酶消化后插入到pUC-19克隆载体中,测序正确后,再亚克隆到融合... 详细信息

目的:克隆、表达藤黄微球菌Rpf结构域多肽,并制备针对该多肽的单克隆抗体(mab)。方法:采用聚合酶链反应(PCR)藤黄微球菌基因组中扩增出藤黄微球菌Rpf结构域基因,用限制性内切酶消化后插入到pUC-19克隆载体中,测序正确后,再亚克隆到融合表达载体pPro-EXHT中,转化大肠杆菌DH5α,目的基因经IPTG诱导,由T7启动子调控表达了氨基端带6个连续组氨酸残基的Rpf结构域基因多肽,在变性条件下纯化目的蛋白。用纯化的目的蛋白作为免疫原免疫BALB/c小鼠,进行细胞融合、克隆化制备抗Rpf domain单抗(mab),用ELISA法初步鉴定其特异性和相对亲和力。结果:在变性条件下用Ni2+-NTA亲和色谱柱纯化目的融合蛋白,纯化获得的蛋白纯度大于90%。用该融合蛋白免疫小鼠后,获得了3株抗Rpfdomain mab,这3株mab均特异性识别Rpf domain多肽。结论:所获得的抗Rpfdomain mab特异性强、效价高,对进一步研究Rpf domain在结核病免疫预防中的作用提供了有力的工具。

关键词：结核分枝杆菌藤黄微球菌 Rpf domain mab